Treatment of Psoriasis with Mesenchymal Stem Cells

2021年1月第28卷第1期 中国中医药信息杂志 ·139· 综述银屑病肠道菌群中西医研究进展徐婉莹1，郭菲2，刘红霞21.新疆医科大学第四临床医学院，新疆乌鲁木齐 830000；2.新疆维吾尔自治区中医医院，新疆乌鲁木齐 830000摘要：银屑病是一种以慢性炎症性为主要发病特征且与免疫相关的疾病。

研究表明，肠道菌群与免疫疾病有密切关系，尤其与炎症性肠道疾病关系密切。

银屑病发病机制与肠道菌群紊乱的机制有相似之处。

通过中医辨治对肠道菌群维持稳态的研究，从炎症性肠道疾病的角度进行探索，为调节银屑病肠道菌群的中医药临床治疗及诊断提供了一定依据。

本文对目前关于银屑病肠道菌群的中西医研究进行了综述。

关键词：银屑病；肠道菌群；综述中图分类号：R275.986.3 文献标识码：A 文章编号：1005-5304(2021)01-0139-03DOI：10.19879/ki.1005-5304.202002365 开放科学（资源服务）标识码（OSID）：Research Progress in TCM and Western Medicine forIntestinal Flora of PsoriasisXU Wanying1, GUO Fei2, LIU Hongxia21. The Fourth Clinical Medical College of Xinjiang Medical University, Urumqi 830000, China;2. TraditionalChinese Medicine Hospital of Xinjiang Uygur Autonomous Region, Urumqi 830000, China Abstract: Psoriasis is a disease characterized by chronic inflammation and related to immunity. Studies have shown that intestinal flora is closely related to immune diseases, especially inflammatory intestinal diseases. The pathogenesis mechanism of psoriasis is similar to that of intestinal flora disorder. From the point of view of inflammatory intestinal diseases, the study on TCM differentiation and treatment to maintain the steady state of intestinal flora provided a certain basis for the clinical treatment and diagnosis of intestinal flora of psoriatic. This article reviewed the current research on TCM and Western medicine for intestinal flora of psoriasis.Keywords: psoriasis; intestinal flora; review银屑病是主要由遗传和环境因素影响的免疫介导的炎性、系统性疾病[1]。

