Associations between microRNA (miR-21,126,155and 221),albuminuria and heavy metals in Hong Kong Chinese adolescents
Alice P.S.Kong a ,1,Kang Xiao a ,1,Kai Chow Choi b ,Gang Wang a ,Michael H.M.Chan c ,Chung Shun Ho c ,Iris Chan c ,Chun Kwok Wong c ,Juliana C.N.Chan a ,d ,Cheuk Chun Szeto a ,?
a
Department of Medicine and Therapeutics,The Chinese University of Hong Kong,Hong Kong SAR,China b
Nethersole School of Nursing,The Chinese University of Hong Kong,Hong Kong SAR,China c
Department of Chemical Pathology,The Chinese University of Hong Kong,Hong Kong SAR,China d
Li Ka Shing Institute of Health Sciences,The Chinese University of Hong Kong,Hong Kong SAR,China
a b s t r a c t
a r t i c l e i n f o Article history:
Received 9January 2012
Received in revised form 27January 2012Accepted 16February 2012Available online 1March 2012Keywords:Arsenic Lead
Cadmium Mercury Albuminuria Adolescents
Background and aim:Pathogenetic mechanisms underlying albuminuria are not completely understood.Heavy metals might lead to atherosclerosis and kidney damage.miR-21,126,155and 221regulated endothelial function and might contribute to the development of albuminuria.To date,no clinical trial has explored the relationship between miRNAs,microalbuminuria and heavy metals in human.In this study,we aimed to examine the association between microalbuminuria,miRNAs and heavy metals in adolescents.Materials and methods:From a cross-sectional,population-recruited study,we identi ?ed 60school children aged 12–19years with microalbuminuria (de ?ned as spot urine albumin –creatinine ratio >3.5mg/mmol).We compared the urine heavy metals (arsenic,mercury,cadmium and lead)and miRNAs levels (miR-21,126,155and 221)with another 60age-and sex-matched normoalbuminuric adolescents as control.Results:Mean age of the study cohort was 15.5±2.1years.43%were boys.Among the four miRNAs tested,only miR-21was associated with microalbuminuria (p =0.02).Urinary arsenic and lead levels had a negative association with both miR-21and miR-221.No signi ?cant association was found between heavy metals examined and microalbuminuria.
Conclusion:The results of our study suggest an association between microalbuminuria,miR-21and heavy metals (arsenic and lead).This might imply that miR-21is involved in the pathogenetic mechanisms linking heavy metals exposure and albuminuria.
?2012Elsevier B.V.All rights reserved.
1.Introduction
Urbanization and technology have led to rapid global transition and may contribute to the pandemic of non-communicable diseases including cardiovascular diseases (CVD)and chronic kidney disease (CKD).Early identi ?cation of at-risk individuals is important to prevent progression of CVD and CKD.Albuminuria,a marker of vascular and renal damage,is a simple,commonly used biochemical tool to identify individuals at risk of development of CVD and CKD.
Apart from changes in habits and lifestyle,exposure to heavy metals is increasingly recognized as a consequence of urbanization.Most heavy metals cannot be metabolized by our body,and excessive accumulation in the body will disturb the normal functions of cells.
Kidney is the key organ to eliminate heavy metals from the body.Heavy metals might lead to albuminuria through inducing oxidative stress to renal tubular cells [1,2].Certain heavy metals have additive effect in inducing nephrotoxicity.For example,synergistic effect of arsenic (As)and cadmium (Cd)in causing renal damage has been demonstrated in Chinese general population [3].In addition,chronic exposure to toxic heavy metals may promote atherosclerosis and contribute to the development of CKD and CVD [2,4].
MicroRNAs (miRNAs)are endogenous,small non-coding RNAs which are involved in regulation of gene expression and many crucial biological processes,including development,differentiation,apoptosis,and proliferation [5].Four miRNAs,miR-21,miR-126,miR-155and miR-221,had been reported to have regulatory functions in gene expression of vascular proliferation factors such as programmed cell death protein 4(PDCD4),phosphatase and tensin(PTEN),vascular endothelial growth factor(VEGF),angiotensin II type 1receptor (AT 1R)and vascular smooth muscle cell (VSMC)[6–9].Whether these four miRNAs are involved in the pathogenetic processes of albuminuria through their roles in regulating gene expressions in vasculature have not been explored.It is plausible that heavy metals induced the
Clinica Chimica Acta 413(2012)1053–1057
?Corresponding author at:Department of Medicine and Therapeutics,The Chinese University of Hong Kong,Prince of Wales Hospital,Shatin,N.T.,Hong Kong SAR,China.Tel.:+852********;fax:+852********.
E-mail address:ccszeto@https://www.doczj.com/doc/d09397765.html,.hk (C.C.Szeto).1
APSK and KX:co-?rst author of the
manuscript.0009-8981/$–see front matter ?2012Elsevier B.V.All rights reserved.doi:
10.1016/https://www.doczj.com/doc/d09397765.html,a.2012.02.014
Contents lists available at SciVerse ScienceDirect
Clinica Chimica Acta
j ou r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c l i n c h i m
development of albuminuria through damage to the vascular functions via these miRNAs.
Against this background,we conducted this study aiming to examine the associations between microalbuminuria,urinary miRNA, namely miR-21,miR-126,miR-155and miR-221which had known regulatory role in vascular functions,and heavy metals levels in a population-recruited cohort of Hong Kong Chinese adolescents.
2.Subjects and method
2.1.Study population
Subjects were recruited from the Hong Kong School Children Project conducted in2003and the methodology had been previously described[10,11].In brief,it was a cross-sectional,population-recruited study including2115Hong Kong Chinese adolescents aged 12–19years who were randomly selected from all secondary schools in Hong Kong.Only healthy school children without any known medical illnesses or on long-term medications were invited to participate in this https://www.doczj.com/doc/d09397765.html,rmed written consents from both the participants and their parents or guardians were sought before they entered the study.The study was approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong.
A team of trained research nurses and research assistants measured the anthropometric parameters and sampled blood and urine specimens from the participants in the school.Anthropometric indices,including body weight(kg),height(m),percentage of body fat by bioimpedance,waist and hip circumstances,were measured. Blood pressure(BP)was measured by Omron blood pressure device (Omron Healthcare Inc.,Tokyo,Japan).Each student had rested for more than5min and the average of two readings was used for the analysis.
After fasting for at least8h,blood samples were collected and spot morning urine samples were also collected.Plasma glucose(PG)and lipid pro?le including total cholesterol(TC),triglyceride(TG),high density lipoprotein cholesterol(HDL-C)and low density lipoprotein cholesterol(LDL-C)were assayed.Urinary miRNAs and heavy metals levels were measured from the stored urine aliquots.
We de?ned microalbuminuria as spot urine albumin creatinine ration(ACR)>3.5mg/mmol[10,12].Among about one tenth of adolescents in the total cohort with microalbuminuria[10],60urine aliquots with microalbuminuria were available for this study.We identi?ed age-and sex-matched controls(i.e.with normal urine ACR levels)from the same cohort for comparison.
https://www.doczj.com/doc/d09397765.html,boratory assays
Serum and urinary creatinine(Jaffe kinetic method),as well as urinary albumin(immunoturbidimetry method)were measured by DP Modular Analytics(Roche Diagnostics Corp,Indianapolis,IN, USA).The urine samples were centrifuged at3000g for30min at 4°C.We used400μL supernatant for total RNA isolation by the mirVanaTM PARISTM Kit(Ambion,Inc.Austin,TX,USA)according to the manufacturer's instruction.Then60μL RNA was collected and stored at?80°C until reverse transcription.
TaqMan?miRNA reverse transcription Kit(Applied Biosystems, Foster City,CA,USA)was used for reverse transcription.In brief, 1.67μL total RNA was mixed with1μL speci?c primers,0.05μL 100mM dNTPs(with dTTP),0.5μL10×reverse transcription buffer, 0.33μL(50U)MultiScribe?Reverse Transcriptase,0.05μL RNase inhibitor(20U/μL)and made up to5μL with H2O.Reverse transcription (RT)was performed at16°C for30min,42°C for30min and85°C for 5min.The resulting cDNA was stored in?80°C until use.