Standards of Medical Care in Diabetes A MERICAN D IABETES A SSOCIATIOND iabetes is a chronic illness that re-quires continuing medical care andpatient self-management education to prevent acute complications and to re-duce the risk of long-term complications. Diabetes care is complex and requires that many issues,beyond glycemic control,be addressed.A large body of evidence exists that supports a range of interventions to improve diabetes outcomes.These standards of care are intended to provide clinicians,patients,research-ers,payors,and other interested persons with the components of diabetes care, treatment goals,and tools to evaluate the quality of care.While individual prefer-ences,comorbidities,and other patient factors may require modification of goals, targets that are desirable for most patients with diabetes are provided.These stan-dards are not intended to preclude more extensive evaluation and management of the patient by other specialists as needed. For more detailed information,refer to Bode(Ed.):Medical Management of Type1 Diabetes(1),Zimmerman(Ed.):Medical Management of Type2Diabetes(2),and Klingensmith(Ed):Intensive Diabetes Management(3).The recommendations included are diagnostic and therapeutic actions that are known or believed to favorably affect health outcomes of patients with diabetes.A grading system(Table1),developed by the American Diabetes Association(ADA) and modeled after existing methods,was utilized to clarify and codify the evidence that forms the basis for the recommenda-tions.The level of evidence that supports each recommendation is listed after eachrecommendation using the letters A,B,C,or E.CLASSIFICATION,DIAGNOSIS,ANDSCREENINGClassificationIn1997,the ADA issued new diagnosticand classification criteria(4);in2003,modifications were made regarding thediagnosis of impaired fasting glucose(IFG)(5).The classification of diabetesincludes four clinical classes:●Type1diabetes(results from␤-cell de-struction,usually leading to absoluteinsulin deficiency).●Type2diabetes(results from a progres-sive insulin secretory defect on thebackground of insulin resistance).●Other specific types of diabetes(due toother causes,e.g.,genetic defects in␤-cell function,genetic defects in insu-lin action,diseases of the exocrine pan-creas,drug or chemical induced).●Gestational diabetes mellitus(GDM)(diagnosed during pregnancy).DiagnosisCriteria for the diagnosis of diabetes innonpregnant adults are shown in Table2.Three ways to diagnose diabetes are avail-able,and each must be confirmed on asubsequent day unless unequivocalsymptoms of hyperglycemia are present.Although the75-g oral glucose tolerancetest(OGTT)is more sensitive and mod-estly more specific than fasting plasmaglucose(FPG)to diagnose diabetes,it ispoorly reproducible and rarely performedin practice.Because of ease of use,accept-ability to patients,and lower cost,theFPG is the preferred screening and diag-nostic test.It should be noted that the vastmajority of people who meet diagnosticcriteria for diabetes by OGTT,but not byFPG,will have an A1C valueϽ7.0%.Theuse of the A1C for the diagnosis of diabe-tes is not recommended at this time.Hyperglycemia not sufficient to meetthe diagnostic criteria for diabetes is cate-gorized as either IFG or impaired glucosetolerance(IGT),depending on whether itis identified through FPG or an OGTT:●IFGϭFPG100mg/dl(5.6mmol/l)to125mg/dl(6.9mmol/l)●IGTϭ2-h plasma glucose140mg/dl(7.8mmol/l)to199mg/dl(11.0mmol/l)Recently,IFG and IGT have been offi-cially termed“pre-diabetes.”Both catego-ries,IFG and IGT,are risk factors forfuture diabetes and cardiovascular dis-ease(CVD).Recent studies have shownthat modest weight loss and regular phys-ical activity can reduce the rate of progres-sion of IGT to type2diabetes(6–8).Drugtherapy(metformin[8],acarbose[9],andorlistat[10]and troglitazone[no longerclinically available][11])has been shownto be effective in reducing progression todiabetes,though generally not as effec-tively as intensive lifestyle interventions.ScreeningGenerally,people with type1diabetespresent with acute symptoms of diabetesand markedly elevated blood glucose lev-els.Type2diabetes is frequently not di-agnosed until complications appear,andapproximately one-third of all peoplewith diabetes may be undiagnosed.Al-though the burden and natural history ofdiabetes is well known and although thereis good evidence for benefit from treatingcases diagnosed in the context of usualclinical care,there are no randomized tri-als demonstrating the benefits of earlydiagnosis through screening of asymp-tomatic individuals(12).Nevertheless,there is sufficient indirect evidence to jus-●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●The recommendations in this paper are based on the evidence reviewed in the following publication: Standards of care for diabetes(Technical Review).Diabetes Care17:1514–1522,1994.Originally approved1988.Most recent review/revision,October2003.Abbreviations:ABI,ankle-brachial index;ARB,angiotensin receptor blocker;CAD,coronary artery disease;CHD,coronary heart disease;CSII,continuous subcutaneous insulin injection;CVD,cardiovascular disease;FPG,fasting plasma glucose;GCT,glucose challenge test;DCCB,dihydropyridine calcium channel blocker;DCCT,Diabetes Control and Complications Trial;DKA,diabetic ketoacidosis;DRS,Diabetic Retinopathy Study;ECG,electrocardiogram;eGFR,estimated GFR;ESRD,end-stage renal disease;ETDRS, Early Treatment Diabetic Retinopathy Study;GDM,gestational diabetes mellitus;GFR,glomerularfiltration rate;HRC,high-risk characteristic;IFG,impaired fasting glucose;IGT,impaired glucose tolerance;MNT, medical nutrition therapy;NPDR,nonproliferative diabetic retinopathy;OGTT,oral glucose tolerance test; PAD,peripheral arterial disease;PDR,proliferative diabetic retinopathy;PPG,postprandidial plasma glu-cose;SMBG,self-monitoring of blood glucose;UKPDS,U.K.Prospective Diabetes Study.©2004by the American Diabetes Association.P O S I T I O N S T A T E M E N Ttify opportunistic screening in a clinical setting of individuals at high risk.Criteria for testing for diabetes in asymptomatic, undiagnosed adults are listed in Table3. The recommended screening test for non-pregnant adults is the FPG.The OGTT is more sensitive for the diagnosis of diabe-tes and pre-diabetes,but is impractical and expensive as a screening procedure.The incidence of type2diabetes in children and adolescents has increased dramatically in the last decade.Consis-tent with screening recommendations for adults,only children and youth at in-creased risk for the presence or the devel-opment of type2diabetes should be tested(13)(Table4).Detection and diagnosis of GDM Risk assessment for GDM should be un-dertaken at thefirst prenatal visit.Women with clinical characteristics consistentwith a high risk for GDM(those withmarked obesity,personal history of GDM,glycosuria,or a strong family history ofdiabetes)should undergo glucose testingas soon as possible(14).An FPGՆ126mg/dl or a casual plasma glucoseՆ200mg/dl meets the threshold for the diagno-sis of diabetes and needs to be confirmedon a subsequent day unless unequivocalsymptoms of hyperglycemia are present.High-risk women not found to have GDMat the initial screening and average-riskwomen should be tested between24and28weeks of gestation.Testing should fol-low one of two approaches:●One-step approach:perform a diagnos-tic100-g OGTT●Two-step approach:perform an initialscreening by measuring the plasma orserum glucose concentration1h after a50-g oral glucose load(glucose chal-lenge test[GCT])and perform a diag-nostic100-g OGTT on that subset ofwomen exceeding the glucose thresh-old value on the GCT.When the two-step approach is used,a glucosethreshold valueՆ140mg/dl identifiesϳ80%of women with GDM,and theyield is further increased to90%by us-ing a cutoff ofՆ130mg/dl.●Diagnostic criteria for the100-g OGTTare as follows:Ն95mg/dl fasting,Ն180mg/dl at1h,Ն155mg/dl at2h,andՆ140mg/dl at3h.Two or more ofthe plasma glucose values must be metor exceeded for a positive diagnosis.The test should be done in the morningafter an overnight fast of8–14h.Thediagnosis can be made using a75-g glu-cose load,but that test is not as wellTable1—ADA evidence grading system for clinical practice recommendationsLevel ofevidence DescriptionA Clear evidence from well-conducted,generalizable,randomized controlled trials that are adequately powered including:●Evidence from a well-conducted multicenter trial●Evidence from a meta-analysis that incorporated quality ratings in the analysis●Compelling nonexperimental evidence,i.e.,“all or none”rule developed by Center for Evidence Based Medicine at OxfordSupportive evidence from well-conducted randomized controlled trials that are adequately powered including:●Evidence from a well-conducted trial at one or more institutions●Evidence from a meta-analysis that incorporated quality ratings in the analysisB Supportive evidence from well-conducted cohort studies●Evidence from a well-conducted prospective cohort study or registry●Evidence from a well-conducted prospective cohort study●Evidence from a well-conducted meta-analysis of cohort studiesSupportive evidence from a well-conducted case-control studyC Supportive evidence from poorly controlled or uncontrolled studies●Evidence from randomized clinical trials with one or more major or three or more minor methodologicalflaws that couldinvalidate the results●Evidence from observational studies with high potential for bias(such ascase series with comparison to historical controls)●Evidence from case series or case reportsConflicting evidence with the weight of evidence supporting the recommendationE Expert consensus or clinical experienceTable2—Criteria for the diagnosis of diabetes1.Symptoms of diabetes and a casual plasma glucose200mg/dl(11.1mmol/l).Casual is defined as any time of day without regard to timesince last meal.The classic symptoms of diabetes include polyuria,polydipsia,and unexplained weight loss.OR2.FPG126mg/dl(7.0mmol/l).Fasting is defined as no caloric intake for at least8h.OR3.2-h PG200mg/dl(11.1mmol/l)during an OGTT.The test should be performed as described by the World Health Organization(4a),using a glucose load containing the equivalent of75-g anhydrous glucose dissolved in water.In the absence of unequivocal hyperglycemia,these criteria should be confirmed by repeat testing on a different day.The OGTT is not recommended for routine clinical use,but may be required in the evaluation of patients with IFG(see text)or when diabetes is still suspected despite a normal FPG.Position Statementvalidated for detection of at-risk infants or mothers as the100-g OGTT.●Low-risk status requires no glucose testing,but this category is limited to those women meeting all of the follow-ing characteristics:•AgeϽ25years.•Weight normal before pregnancy.•Member of an ethnic group with alow prevalence of GDM.•No known diabetes infirst-degreerelatives.•No history of abnormal glucose tol-erance.•No history of poor obstetric out-come.Recommendations●The FPG is the preferred test to screen for and diagnose diabetes in children and nonpregnant adults.(E)●Screen for diabetes in high-risk,asymp-tomatic,undiagnosed adults and chil-dren within the health care setting.(E)●In those with pre-diabetes(IFG/IGT), lifestyle modification should bestrongly recommended and progres-sion of glycemic abnormalities followedby screening at least yearly.(A)●Screen for diabetes in pregnancy usingrisk factor analysis and screening testsas noted;the OGTT is the preferredscreening test in pregnancy.(E)INITIAL EVALUATIONA complete medical evaluation should beperformed to classify the patient,detectthe presence or absence of diabetes com-plications,assist in formulating a manage-ment plan,and provide a basis forcontinuing care.If the diagnosis of diabe-tes has already been made,the evaluationshould review the previous treatment andthe past and present degrees of glycemicboratory tests appropriate tothe evaluation of each patient’s generalmedical condition should be performed.A focus on the components of compre-hensive care(Table5)will assist thehealth care team to ensure optimal man-agement of the patient with diabetes.MANAGEMENTPeople with diabetes should receivemedical care from a physician-coordi-nated team.Such teams may include,but are not limited to,physicians,nurse practitioners,physician’s assis-tants,nurses,dietitians,pharmacists,andmental health professionals with exper-tise and a special interest in diabetes.It isessential in this collaborative and inte-grated team approach that individualswith diabetes assume an active role intheir care.The management plan should be for-mulated as an individualized therapeuticalliance among the patient and family,thephysician,and other members of thehealth care team.Any plan should recog-nize diabetes self-management educationas an integral component of care.In de-veloping the plan,consideration shouldbe given to the patient’s age,school orwork schedule and conditions,physicalactivity,eating patterns,social situationand personality,cultural factors,andpresence of complications of diabetes orTable3—Criteria for testing for diabetes in asymptomatic adult individuals1.Testing for diabetes should be considered in all individuals at age45years and above,particularly in those with a BMI25kg/m2*,and,ifnormal,should be repeated at3-year intervals.2.Testing should be considered at a younger age or be carried out more frequently in individuals who are overweight(BMI25kg/m2*)andhave additional risk factors:●are habitually physically inactive●have afirst-degree relative with diabetes●are members of a high-risk ethnic population(e.g.,African American,Latino,Native American,Asian American,Pacific Islander)●have delivered a baby weighingϾ9lb or have been diagnosed with GDM●are hypertensive(140/90mmHg)●have an HDL cholesterol level35mg/dl(0.90mmol/l)and/or a triglyceride level250mg/dl(2.82mmol/l)●have PCOS●on previous testing,had IGT or IFG●have other clinical conditions associated with insulin resistance(e.g.PCOS or acanthosis nigricans)●have a history of vascular disease*May not be correct for all ethnic groups.PCOS,polycystic ovary syndrome.Table4—Testing for type2diabetes in children●CriteriaOverweight(BMIϾ85th percentile for age and sex,weight for heightϾ85th percentile,or weightϾ120%of ideal for height)PlusAny two of the following risk factors:Family history of type2diabetes infirst-or second-degree relativeRace/ethnicity(Native American,African American,Latino,Asian American,Pacific Islander)Signs of insulin resistance or conditions associated with insulin resistance(acanthosis nigricans,hypertension,dyslipidemia,or PCOS)●Age of initiation:age10years or at onset of puberty,if puberty occurs at a younger age●Frequency:every2years●Test:FPG preferredClinical judgment should be used to test for diabetes in high-risk patients who do not meet these criteria.PCOS,polycystic ovary syndrome.Standards of Medical Careother medical conditions.A variety of strategies and techniques should be used to provide adequate education and devel-opment of problem-solving skills in the various aspects of diabetes management.Implementation of the management planrequires that each aspect is understoodand agreed on by the patient and the careproviders and that the goals and treat-ment plan are reasonable.Glycemic controlGlycemic control is fundamental to themanagement of diabetes.Prospective ran-domized clinical trials such as the Diabe-tes Control and Complications TrialTable5—Components of the comprehensive diabetes evaluationMedical history●Symptoms,results of laboratory tests,and special examination results related to the diagnosis of diabetes●Prior AIC records●Eating patterns,nutritional status,and weight history;growth and development in children and adolescents●Details of previous treatment programs,including nutrition and diabetes self-management education,attitudes,and health beliefs●Current treatment of diabetes,including medications,meal plan,and results of glucose monitoring and patients’use of data●Exercise history●Frequency,severity,and cause of acute complications such as ketoacidosis and hypoglycemia●Prior or current infections,particularly skin,foot,dental,and genitourinary infections●Symptoms and treatment of chronic eye;kidney;nerve;genitourinary(including sexual),bladder,and gastrointestinal function(includingsymptoms of celiac disease in type1diabetic patients);heart;peripheral vascular;foot;and cerebrovascular complications associated with diabetes●Other medications that may affect blood glucose levels●Risk factors for atherosclerosis:smoking,hypertension,obesity,dyslipidemia,and family history●History and treatment of other conditions,including endocrine and eating disorders●Assessment for mood disorder●Family history of diabetes and other endocrine disorders●Lifestyle,cultural,psychosocial,educational,and economic factors that might influence the management of diabetes●Tobacco,alcohol,and/or controlled substance use●Contraception and reproductive and sexual historyPhysical examination●Height and weight measurement(and comparison to norms in children and adolescents)●Sexual maturation staging(during pubertal period)●Blood pressure determination,including orthostatic measurements when indicated,and comparison to age-related norms●Fundoscopic examination●Oral examination●Thyroid palpation●Cardiac examination●Abdominal examination(e.g.,for hepatomegaly)●Evaluation of pulses by palpation and with auscultation●Hand/finger examination●Foot examination●Skin examination(for acanthosis nigricans and insulin-injection sites)●Neurological examination●Signs of diseases that can cause secondary diabetes(e.g.,hemochromatosis,pancreatic disease)Laboratory evaluation●A1C●Fasting lipid profile,including total cholesterol,HDL cholesterol,triglycerides,and LDL cholesterol●Test for microalbuminuria in type1diabetic patients who have had diabetes for at least5years and in all patients with type2diabetes;some advocate beginning screening of pubertal children before5years of diabetes●Serum creatinine in adults(in children if proteinuria is present)●Thyroid-stimulating hormone(TSH)in all type1diabetic patients;in type2if clinically indicated●Electrocardiogram in adults,if clinically indicated●Urinalysis for ketones,protein,sedimentReferrals●Eye exam,if indicated●Family planning for women of reproductive age●MNT,as indicated●Diabetes educator,if not provided by physician or practice staff●Behavioral specialist,as indicated●Foot specialist,as indicated●Other specialties and services as appropriatePosition Statement(DCCT)(15)and the U.K.Prospective Di-abetes Study(UKPDS)(16,17)have shown that improved glycemic control is associated with sustained decreased rates of retinopathy,nephropathy,and neu-ropathy(18).In these trials,treatment regimens that reduced average A1C to ϳ7%(ϳ1%above the upper limits of normal)were associated with fewer long-term microvascular complications;how-ever,intensive control was found to increase the risk of severe hypoglycemia and weight gain(19,20).Epidemiological studies support the potential of intensive glycemic control in the reduction of CVD (15–20).Recommended glycemic goals for nonpregnant individuals are shown in Ta-ble6.A major limitation to the available data are that they do not identify the op-timum level of control for particular pa-tients,as there are individual differences in the risks of hypoglycemia,weight gain, and other adverse effects.Furthermore, with multifactorial interventions,it is un-clear how different components(e.g.,ed-ucational interventions,glycemic targets, lifestyle changes,and pharmacological agents)contribute to the reduction of complications.There are no clinical trialdata available for the effects of glycemiccontrol in patients with advanced compli-cations,the elderly(Ն65years of age),oryoung children(Ͻ13years of age).Lessstringent treatment goals may be appro-priate for patients with limited life expect-ancies,in the very young or older adults,and in individuals with comorbid condi-tions.Severe or frequent hypoglycemia isan indication for the modification of treat-ment regimens,including setting higherglycemic goals.More stringent goals(i.e.,a normalA1C,Ͻ6%)can be considered in individ-ual patients based on epidemiologicalanalyses that suggest that there is no lowerlimit of A1C at which further loweringdoes not reduce risk of complications,atthe risk of increased hypoglycemia(par-ticularly in those with type1diabetes).However,the absolute risks and benefitsof lower targets are unknown.The risksand benefits of an A1C goal ofϽ6%arecurrently being tested in an ongoing study(ACCORD[Action to Control Cardiovas-cular Risk in Diabetes])in type2diabetes(21).Elevated postchallenge(2-h OGTT)glucose values have been associated withincreased cardiovascular risk indepen-dent of FPG in some epidemiologicalstudies.Postprandial plasma glucose(PPG)levelsϾ140mg/dl are unusual innondiabetic individuals,although largeevening meals can be followed by plasmaglucose values up to180mg/dl.There arenow pharmacological agents that primar-ily modify PPG and thereby reduce A1Cin parallel.Thus,in individuals who havepremeal glucose values within target butwho are not meeting A1C targets,consid-eration of monitoring PPG1–2h after thestart of the meal and treatment aimed atreducing PPG valuesϽ180mg/dl maylower A1C.However,it should be notedthat the effect of these approaches on mi-cro-or macrovascular complications hasnot been studied(22).For information on glycemic controlfor women with GDM,refer to the ADAposition statement“Gestational DiabetesMellitus”(14).For information on glyce-mic control during pregnancy in womenwith preexisting diabetes,refer to MedicalManagement of Pregnancy Complicated byDiabetes(3rd ed.)(23).Referral for diabetes managementFor a variety of reasons,some people withdiabetes and their health care providersdo not achieve the desired goals of treat-ment(Table6).In such instances,addi-tional actions suggested includeenhanced diabetes self-management edu-cation,comanagement with a diabetesteam,change in pharmacological therapy,initiation of or increase in self-monitoringof blood glucose(SMBG),more frequentcontact with the patient,and referral to anendocrinologist.Intercurrent illnessThe stress of illness frequently aggravatesglycemic control and necessitates morefrequent monitoring of blood glucose andurine or blood ketones.A vomiting illnessaccompanied by ketosis may indicate di-abetic ketoacidosis(DKA),a life-threatening condition that requiresimmediate medical care to prevent com-plications and death;the possibility ofDKA should always be considered(24).Marked hyperglycemia requires tempo-rary adjustment of the treatment programand,if accompanied by ketosis,frequentinteraction with the diabetes care team.The patient treated with oral glucose-lowering agents or medical nutrition ther-apy(MNT)alone may temporarily requireTable6—Summary of recommendations for adults with diabetesGlycemic controlAICϽ7.0%*Preprandial plasma glucose90–130mg/dl(5.0–7.2mmol/l)Postprandial plasma glucose†Ͻ180mg/dl(Ͻ10.0mmol/l)Blood pressureϽ130/80mmHgLipids‡LDLϽ100mg/dl(Ͻ2.6mmol/l)TriglyceridesϽ150mg/dl(Ͻ1.7mmol/l)HDLϾ40mg/dl(Ͼ1.1mmol/l)§Key concepts in setting glycemic goals:●Goals should be individualized●Certain populations(children,pregnant women,and elderly)require special considerations●Less intensive glycemic goals may be indicatedin patients with severe or frequenthypoglycemia●More stringent glycemic goals(i.e.a normalA1C,Ͻ6%)may further reduce complicationsat the cost of increased risk of hypoglycemia(particularly in those with type1diabetes)●Postprandial glucose may be targeted if AICgoals are not met despite reaching preprandialglucose goals*Referenced to a nondiabetic range of4.0–6.0%using a DCCT-based assay.†Postprandial glucose mea-surements should be made1-2h after the beginning of the meal,generally peak levels in patients withdiabetes.‡Current NCEP/ATP III guidelines suggest that in patients with triglycerides200mg/dl,the“non-HDL cholesterol”(total cholesterol minus HDL)be utilized.The goal is130mg/dl(61).§For women,it has been suggested that the HDL goal be increased by10mg/dl.Standards of Medical Careinsulin.Adequatefluid and caloric intake must be assured.Infection or dehydration is more likely to necessitate hospitaliza-tion of the person with diabetes than the person without diabetes.The hospitalized patient should be treated by a physician with expertise in the management of dia-betes,and recent studies suggest that achieving very stringent glycemic control may reduce mortality in the immediate postmyocardial infarction period(25). Aggressive glycemic management with insulin may reduce morbidity in patients with severe acute illness(26).For information on management of patients in the hospital,refer to the ADA position statement titled“Hyperglycemic Crises in Diabetes”(24).Recommendations●Lowering A1C has been associated witha reduction of microvascular and neu-ropathic complications of diabetes.(A)●Develop or adjust the management plan to achieve normal or near-normal glycemia with an A1C goal ofϽ7%.(B)●More stringent goals(i.e.,a normal A1C,Ͻ6%)can be considered in indi-vidual patients.(B)●Lowering A1C may lower the risk of myocardial infarction and cardiovascu-lar death.(B)●Aggressive glycemic management with insulin may reduce morbidity in pa-tients with severe acute illness,periop-eratively and following myocardial infarction.(B)●Less stringent treatment goals may be appropriate for patients with a history of severe hypoglycemia,patients with limited life expectancies,very young children or older adults,and individu-als with comorbid conditions.(E)ASSESSMENT OF GLYCEMIC CONTROLTechniques are available for health pro-viders and patients to assess the effective-ness of the management plan on glycemic control.SMBGThe ADA’s consensus statements on SMBG provide a comprehensive review of the subject(27,28).Major clinical trials assessing the impact of glycemic control on diabetes complications have included SMBG as part of multifactorial interven-tions,suggesting that SMBG is a compo-nent of effective therapy.SMBG allows patients to evaluate their individual re-sponse to therapy and assess whether gly-cemic targets are being achieved.Resultsof SMBG can be useful in preventing hy-poglycemia and adjusting medications,MNT,and physical activity.The frequency and timing of SMBGshould be dictated by the particular needsand goals of the patients.Daily SMBG isespecially important for patients treatedwith insulin to monitor for and preventasymptomatic hypoglycemia.For mostpatients with type1diabetes and preg-nant women taking insulin,SMBG is rec-ommended three or more times daily.Theoptimal frequency and timing of SMBGfor patients with type2diabetes is notknown but should be sufficient to facili-tate reaching glucose goals.When addingto or modifying therapy,type1and type2diabetic patients should test more oftenthan usual.The role of SMBG in stablediet-treated patients with type2diabetesis not known.Because the accuracy of SMBG is in-strument-and user-dependent(29),it isimportant for health care providers toevaluate each patient’s monitoring tech-nique,both initially and at regular inter-vals thereafter.In addition,optimal use ofSMBG requires proper interpretation ofthe data.Patients should be taught how touse the data to adjust food intake,exer-cise,or pharmacological therapy toachieve specific glycemic goals.Healthprofessionals should evaluate at regularintervals the patient’s ability to use SMBGdata to guide treatment.Recommendations●SMBG is an integral component of dia-betes therapy.(B)●Include SMBG in the managementplan.(E)●Instruct the patient in SMBG and rou-tinely evaluate the patient’s techniqueand ability to use data to adjust therapy.(E)A1CBy performing an A1C test,health provid-ers can measure a patient’s average glyce-mia over the preceding2–3months(29)and,thus,assess treatment efficacy.A1Ctesting should be performed routinely inall patients with diabetes,first to docu-ment the degree of glycemic control atinitial assessment and then as part of con-tinuing care.Since the A1C test reflectsmean glycemia over the preceding2–3months,measurement approximately ev-ery3months is required to determinewhether a patient’s metabolic control hasbeen reached and maintained within thetarget range.Thus,regular performanceof the A1C test permits detection of de-partures from the target(Table6)in atimely fashion.For any individual patient,the frequency of A1C testing should bedependent on the clinical situation,thetreatment regimen used,and the judg-ment of the clinician.Glycemic control is best judged bythe combination of the results of the pa-tient’s SMBG testing(as performed)andthe current A1C result.The A1C shouldbe used not only to assess the patient’scontrol over the preceding2–3monthsbut also as a check on the accuracy of themeter(or the patient’s self-reported re-sults)and the adequacy of the SMBG test-ing schedule.Table7contains thecorrelation between A1C levels and meanplasma glucose levels based on data fromthe DCCT(30).Recommendations●Perform the A1C test at least two timesa year in patients who are meeting treat-ment goals(and who have stable glyce-mic control)and quarterly in patientswhose therapy has changed or who arenot meeting glycemic goals.(E)MNTMNT is an integral component of diabetesmanagement and diabetes self-manage-ment education.A review of the evidenceand detailed information can be found inthe ADA technical review and positionstatement in this area(31,32).Peoplewith diabetes should receive individual-ized MNT as needed to achieve treatmentgoals,preferably provided by a registereddietitian familiar with the components ofTable7—Correlation between A1C level andmean plasma glucose levels(30)AIC(%)Mean plasma glucosemg/dl mmol/l61357.571709.5820511.5924013.51027515.51131017.51234519.5Position Statement。