For RT-QPCR(reverse transcription-quantitative polymerase chain reaction),we added0.25μL primer,0.33μL RT production, 2.5μL TaqMan Universal PCR master mix and1.92μL nuclease-free water together to get5μL?nal reaction volume.The expression of miR-21,miR-126,miR-155,and miR-221were quanti?ed by RT-QPCR.ABI Prism7900Sequence Detection System(Applied Biosystems, Foster City,CA)was used for the RT-QPCR reaction.All primers and probes are commercially available from the manufacturer.Each sample was run in triplicate.RT-QPCR was performed at50°C for2min,95°C for10min,followed by40cycles at95°C for15s and60°C for1min. The small nucleolar RNA RNU48(Applied Biosystems)was used as housekeeping gene to normalize the miRNA expression.Data were analyzed with SDS relative quanti?cation software version 2.2.2 (Applied Biosystems).The same baseline and threshold of threshold cycle were set for each target.
Arsenic(As),mercury(Hg),cadmium(Cd)and lead(Pb)levels in urine were analyzed in stored urine samples using the state-of-the-art inductively coupled plasma mass spectrometry(ICPMS)technique (ICPMS7500c,Agilent Technologies,Tokyo,Japan)[13].
2.3.Statistical analysis
Continuous variables with skewed and normal like distribution were presented as medians(inter-quartile ranges)and means(standard deviations)respectively.Categorical data were presented as counts and percentages.The normality of the continuous variables was assessed using skewness statistic and normal Q–Q plot graphically. BMI and TG were positively skewed and therefore log-transformed to correct their skewness before subjected to statistical analysis. Log-transformation of miRNA data did not render their normality satis-factorily;they were analyzed with non-parametric methods.Since undetectable urine heavy metal level was set to zero,the urine levels of the four heavy metals were thus again analyzed with non-parametric methods.The comparisons between the normoalbuminuric and albuminuric groups on demographic and clinical characteristics, cardiovascular risk factors,miRNA data and urine heavy metals were done using t-test,Mann–Whitney test,Pearson Chi-square test and Fisher's exact test,as appropriate.Inter-correlations between cardiovas-cular risk factors,ACR,miRNA data and urine heavy metals were assessed by Pearson or Spearman correlation coef?cients,depending on whether miRNA data and urine heavy metals were involved or not.All statistical analyses were performed using SPSS17.0(SPSS Inc., Chicago,IL).All statistical tests were two-sided and a p value b0.05 was considered statistically signi?cant.
3.Results
Baseline clinical demographic data of the albuminuric and control groups are summarized and compared in Table1.In short,there was no signi?cant difference between any baseline biochemical nor anthropometric characteristics between the albuminuric and control groups.
3.1.Relation between microalbuminuria and urinary miRNA
Urinary miRNA levels were compared between albuminuric and control groups and summarized in Fig.1.We found that urinary miR-21level was signi?cantly higher in the albuminuric than the control group(3234.3[772.8–5248.7]versus1028.7[265.6–2999.6] copy per100,000copies of the housekeeping gene,p=0.020).Similarly, urinary miR-221level was marginally higher in the albuminuric than the control group(102.1[29.2–324.9]versus66.6[27.8–129.5]copy per100,000copies of the housekeeping gene,p=0.077),but the result did not reach statistical signi?cance.In contrast,urinary miR-126and miR-155levels were similar between the albuminuric and control groups.The actual level of urinary albumin excretion also correlated with the urinary level of miR-21(Spearman's r=0.223,p=0.015), but not miR-221(r=0.155,p=0.09),miR-126(r=0.040,p=0.7)or miR-155(r=0.081,p=0.4).
1054 A.P.S.Kong et al./Clinica Chimica Acta413(2012)1053–1057
3.2.Urinary heavy metal
Urinary heavy metal levels were compared between albuminuric and control groups and summarized in Table 2.In short,there was no signi ?cant difference in urinary arsenic,mercury,cadmium,or lead level between albuminuric and control groups.Similarly,the actual level of urinary albumin excretion did not correlate with the urinary level of any of the heavy metal (details not shown).
We observed signi ?cant correlations between the urinary level of some heavy metals and miRNA targets in the total cohort (Table 3).Speci ?cally,urinary arsenic level signi ?cantly correlated with urinary level of miR-21(r=?0.288,p=0.001)and miR-221(r=?0.253,p=0.005);urinary lead level also correlated with urinary level of miR-21(r=?0.184,p=0.048)and miR-221(r=?0.303,p=0.001)(Table 3).4.Discussion
From the results of this study,we had found that urinary miR-21was associated with microalbuminuria,and miR-21was associated with urinary arsenic and lead levels.To our knowledge,our study is the ?rst clinical study to examine the relationship of miRNAs,micro-albuminuria and heavy metals in human.