银屑病与真菌感染的关系综述-传染病学论文-基础医学论文-医学论文——文章均为WORD文档，下载后可直接编辑使用亦可打印——摘要：随着近年来银屑病的发病率逐步增加，关于银屑病病因的研究更加深入。

除了遗传、免疫和环境等相关因素，微生物与银屑病的关系也备受关注。

关于真菌感染与银屑病的相关性研究始于19世纪，认为糠秕马拉色菌可能是银屑病的致病因子，与银屑病的发生发展密切相关。

随后也有研究指出白色念珠菌通过超抗原等作用发或加重银屑病。

但银屑病患者依据自身IL-17增高的特点可以起到一定抵抗微生物感染的作用，因此真菌感染与银屑病的关系暂无定论。

随着近年来关于两者之间的研究逐渐增多，真菌感染仍是银屑病患者不可忽视的发或加重因素之一。

明确真菌感染在银屑病患者体内的作用有助于更好的研究银屑病的发病机制以及改善预后。

关键词：银屑病; 马拉色菌属; 念珠菌属; 真菌; 感染;Abstract：With the increasing incidence of psoriasis in recent years, research on the etiology of psoriasis has become deepgoing. Thestudy on the correlation between fungal infection and psoriasis began in the 19th century, and it was believed that malasszia infection might be the pathogenesis of psoriasis, and the fungus was closely related to the occurrence and development of psoriasis. Some studies indicated that candida albicans induced or aggravated psoriasis through superantigen. However, relying on the characteristics of increasing Il-17, psoriasis patients can resist microbial infection to a certain extent. The relationship between fungal infection and psoriasis is not conclusive yet. With the deepening research, fungal infection is still regarded as one of the factors that can not be ignored in inducing or aggravating psoriasis.Keyword：psoriasis; Malassezia; Candida; fungi; infection;银屑病是一种多因素参与的慢性免疫异常性的炎症性皮肤病，其特征是由T细胞介导的角质形成细胞过度增殖。

第三部分医学英语的写作任务一标题的写作（Title）标题的结构1. 名词+介词Blindness（视觉缺失）after Treatment for Malignant Hypertension 2. 名词+分词Unilateral Neurogenic Pruritus Following Stroke中风后单侧神经性瘙痒3. 名词+不定式Suggestion to Abolish Icterus Index Determination（黄疸指数测定）where Quantitative Bilirubin Assay（胆红素定量）is Available建议能做胆红素定量的化验室不再做黄疸指数测定4. 名词+同位语Gentamicine, a Selelctive Agent for the isolation of Betahemolytic Streptocc ociβ-溶血性链球菌庆大霉素是分离β-溶血性链球菌的选择性药物5. 名词+从句Evidence that the V-sis Gene Product Transforms by Interaction with the Receptor for Platelet-derived Growth Factor血小板源性生长因子.V-sis 基因产物由血小板生成因子受体相互作用而转化的依据6. 动名词短语Preventing Stroke in patients with Atrial Fibrillation心房纤维性颤动心旁纤颤患者中风预防Detecting Acute Myocardial Infarction（急性心肌梗死）byRadio-immunoassay for Creative Kinase（酐激酶）用放射免疫法测定酐激酶诊断急性心肌梗死7. 介词短语On Controlling Rectal Cancer8. 陈述句Dietary Cholesterol is Co-carcinogenic协同致癌因素for Human Colon Cancer9. 疑问句Home or Hospital BirthsIs Treatment of Borderline Hypertension Good or Bad?注意副标题的作用1．数目：Endoluminal Stent-graft 带支架腔内搭桥for Aortic Aneurysms动脉瘤: A report of 6 cases带支架腔内搭桥治疗动脉瘤的六例报告2．重点：Aorto-arteritis 大动脉炎Chest X-ray Appearance and Its Clinical Significance大动脉炎胸部X线表现及临床意义3．方法：Gallstone Ileus（胆结石梗阻）: A Retrospective Study 4．作用：Carcinoembryonic Antigen in Breast-cancer Tissue: A useful prognostic indictor乳腺癌组织中癌胚抗原——一种有用的预后指示5．疑问：Unresolved—Do drinkers have less coronary heart disease? 6．连载顺序：Physical and Chemical Studies of Human Blood Serum: II. A study of miscellaneous Disease conditions人类血清的理论研究：II. 多种病例的研究7．时间：A Collaborative 综合Study of Burn Nursing in China: 1995-1999常见标题句式举例1. 讨论型：Discussion of/ on; An approach to; A probe into; Investigation of; Evaluation of / on汉语中的“初步体会”、“试论”、“浅析”之类的谦辞可以不译。

第24卷 第9期 2022 年 9 月辽宁中医药大学学报JOURNAL OF LIAONING UNIVERSITY OF TCMVol. 24No. 9Sep .，2022中药过敏康方对银屑病小鼠模型皮损及免疫因子的调节作用王蒙蒙，李欣坪，薛咏梅，乔雪，方琼莲，尹逊青，曾永成，黄丰，林玉萍（云南中医药大学，云南 昆明 650500）摘要：目的 研究中药过敏康方对银屑病小鼠模型皮损组织及免疫因子的调节作用，评价中药过敏康方（Guominkang Decoction，GMK）干预银屑病小鼠模型的作用及机制。

方法 将50只Balb/c雄性小鼠分为5组，分别为正常组（生理盐水）、模型组（咪喹莫特）、甲氨蝶呤组（咪喹莫特+1 mg/kg）、过敏康方高剂量组[咪喹莫特+5.6 g/kg，GMK（H）]、过敏康方低剂量组[咪喹莫特+2.8 g/kg，GMK（L）]，每日涂抹咪喹莫特、灌胃给药各1次，连续给药6 d。

给药开始每天对各组小鼠进行表皮厚度及银屑病皮损面积和严重程度指数（PASI）评分；实验结束后，剪取小鼠造模皮肤进行形态学、组织病理学观察；并用酶联免疫吸附试验法（ELISA）检测各组小鼠皮损组织中免疫因子白细胞介素（Interleukin，IL）-6、IL-1β、IL-23、IL-8、IL-10、IL-22、IL-17A的表达。