From our results,there were higher levels of miR-21in the micro-albuminuric group compared to the normoalbuminuric group,signi-fying the increased expression of miR-21in school children with microalbuminuria.miR-21was postulated to be protective against the development of albuminuria because it might inhibit apoptosis
Table 1
Clinical and biochemical characteristics of the study cohort.
Albuminuric group
Control group p value Number of case 6060Sex (M:F)26:3426:34–Age (years)
15.5(2.1)
15.5(2.1)
–Urine ACR (mg/mmol)?7.49(5.58–16.75)0.43(0.30–0.82)–
Weight (kg)48.7(9.7)50.9(10.3)p=0.23Height (cm)
160.7(8.6)
160.2(8.5)
p=0.79Body Mass Index (kg/m 2)?18.4(17.3–19.8)19.3(18.0–21.2)p=0.07Waist circumference (cm)65.1(5.8)67.1(7.6)p=0.10Hip circumference (cm)86.6(7.0)88.4(7.1)p=0.17Waist to hip ratio
0.75(0.04)0.76(0.05)p=0.39Blood pressure (mmHg)Systolic 114.0(13.2)115.2(13.8)p=0.63Diastolic
71.2(9.8)73.8(10.3)p=0.15Serum creatinine (μmol/L)71.4(14.9)73.7(13.6)p=0.38HDL-cholesterol (mmol/L) 1.7(0.3) 1.7(0.4)p=0.45LDL-cholesterol (mmol/L) 2.3(0.6) 2.2(0.5)p=0.56Triglyceride (mmol/L)?0.7(0.6–1.0)0.7(0.6–1.0)p=0.76FPG (mmol/L)
4.7(0.3)
4.7(0.3)
p=0.32
Data marked with ?were presented as median (inter-quartile range);all others were presented as mean (standard deviation)and compared by Student's t test.ACR,albumin –creatinine ratio;FPG,fasting plasma
glucose.
https://www.doczj.com/doc/d09397765.html,parison of urinary miRNA levels between albuminuric group and control group:(A)miR-21;(B)miR-126;(C)miR-155;and (D)miR-221.Data are compared by Mann –Whitney U test.
Table 2
Comparison of urinary heavy metal levels between groups.
Albuminuric group Control group p value No.of patient 6060Arsenic (nmol)54.2(32.8–102.8)59.4(36.1–117.0)p=0.60Mercury (nmol)0.26(0.10–0.60)0.29(0.19–0.52)p=0.71Cadmium (nmol)0.18(0.13–0.29)0.20(0.16–0.27)p=0.83Lead (nmol)
0.36
(0.25–0.54)
0.41
(0.28–0.52)
p=0.70
Data are presented in concentration per mmol of creatinine,as median (inter-quartile range),and compared using Mann –Whitney U test.
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of podocytes[14].miR-21also inhibits mesangial cells proliferation in animal model and in vitro experiments[15].Contrary to the results from animal study[15],in which reduced expression of miR-21in db/db diabetic nephropathy(DN)mice were found,our results in human subjects found that adolescents with microalbuminuria had increased expression of miR-21level compared to normoalbuminuric control.Our results suggest that miR-21might be involved in the pathogenetic mechanisms of albuminuria.Since more than1000 miRNAs have been identi?ed in human beings,it is plausible that other miRNAs,rather than the4tested in our experiments,are involved in the process of development of albuminuria.For examples, miR-216a and miR-217also target PTEN and may be the players in linking albuminuria,CKD and CVD[16].Other example includes miR-377which decreases the expression of Superoxide dismutase (SOD)1,SOD2and p21-Activated protein kinase(PAK)1,and in turn leads to enhanced susceptibility to oxidant stress and accumulation of the extracellular matrix protein and?bronectin[17].