结果 与正常组相比，模型组皮损部位的鳞屑较多，出现红斑，皮肤厚度显著性增高（P<0.001）；皮损组织中IL-6、IL-1β、IL-23、IL-8、IL-22、IL-17A 显著性升高（P<0.001）；与模型组相比，各给药组皮损部位的鳞屑、皮肤厚度等具有明显的改善；病理学观察皮损组织切片，各给药组均变薄，具有显著性差异（P<0.001）；皮损组织中IL-6、IL-1β、IL-23、IL-8、IL-22、IL-17A，各给药组均具有显著性降低。

结论 过敏康方高、低剂量组均具有抑制银屑病小鼠模型的作用，可能与降低IL-6、IL-1β、IL-23、IL-8、IL-22、IL-17A，升高IL-10相关。

肿瘤坏死因子α与白介素17在银屑病中的表达吴方毅;任妮丽【摘要】目的：：检测TNF-α与IL-17在银屑病皮损中的表达。

方法： ELISA检测68例银屑病患者与28例对照(非银屑病患者)血清TNF-α和IL-17的表达；免疫印迹法检测局部皮损、患者正常皮肤及对照者皮肤中TNF-α和IL-17的表达；qT-PCR检测TNF-α和IL-17的mRNA表达。

结果：银屑病患者血清TNF-α、IL-17的表达均高于对照组( P<0.05)；皮损组织中TNF-α、IL-17蛋白及mRNA 的表达均高于患者正常皮肤及对照组皮肤(均P<0.05)。

结论： TNF-α与IL-17在银屑病的发展中可能相互作用。

%Objective:To detect the expression of TNF-αand IL-17 in the patients with psoriasis. Meth-ods:The serum levels of TNF-α and IL-17 were detected in 68 patients with psoriasis and 28 controls from cosmetic surgery department. The expression of protein TNF-αand IL-17 in the patients’ lesions and normal skin of the patients as well as the skin of normal controls was detected by ELISA and the mRNA of TNF-αand IL-17 was detected by qT-PCR. Results:The levels of serum TNF-αand IL-17 were higher in the patients than in controls. The levels of protein and mRNA expression of TNF-αand IL-17 were higher in the patients’ lesions than in the normal skin of the patients and controls. Conclusion:TNF-αand IL-17 may be interacted in the progress of psoriasis.【期刊名称】《中国麻风皮肤病杂志》【年(卷),期】2015(000)009【总页数】4页(P551-554)【关键词】TNF-α;IL-17;银屑病【作者】吴方毅;任妮丽【作者单位】湖北医药学院附属人民医院皮肤科，十堰，442000;湖北医药学院附属人民医院医务科，442000【正文语种】中文银屑病的发病率约1～5%，1，2银屑病的病因还不完全清楚，普遍认为是一种自身免疫性的炎性疾病。

含有卡泊三醇及二丙酸倍他米松的复方制剂可快速有效治疗各种严重程度的寻常型银屑病Van De Kerkhof P.C.M;Wasel N;Kragballe K;P.C.M. Van De Kerkhof;潘敏【期刊名称】《世界核心医学期刊文摘：皮肤病学分册》【年(卷),期】2005(000)010【摘要】Background:A two-compound product containing calcipotriol and betamethasone dipropionate (Daivobet./Dovobet.) has been evaluated in a large clinical trial programme, providing a wealth of data on the treatment of psoriasis vulgaris. Objective:To determine the effectiveness of the two-compound product in patients with mild, moderate and severe psoriasis vulgaris. Methods:Data from over 1,534 patients with psoriasis vulgaris who received the two-compound product once daily for at least 4 weeks in four randomised, double-blind studies were pooled. A meta-analysis of the pooled data is presented. Severity of psoriasis at baseline was determined by investigator assessment and Psoriasis Area and Severity Index (PASI) score. Results:For patients with severe disease defined by PASI score (PASI baseline ≥17), the mean reduction in PASI after up to 4 week s of treatment was 71.6%compared with 68.9 and 67.2%for those with moderate (PASI baseline 5.1-16.0) and mild disease (PASI baseline ≤5). Corresponding reductions for investigator-assessed severity were 72.6, 69.1 and 68.7%, respectively. Conclusion:Although the metaanalysis of the data from these four studies was performed post hoc, we may conclude that the two-compound product provided highly effective treatment ofpsoriasis,regardless of the category of baseline disease severity.【总页数】2页(P54-55)【作者】Van De Kerkhof P.C.M;Wasel N;Kragballe K;P.C.M. Van De Kerkhof;潘敏【作者单位】University Hospital Nijmegen PO Box 9101 NL-6500 HB Nijmegen Netherlands.Dr【正文语种】中文【中图分类】R758.63【相关文献】1.卡泊三醇与恩肤霜复方制剂治疗寻常型银屑病疗效观察 [J], 王鹏;陈波;赵光;欧丽嫦;练霭云2.卡泊三醇与恩肤霜复方制剂治疗寻常型银屑病疗效观察 [J], 王鹏;陈波;赵光;欧丽嫦;练霭云3.复方丙酸氯倍他索软膏与卡泊三醇软膏在退行期寻常型银屑病维持治疗中疗效及安全性的自身对照研究 [J], 于秋月;张胡莲;张芬;陈玉平;穰真;崔凡4.随机对照研究复合制剂卡泊三醇和倍他米松二丙酸盐(Daivobet~/Dovobet~)对银屑病患者生活质量的影响 [J], Van De Kerkhof P.C.M.;崔荣5.复方丙酸氯倍他索软膏与卡泊三醇软膏序贯疗法治疗轻、中度寻常型银屑病的临床观察 [J], 陈小英;莫伟英;曾华蓉;肖卫棉;马腾驹;张冠文;黄润安因版权原因，仅展示原文概要，查看原文内容请购买。

高脂血症性胰腺炎的诊治进展于浩【摘要】高脂血症是临床上较为常见的引起急性胰腺炎的病因之一,常导致AP反复恶化.其发病机制主要与高三酰甘油有关,故降低血三酰甘油水平是治疗的核心,只有与急性胰腺炎综合治疗中的其他手段相结合,同时控制引发高脂血症的原发及继发因素,才能明显改善预后,防止复发.【期刊名称】《医学综述》【年(卷),期】2010(016)016【总页数】3页(P2449-2451)【关键词】急性胰腺炎;高脂血症;三酰甘油【作者】于浩【作者单位】安徽医科大学,合肥,230032【正文语种】中文【中图分类】R576高脂血症(hyperlipidaemia，HL)是临床上除胆源性、酒精性之外最为常见的引起急性胰腺炎(acute pancreatitis，AP)的病因。

国外资料显示，1%～4%的HL患者能继发AP，并且是50%以上妊娠期胰腺炎的病因［1］。

我国台湾地区的多中心临床研究认为［2］，HL占AP全部病因的12.3%。

大陆的一份多中心的研究也表明［3］，HL引起的胰腺炎占总胰腺炎病因的 8.3%。

另有研究表明［4］，13%～38% 的 AP 伴有HL，HL经常加重AP的临床表现。

所以，HL既是AP的病因，又是AP常见的并发症。

随着我国生活水平的提高和饮食结构的不合理，HL相关性胰腺炎的发病率有明显升高的趋势。

现就HL性胰腺炎的相关现状与进展予以综述。

1 病因HL是AP的病因之一，然而，胰腺炎本身也可引起血脂升高，二者形成恶性循环，从而使胰腺炎临床症状加重。

动物实验表明［5］，HL能加重胰腺炎的进程。

在HL相关性AP中，HL既可表现为原发性，又可表现为继发性。

原发性 HL 中，按 Frederickson 分型［6］，Ⅰ、Ⅳ和Ⅴ型更易引发AP，Ⅰ和Ⅴ型导致AP通常没有继发因素，而Ⅳ型通常需要有一个继发因素(如糖尿病、酗酒等)去充分提高血脂水平才能引发AP［7］。

继发性HL主要有:①糖尿病未治或控制不佳。

2024年4月第14卷第8期·综 述·[基金项目] 郑州大学药学院国药科技创新创业基金项目（2022yxy018）。

倍他米松磷酸钠注射液治疗早产有效性和安全性研究进展王青宇1 李丝雨1 刘林凤1 刘　倩1 钟　晴1 周红建2 李俊霞21.郑州大学药学院，河南郑州　450001；2.遂成药业股份有限公司，河南新郑　451150[摘要]倍他米松是地塞米松的同分异构体，具有抗炎、抗过敏、抗内毒素和免疫抑制等功能。

倍他米松磷酸钠（BSP）注射液是倍他米松的磷酸盐制剂，作为一种糖皮质激素（ACS）类药物，其临床应用较广。

BSP 治疗早产已有充分的证据并被纳入各国指南，然而这些证据主要集中在高收入国家，中低收入国家相关证据则较少。

我国关于BSP 在产科应用疗效和安全性临床研究方面存在不足。

关于倍他米松和地塞米松疗效和安全性直接比较的临床研究较少，BSP 治疗早产后儿童期和成年期随访研究尚不足。

因此，需进一步开展相关研究，为早产临床科学合理用药提供决策依据。

[关键词]倍他米松磷酸钠注射液；早产；呼吸窘迫综合征；败血病；脑室内出血[中图分类号] R714.21 [文献标识码] A [文章编号] 2095-0616（2024）08-0058-05DOI:10.20116/j.issn2095-0616.2024.08.14Research progress on the efficacy and safety of betamethasonesodium phosphate injection in the treatment of premature birthWANG　Qingyu 1 LI　Siyu 1 LIU　Linfeng 1 LIU　Qian 1 ZHONG　Qing 1 ZHOU　Hongjian2LI　Junxia21. School of Pharmaceutical Sciences, Zhengzhou University, Henan, Zhengzhou 450001, China;2. Suicheng Pharmaceutical Co., Ltd., Henan, Xinzheng 451150, China[Abstract] Betamethasone is an isomer of dexamethasone, which has anti-inflammatory, anti-allergic, anti-endotoxin and immunosuppressive functions. Betamethasone sodium phosphate (BSP) injection is a phosphate preparation of betamethasone, which is widely used as a antenatal corticostemids (ACS) drug in clinical practice. There is sufficient evidence for the treatment of premature birth with BSP, and BSP has been included in national guidelines. However, the evidence related to its treatment of premature birth is mainly in high-income countries, and there is less evidence in low to middle-income countries. There is insufficient clinical research on the efficacy and safety of BSP in obstetric applications in China. There are few clinical studies on the direct comparison of efficacy and safety between betamethasone and dexamethasone, and the follow-up study of BSP in treating premature in childhood and adulthood is still insufficient. Further research is needed to provide a decision-making basis for the scientific and rational use of medication in preterm birth.[Key words] Betamethasone sodium phosphate injection; Premature birth; Respiratory distress syndrome; Septicemia; Intraventricular hemorrhage倍他米松磷酸钠（betamethasone sodium phosphate，BSP）是倍他米松的水溶性衍生物，也是地塞米松磷酸钠的16位差向异构体[1]。