Arsenic might decrease kidney function by inducing oxidative stress in kidney.In animal experiment,mice with subchronic exposure to arsenic had oxidative damage to DNA in kidney tissue,especially in the proximal tubular cells and Bowman's capsule,and these led to more8-OHdG expression[18].It had also been demonstrated in290 adults(86men and204women)that urinary arsenic level was positively correlated with both kidney biomarker of toxicity,such as β2-microglobulin and N-acetyl-β-D-glucosaminidase(NAG),and oxidative stress indices,such as urinary malondialdehyde(MDA)and 8-hydroxy-2′-deoxyguanosine(8-OHdG)[1].In a case-control, hospital-based study in Taiwan,including125CKD patients and229 controls,urinary arsenic level was found to positively associated with CKD[19].Contrary to these previous reported studies,our results did not show any signi?cant correlation between arsenic and https://www.doczj.com/doc/d09397765.html,paring microalbuminuric to normoalbuminuric group,there was no difference between urinary arsenic levels.We also did not?nd any signi?cant association between microalbuminuria and other heavy metals including mercury,lead and cadmium.Indeed,the associ-ation between mercury and albuminuria was unclear and the results from previous published literatures were con?icting.Our negative results are in keeping with the negative?ndings from other published studies[20].Occupational exposure to mercury had insigni?cant impact on urine albumin excretion[20].Among89chloralkali workers who had been exposed to mercury vapor and75controls,whose median urinary mercury levels being14.3nmol/mmol creatinine(25.4μg/g creatinine)and1.1nmol/mmol creatinine(1.9μg/g creatinine)respec-tively,their urinary albumin levels had no difference[20].In contrast, some research found an association between urinary mercury and urinary albumin levels[21].Dental amalgam is a source of mercury exposure.In a research involving534children aged6–10years, children in the dental amalgam group had a signi?cantly increased prevalence of microalbuminuria compared to children without prior dental amalgam restorations[22].A positive association between lead and albuminuria had previously been described[23].In NHANES (1999–2006),among14,778participants analyzed,compared subjects from the top quartiles(>2.4μg/dL,>1.6–2.4μg/dL,>1.1–1.6μg/dL) to the lowest quartiles(b1.1μg/dL)of blood lead levels,the adjusted odd ratios for the subjects to have albuminuria(≥30mg/g creatinine) were 1.19(95%CI=0.96–1.47),0.92(95%CI=0.76–1.12),0.83 (95%CI=0.66–1.04)respectively[23].Our negative?nding of the association between urinary cadmium and microalbuminuria echoes with the?nding of non-diabetic subjects in an Australian study[24]. However,some studies reported a positive association between cadmium and albuminuria.In NHANES(1999–2006)involving14,778 participants,compared subjects from the top quartiles(>0.6μg/L, >0.4–0.6μg/L,>0.2–0.4μg/L)to the lowest quartile of blood cadmium levels(≤0.2μg/L),the adjusted odd ratios for the subjects to have albuminuria were1.92(95%CI=1.53–2.43),1.32(95%CI=1.07–1.64), 1.10(95%CI=0.89–1.36)respectively[23].According to an Australian study(n=182,43with diabetes,139without diabetes,37.19±0.92years of age),among people with diabetes,urinary cadmium level was associated with albuminuria,while in people without diabetes,there was no such association[24].In a study in China,people who worked and lived in cadmium-polluted area had higher urine cadmium level(11.86μg/g creatinine,range:1.69–55.72)and higher albumin(6.4mg/g creatinine,range:1.1–85.3)comparing to people in non–cadmium-polluted area(urine cadmium1.81μg/g creatinine, range:0.09–5.72;albumin2.9mg/g creatinine,range:0.1–37.8)[25]. In another study of361people aged from6to74years,for children aged from6to17years,urinary albumin level was associated with urine cadmium.However,after adjusted for creatinine and other poten-tial confounders,these association became insigni?cant,except for adults aged18years or older[26].
Several limitations of this study have to be addressed.First,our study subjects were community recruited and did not have toxic range of urinary heavy metal levels.Therefore,it might because of these low levels of exposure to heavy metals,no clinically signi?cant alteration in kidney function is found or the renal effect is too subtle to be detected.Second,only one spot urine aliquot was sampled from each subject.It might be possible that subjects had no exposure to heavy metals at the juncture of urine collection.Repeated and serial urine collection would be more informative about prolonged exposure to heavy metals and its effect on microalbuminuria.Consecu-tive urine sampling in a prospective cohort for heavy metals poisoning would provide more information regarding the examination of the association between long-term exposure to heavy metals and https://www.doczj.com/doc/d09397765.html,stly,the cross-sectional design of this study precludes the exam-ination of causal relationships between albuminuria,miRNAs targets and urinary heavy metal levels.