Standard Operating Procedure for the Good Manufacturing Practice-Compliant Production of Human Bone MarrowMesenchymal Stem CellsLivia Roseti,Marta Serra,and Alessandra Bassi AbstractAccording to the European Regulation (EC 1394/2007),Mesenchymal Stem Cells expanded in culture forclinical use are considered as Advanced Therapy Medicinal Products.As a consequence,they must be produced in compliance with Good Manufacturing Practice in order to ensure safety,reproducibility,and efficacy.Here,we report a Standard Operating Procedure describing the Good Manufacturing Practice-compliant production of Bone Marrow-derived Mesenchymal Stem Cells suitable for autologous implan-tation in humans.This procedure can be considered as a template for the development of investigational medicinal Mesenchymal Stem Cells-based product protocols to be enclosed in the dossier required for a clinical trial approval.Possible clinical applications concern local uses in the regeneration of bone tissue in nonunion fractures or in orthopedic and maxillofacial diseases characterized by a bone loss.Keywords:Bone marrow,Mesenchymal stem cells,Good manufacturing practice,Quality control,Advanced therapy medicinal products,Bone regeneration1IntroductionMesenchymal Stem Cells (MSCs)display a low density in bonemarrow,thus an in vitro expansion step is required in order to obtain a cell number suitable for clinical applications (1–3).Euro-pean regulations define such expanded cells as Advanced Therapy Medicinal Products (ATMPs)that must be produced in accordance with the current Good Manufacturing Practice (GMP)(4,5).Those rules,which have been already encoded for conventional drugs,allow for the manufacture of medicinal products in a stan-dardized and controlled way minimizing,at the same time,con-tamination risks.Here,we report a Standard Operating Procedure (SOP)describing the multistep GMP-compliant manual production of Bone Marrow-derived MSCs to be used for autologous implanta-tion in humans (6–8).MSCs,isolated from an eparinated (1,000I.U.of heparin)bone marrow sample harvested from the iliac crest of adult donors (9,10)(about 10–20ml),are expanded in monolayerMethods in Molecular Biology (2015)1283:171–186DOI 10.1007/7651_2014_103©Springer Science+Business Media New York 2014Published online:05August 2014w w w wc el l 8.c n w w w .s t e mc e l l 8.c n w w w .s t em c c e l l 8.c n ww w .s t e m c e l l 8.c n w ww.s t e mc c el l 8.c n w w w.s t e mc e l l 8.c n ww w .s t e m c c el l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc cel l 8.c n w .s t e mc e l l 8.c n w .s t em cup to 3weeks.The final product is a small volume MSC suspension suitable for the clinical use only after passing proper quality con-trols.In fact,the procedure involves several GMP’s mandatory samplings for environmental and cell quality control analyses (3,11)that must be carried out by specialized laboratories and will not be described in detail.Previous validation studies have stated final product’s shelf-life (properly named stability):we demonstrated that MSC’s features (sterility,viability,and phenotype)are maintained at acceptable levels when the MSC suspension is stored at room temperature for a maximum of 72h.After this time the product expires and can no longer be transplanted.To reach full GMP-compliance,the process generates two types of reference samples collected from the final product—cells and supernatant—which should be both stored for at least 1year after implantation and analyzed if issues arise (12).If implantation is delayed for organizational reasons or patient’s illness or if repeated doses at different times are required,cells are stored in liquid nitrogen as intermediate product.This SOP can be utilized as a template for the development of investigational medicinal Mesenchymal Stem Cells-based product protocols to be enclosed in the dossier required for a clinical trial approval.Possible clinical applications concern local uses (cells alone or in combination with biomaterials and/or growth factors)for the regeneration of bone traumatic or degenerative damages,pseudoarthrosis and defects of consolidation,congenital disorders or maxillofacial injuries (13–15).2Materials2.1Raw MaterialsCell culture reagents suitable for cell therapy applications should bepurchased from Companies that guarantee their GMP compliance production.Sterility should be certified by specific analyses,pre-formed in compliance with the requirements of the European Pharmacopoeia,current edition (16).All reagents used in this protocols are:Dulbecco’s Modified Eagle Medium (DMEM)low glucose (1g/l)(basal medium);Dul-becco’s Phosphate Buffered Saline (PBS)(1Â);Fetal Bovine Serum (FBS)Pharma Grade,Australian Origin;L -Glutamine 200mM (recombinant origin)and Trypsin-Ethylenediaminetetraacetic acid (EDTA)(1:250)1Â(porcine source).Trypsin-EDTA and FBS must be also certified to be free from porcine and bovine mycoplasmas and viruses,respectively.In addi-tion,FBS must be attested to be produced in a Bovine Spongiform Encephalopathy/Transmissible Spongiform Encephalopathy172Livia Roseti et al.c el l 8.c n w ww .s t e mc e l l 8.c n www .s t em c cel l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc c el l 8.c n w w w.s t e mc e l l 8.c n ww w .s t e m c c el l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc cel l 8.c n w .s t e mc e l l 8.c n w .s t em c(BSE/TSE)free Country,such as Australia or New Zealand,andscreened for prion absence (17).L -Glutamine’s recombinant origin must be clearly indicated and certified too.CryoSure-Dimethyl Sulfoxide (DMSO)is purchased from WAK–Chemie Medical GmbH,Germany.Plastic material must be certified to be sterile,pyrogen free (latex free whenever necessary)and tested for cell culture (or stem cell tested).Vented culture flasks are recommended.To manage high cell numbers we use a multilayer flasks system (Hyperflask vessels,Corning Life Sciences).Double or triple packages should be preferred to eliminate/reduce the need for alcohol wipe downs and similar procedures required in clean room production facilities.Quality control sampling:–BacT/ALERT 1Aerobic Culture Bottles (SA)(BioMerieuxInc.,Marcy L’Etoile,Craponne,France)(18).–BacT/ALERT 1Anaerobic Culture Bottles (SN)(BioMerieuxInc.,(Biomerieux Industry,Marcy L’Etoile,Craponne,France)(18).–Apirogenic Bottles for endotoxin testing (Charles River Labor-atoires,l’Arbresle,France).–PCR tubes for Real time PCR detection of Mycoplasma contam-ination (Venor 1GeM-qDual Mycoplasma Detection Kit for Real-Time PCR,Minerva Biolabs GmbH,Berlin,Germany).–Settle and contact plates for environmental microbiological con-trol and glove prints (Heipha Dr M €uller GmbH,Eppelnheim,Germany).–Particle counter for environmental particle control.2.2LocationIn Europe,a GMP-facility (Cell Factory)allowed to ATMP pro-duction by the competent authority is required (5).A GMP-facilityconsists in environments where specific parameters such as air filtration and ventilation,temperature,relative humidity,differen-tial pressure,number of air particles,and bacterial colony forming units are standardized and continuously monitored.The structure includes clean rooms of different classification up to A (the local zone for high-risk operations i.e.biohazard hood)in B work places (the background environment for the grade A zone),according to European current GMP (5).High-risk operations include manip-ulations where the cells are exposed to environment such as trypsi-nization and medium change.For lower-risk operations,such as medium warming or centrifugation,grade B areas are appropriate.Only trained and equipped personnel are admitted to the facility.GMP Production of Mesenchymal Stem Cells173c el l 8.c n w ww .s t e mc e l l 8.c n www .s t em c cel l 8.c n w w w.s t e mc e l l 8.c n w ww.s t e mc ce l l 8.c n w ww.s t e mc e l l 8.c n ww w .s t e m c c el l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc cel l 8.c n w .s t e mc e l l 8.c n w .s t em cOperator’s equipment includes protective and disposable clothes towear(Fig.1).Raw materials must be introduced separately frompersonnel,through a clean pass box.2.3Equipment Biohazard hood;CO2incubator(set at37 C temperature,5%CO2and95%relative humidity);centrifuge;phase contrast micro-scope;incubator(work chamber temperature of37 C);vortex;chilling plate/refrigerated racks;cell counter;refrigerator/freezer;liquid nitrogen tank.3Method3.1Cell Isolation and Seeding (See Notes1–4)1.Warm/thaw basal medium,L-glutamine and FBS in theincubator.2.Under the biohazard hood prepare complete medium(basalmedium supplemented with10%FBS and4mM L-glutamine): discard by aspiration60ml from a500ml bottle of basal medium,add10ml L-glutamine and50ml FBS;resuspend with a pipette.Fig.1Disposable sterile garments for cell manipulation.Cell manipulating operators as well as personnel must wear disposable garments that ensure biological products protection.Only trained and equipped operators can enter the GMP-facility174Livia Roseti et al.c e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw.s t e mc e l l8.c nw.st e mc3.Aspirate the eparinated bone marrow with a 18-G needle inserted in a syringe and transfer in sterile tube/s.4.Resuspend and measure bone marrow amount with a pipette.5.Dilute the bone marrow samples 1:4with complete mediumand resuspend.6.Harvest samples for microbiological control:–1ml with a sterile syringe to inoculate in a BacT/ALERT 1Aerobic Culture Bottle (SA)(Fig.2);–1ml with a sterile syringe to inoculate in a BacT/ALERT 1Anaerobic Culture Bottle (SN)(Fig.2);–2ml to collect in PCR tube for mycoplasma detection.Collected samples are then to be sent to the proper specializedquality control laboratories that will perform the analyses (see Note 5).7.Seed cells in culture flasks,typically T75(7ml/T25;14ml/T75;28ml/T150;560ml/multilayer flasks).8.Place the flasks in the CO 2incubator.9.Store the complete medium at 4 C.Fig.2Quality control sampling on Mesenchymal Stem Cells.To harvestsamples for microbiological control during critical steps,1ml of supernatant is inoculated into a BacT/ALERT ®Aerobic Culture Bottle (SA)and 1in a BacT/ALERT ®Anaerobic Culture Bottle (SN)with a sterile syringe (see Note 5).The bottles are then sent to a GMP-compliant Quality Control Laboratory for the analysisGMP Production of Mesenchymal Stem Cells175c el l 8.c n w ww .s t e mc e l l 8.c n w ww .s t em c cel l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc c el l 8.c n w w w.s t e mc e l l 8.c n ww w .s t e m c c el l 8.c n w w w.s t e mc e l l 8.c n w ww.s t e mc ce l l 8.c n w .s t e mc e l l 8.c n w .s t em c3.2First Medium Change(See Notes 1and2)1.After3days,warm complete medium and PBS in the incubator.2.Pull out theflasks from the CO2incubator and observe themacroscopic appearance of the culture medium:–if the medium is opaque or dense and yellow there is the suspect of a contamination.In this case the procedure isstopped and sampling for microbiological control isneeded(see Section3.1);–if the appearance is clear and the color red or dark red go on with this procedure.3.Under the biohazard hoodfilter the amount of completemedium required for processing with a vacuumfiltration sys-tems(or with afilter and a syringe).4.Aspirate medium from theflasks and place it in sterile tubes.5.Centrifuge the medium at500Âg for7min,at roomtemperature.6.In the meantime add to theflasks the PBS solution(5ml forT25flasks,10ml for T75flasks,20ml for T150flasks,100ml for multilayerflasks)in order to wash out stromal matrix debris and red blood cells(see Note6and Fig.3a).7.Close theflasks and placed them horizontally for1min,gentlyshaking.8.Aspirate and discard the PBS.9.Run a second wash(only steps7and8).10.Evaluate the cultures under a phase-contrast microscope(Fig.3b),noting the following parameters:cell adhesion (absent,present,partial,poor);cell density(confluence,non-confluence,next to the confluence);cells in the supernatant (presence,absence);matrix/red blood cells(presence, absence);unusual morphology.11.Transfer theflasks under the biohazard hood and discardthe PBS.12.Transfer the centrifuged tubes under the biohazard hood,aspirate half supernatant volume of that normally used to eachflask(3.5ml for the T25flasks,7ml for T75flasks, 14ml for T150flasks,280ml for multilayerflasks)(see Note6).13.Add to theflasks the same amount of complete fresh mediumand resuspend.14.Place theflasks in the CO2incubator.15.Store reagents at4 C.176Livia Roseti et al.c e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw.s t e mc e l l8.c nw.st e mc3.3Trypsinizationand Intermediate Product Freezing(See Notes 1,2,and 7)1.After 3–4days,macroscopically evaluate culture medium,not-ing the following parameters:clarity/turbidity,color (red orange,red violet,yellow).2.Check the cells under a phase-contrast microscope noting thefollowing parameters:cell adhesion (present,absent,poor);confluence (70–80%confluent,nonconfluent,next to the confluence);cell presence or absence in the supernatant;matrix/red blood cells (presence,absence);unusual morphology.3.Make a decision based on both observations (see steps 4,5,6,and 7).Fig.3Phase-contrast microscope morphological observation of Mesenchymal Stem Cells at different steps of the Standard Operating Procedure.(a )Three days after seeding (First medium change )red blood cells and matrix hamper observation.(b)Adherent cells become more visible only after two PBS washes .Matrix debris and/or red blood cells are still evident and should disappear in a few days.(c)Cells have reached 70–80%confluence and trypsinization is recommended.(d)Full cell detachment occurs as a suspension of cells with round morphology after Trypsin-EDTA incubation.All images were taken with a digital camera (magnification Â4)GMP Production of Mesenchymal Stem Cells177c el l 8.c n w ww .s t e mc e l l 8.c n w ww .s t em c cel l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc c el l 8.c n w w w .s t e mc e l l 8.c n ww w .s t e m c c el l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc cel l 8.c n w .s t e mc e l l 8.c n w .s t em c4.If 70–80%confluence is not reached,trypsinization should be delayed until reached and a medium change is needed.5.In particular,change the culture medium with complete medium in the case of red medium color and/or adhesion;change the culture medium with a 20%FBS supplement in the case of violet medium color and/or poor adhesion:–warm complete medium and FBS (if necessary);–prepare 10or 20%FBS medium;–transfer the cultures under the biohazard hood;–filter the needed amount of medium,depending on flasks size and number;–aspirate the medium from the flasks and add propermedium—complete or with 20%FBS—(7ml for T25flasks,14ml for T75flasks,28ml for T150flasks;560ml for multilayer flasks);–close and place flasks in the CO 2incubator.6.Trypsinization and freezing can be performed in case of about70–80%confluence (Fig.3c ):–prepare complete medium if necessary;–warm complete medium and PBS in the incubator;–thaw Trypsin-EDTA in the incubator;–filter the amount of reagents required for processing;–transfer the cultures under the biohazard hood;–aspirate and discard the supernatants;–add the PBS wash solution (5ml for T25flasks,10ml for T75flasks,20ml for T150flasks,and 100ml for multilayer flasks);–close and place horizontally the flasks,gently shaking for 30–60s;–run a second wash;–add the Trypsin-EDTA solution (4ml for T25flasks,7ml for T75flasks,14ml for T150flasks,and 100ml for multilayer flasks);–close and place the flasks in the CO 2incubator for 15min toallow cell detachment;–pick the flasks and check the cells under the phase-contrast microscope to verify cell detachment (which occurs as a suspension of cells with round morphology)(Fig.3d );–if some cells are still adherent to the substrate,put the flasksagain in the CO 2incubator for 3–5min to allow full detachment;178Livia Roseti et al.c el l 8.c n w w w .s t e mc e l l 8.c n www .s t em c ce l l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc c el l 8.c n w w w.s t e mc e l l 8.c n ww w .s t e m c c el l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc cel l 8.c n w .s t e mc e l l 8.c n w .s t em c–if detachment is completed,inactivate the Trypsin-EDTA solution by adding complete medium to theflasks under the biohazard hood(8ml for T25flasks,14ml for T75flasks, 28ml for T150flasks,and200ml for multilayerflasks);–resuspend and transfer to50ml tubes;–collect a0.5ml sample for cell counting;–centrifuge7min at500Âg,at room temperature;–in the meantime count the cells;–under the biohazard hood open the centrifuged tubes,care-fully discard the supernatants and gently shake the pellets (see Note8);–set the number of cryovials to use for freezing:1cryovial up to3Â106Æ0.5Â106MSCs;–place the empty cryovials in a refrigerated rack;–prepare the cell freezing mixture in a tube:0.9ml of com-plete medium,0.2ml of DMSO and0.9ml of FBS(mixture for one cryovial)(see Note7);–resuspend,filter,and place the mixture in the refrigerated rack;–carefully discard the supernatant of the centrifuged tubes and gently resuspend the cell pellet(see Note8);–add1.6ml of the freezing mix to each pellet,drop by drop, using a sterile pipette while gently shaking the tubes with the other hand;–gently resuspend and transfer into the cryovials;–close the cryovials and house them in a storage box that enables a slow decrease of the temperature(about1 C per minute);–exit the cleanroom and place the container into a freezer set at a75 C temperature;–after about24h place the cryovials in the vapor phase of a liquid nitrogen tank(see Note7).7.Collect samples for microbiological tests in case of suspectedcontamination(yellow color,unusual morphology)as in Section3.1and stop the procedure until results(see Note5).3.4Intermediate Product Thawing and Seeding(See Notes1, 2,5,and7)1.Open the liquid nitrogen tank and harvest the cryovials,check-ing their integrity and identity.2.Get samples in the cleanroom(through the pass box)within arefrigerated rack.3.Prepare and warm a20%FBS supplemented medium.4.Place the cryovials in the incubator.GMP Production of Mesenchymal Stem Cells179c e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw.s t e mc e l l8.c nw.st e mc5.After3–4min check if the cell suspension appears thawed.6.If small ice chunks are still evident put the samples again in theincubator for30–60s.7.When no more ice residues are visible,place the cryovials underthe biohazard hood and transfer each cell suspension in a tube containing18ml of the20%FBS medium.8.Close the tubes and vortex the solution for about10s.9.Centrifuge at500Âg for7min,at room temperature.10.Transfer the supernatant into sterile tubes for quality controlsampling as in Section3.1.11.Gently shake the pellets,add20%FBS medium(7ml for T25flasks,14ml for T75flasks,28ml for T150flasks,560ml for multilayerflasks)and resuspend(see Note8).12.Seed at low density in an adequate number of cultureflasks(seeNote9).13.Check cell presence under the phase-contrast microscope.14.Place theflasks in the CO2incubator.3.5Expansion(See Notes1,2,and10)1.Pull outflasks from the CO2incubator.2.Macroscopically evaluate culture medium,noting the followingparameters:clarity/turbidity,color(red orange,red violet, yellow).3.Check the cells under a phase-contrast microscope noting thefollowing parameters:cell adhesion(present,absent,poor);confluence(confluent,nonconfluent,next to the confluence);cell presence or absence in the supernatant;matrix/red blood cells(presence,absence);unusual morphology.4.Make a decision based on both observations as detailed below.5.Change the culture medium with complete medium in the caseof red medium color and/or cell adhesion;change the culture medium with a20%FBS supplement in the case of violet medium color and/or poor adhesion,as described in Section3.3.6.Trypsinization in case of about70–80%confluence(seeNote10)(Fig.3c)or if matrix debris and/or red blood cells are still evident(see Note6)(Fig.3a,b):–trypsinize(until centrifugation step)and count the cells,as detailed in Section3.3–add to the pellets the necessary amount of complete medium in order to allow a low density seeding,resuspend and place in cultureflasks(14ml for T75flasks,28ml for T150flasks,560for the multilayerflasks)(see Note9);180Livia Roseti et al.c e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw.s t e mc e l l8.c nw.st e mc–check cell presence in theflasks under the phase-contrast microscope;–place theflasks in the CO2incubator.7.Collect samples for microbiological tests in case of suspectedcontamination(yellow color,unusual morphology)as in Sec-tion3.1and stop the procedure until results(see Note5).3.6Final Product Packaging and Reference Samples Generation(See Notes 1and2)1.Warm reagents and preparefinal medium:basal medium sup-plemented with L-glutamine(no FBS)(see Note11).2.Trypsinize and count the cells as detailed in Section3.3,untilcentrifugation step.3.Gently shake the pellets and add40mfinal medium(see Note8).4.Centrifuge7min at500Âg,at room temperature.5.Repeat steps3and4(see Note11).6.Under the biohazard hood transfer the supernatant into steriletubes from which to collect the following samples that are then to be sent to the proper specialized laboratories of quality control:–1ml with a sterile syringe to inoculate in a BacT/ALERT1 Aerobic Culture Bottle(SA);–1ml with a sterile syringe to inoculate in a BacT/ALERT1 Anaerobic Culture Bottle(SN)(see Note5);–2ml with a sterile pipette to be placed in a proper tube for PCR mycoplasma detection;–0.5ml with a sterile pipette to inoculate in an a-pyrogenic Bottle for endotoxin testing;–0.5ml with a sterile pipette to be placed in a tube and frozen as counter sample atÀ80 C.7.Gently shake the pellets,addfinal medium in order to reach a1–2Â106cell concentration.Resuspend and collect the following samples which are then to be sent to the proper specialized laboratories of quality control (see Notes5and12):– 1.2Â106cells in micro-tubes for the phenotypical analysis (immuno-phenotype and tri-lineage ability);– 2.0Â106cells for karyotyping;– 1.0Â106cells in a tube as a counter sample to be frozen as in Section3.3.8.Transfer cell suspension into proper,identified tubes(primarycontainer)(Fig.4a)GMP Production of Mesenchymal Stem Cells181c e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw w w .s t em c el l8.cnw w w.s t em cc e l l8.c nw.s t e mc e l l8.c nw.st e mc9.Close each tube and put it in a secondary plastic sterile bag(secondary container)(Fig.4a ).10.Close and insert in outer jar container (Fig.4b ).11.Put a label on the outer container,which must contain(Fig.4b ):–identification of the Manufacturer;–identification of the product;–indication of the use (i.e.autologous use only);–batch number;–identification code of the patient;–expiration date;–signature of the person in charge.12.Exit the Cleanroom and store the final product at room tem-perature for a maximum of 72h.13.For the shipment use a container with lock (Fig.4c ).4Notes1.To avoid the risk of cross-contamination during manipulation it is required to use disposable materials and not to share reagents between cultures.Moreover,it is not possible to simultaneously process cultures from different patient,but only sequentially and after decontamination of the biohazard hood and relative equipment.2.Sampling for environmental (microbiological and particles)control is required during each phase of the process.At theFig.4Final product packaging.The final cellular products is transferred into proper tubes (primary container,a ),then put it into secondary sterile bags (secondary container,a ).The product is then inserted in a plastic jar,properly labeled (a andb ).For the shipment a container with lock is utilized (c )182Livia Roseti et al.c el l 8.c n w ww .s t e mc e l l 8.c n www .s t em c cel l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc c el l 8.c n w w w .s t e mc e l l 8.c n ww w .s t e m c c el l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc cel l 8.c n w .s t e mc e l l 8.c n w .s t em cend of cell manipulation,each operator should take glove printson agar plates.Recommended limits for contamination are indicated in EudraLex-Volume 4GMP Guidelines (5).3.A peripheral blood sample from each patient is needed in order to evaluate the presence of transmissible pathologies (Acquired Immune Deficiency Syndrome,Hepatitis B and C,and syphilis)(12).If a GMP-facility does not have the possibility to manipu-late the infected cells in separated areas,positive patients must be excluded.Therefore,manipulation can start only after nega-tivity is stated.This usually takes 1or 2days.In the meantime the bone marrow samples can be stored at 5Æ3 C.4.Density-gradient separation method is considered a standardstep for MSC isolation from bone marrow.However,such a procedure is time consuming and reagents are not often approved for clinical use.Therefore many GMP-facilities are trying to develop alternative methods,such as direct or diluted bone marrow seeding (9).5.Microbiological control and Mycoplasma analysis are per-formed in the critical steps of isolation,thawing,and final product packaging.Isolation and thawing criticism have been established by our GMP-facility on the basis that cell material is newly introduced or reintroduced in the cleanroom,respec-tively.Being “final product packaging”the most critical step since cells are then implanted in the patient,additional sam-pling for quality control analyses are required,such as for endotoxin detection,viability,phenotype,and genotype stabil-ity evaluation (4,5,7).If a cell contamination is detected,the process must be stopped.Since the here described orthopedic applications are not lifesaving,contaminated cells cannot be sterilized by irradiation or other treatments and must be discarded.6.In the “first medium change step”matrix and red blood cellspresence (Fig.3a )should be reduced by two PBS washes.This makes also possible to check cell presence under the phase-contrast microscope (Fig.3b ).Usually,debris gradually reduces until disappearance in 7-8days.However,if residues still persist in the cultures,a trypsinization,which in general dissolves all of them,is recommended.The first medium change is then per-formed using half of the centrifuged supernatants of the isola-tion step and half fresh complete medium.The supernatants,centrifuged in order to remove matrix debris and red blood cells,are utilized since rich of soluble factor produced by the cells themselves that can be useful for adhesion and growth.On the other hand,since it can carry also catabolic factors,fresh medium is added to enhance cell metabolism.GMP Production of Mesenchymal Stem Cells183c el l 8.c n w ww .s t e mc e l l 8.c n www .s t em c cel l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc c el l 8.c n w w w.s t e mc e l l 8.c n ww w .s t e m c c el l 8.c n w ww.s t e mc e l l 8.c n w ww.s t e mc cel l 8.c n w .s t e mc e l l 8.c n w .s t em c。