In conclusion,from this case-control cohort recruited from a population wide survey,we found that miR-21was associated with microalbuminuria.Increased expression of miR-21level in the micro-albuminuric group might be the reaction secondary to the develop-ment of microalbuminuria.Furthermore,miR-21was also associated with urinary arsenic and lead levels.The results might imply that miR-21is involved in the pathogenetic mechanisms linking heavy metals exposure and microalbuminuria.Further studies and experi-ments are required to examine the causal relationship between miR-21,microalbuminuria and heavy metals.
Con?ict of interest
All authors declare no con?ict of interest.
Acknowledgements
We thank all school personnel,parents and participants for making this study possible.This study was supported by funding from the Research Grant Committee(CUHK4055/01M and CUHK 4465/06M),Li Ka Shing Institute of Health Science and Hong Kong Institute of Diabetes and Obesity,under the auspices of The Chinese
Table3
Relation between urinary heavy metal and miRNA levels in the total cohort.a Arsenic Mercury Cadmium Lead
miR-21r=?0.288,
p=0.001r=?0.177,
p=0.08
r=?0.098,
p=0.29
r=?0.184,
p=0.048
miR-126r=?0.128,
p=0.16r=?0.146,
p=0.15
r=?0.168,
p=0.07
r=?0.037,
p=0.70
miR-155r=0.049,
p=0.59r=?0.006,
p=0.95
r=?0.094,
p=0.31
r=?0.118,
p=0.21
miR-221r=?0.253,
p=0.005r=?0.181,
p=0.075
r=?0.114,
p=0.22
r=?0.303,
p=0.001
a Spearman's correlation coef?cients are presented.
1056 A.P.S.Kong et al./Clinica Chimica Acta413(2012)1053–1057
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实验三炎症 一、目的要求 （一）掌握炎症三种基本病理变化。 （二）掌握炎症的分类，注意各类型炎症的病变特点。 （三）掌握下列名词概念：化脓、脓肿、蜂窝织炎、炎细胞浸润、窦道、瘘管、脓毒血症等。 二、实习内容 （一）本章幻灯及课件 （二）大体标本 1.纤维素性心包炎（绒毛心）：心脏体积增大，心外膜附着灰白色絮状物，呈绒毛状（镜下观系何种渗出物？） 2.细菌性痢疾（示假膜性炎）：为结肠标本，已剪开，可见肠粘膜表面复有一层白色假膜，呈糠皮样。假膜脱落处有大小不等、形状不一的地图状浅表溃疡。 3.脑脓肿：标本系脑之矢状切面，可见一鸡蛋大小、边界清楚的脓腔（脓液在标本切开时已流失）。 4.蜂窝织性阑尾炎：见实验一。 5.结肠息肉：肠腔面可见一花生米大小的带蒂的肿物向粘膜表面突起，即息肉（注意不要把肠壁外表的肠脂垂误为息肉）。 6.慢性胆囊炎：此胆囊炎系胆结石所致，由于结石嵌顿，使胆囊体积增大，囊壁膨胀变薄，囊腔充满由囊壁分泌的稀薄粘液（已流失），囊腔内可见结石（已取出）。（胆结石、胆囊炎常互为因果，二者常同时存在）。 7.炎性假瘤：标本为肺组织，其内可见一个境界清楚清的肿瘤样团块。 （三）病理切片 1.急性蜂窝织性阑尾炎复习正常阑尾组织学：阑尾分粘膜层、粘膜下层、肌层和浆膜四层，前二层较厚，管腔狭窄，粘膜固有膜有丰富的淋巴组织是阑尾的特点（见示教片）。（1）肉眼：本片为阑尾的横切面，中间空白区为管腔，内层兰色部分是粘膜层，周围红染部分包括粘膜下层、肌层和浆膜层。（2）低倍：辨认阑尾的四层结构，其粘膜层可有组织缺损，即溃汤形成，各层均有中性粒细胞浸润，故称为蜂窝织炎。各层血管充血，组织间隙内有炎性水肿（水肿液被染成
实验四炎症 一、实验目的 1．熟悉变质性炎、出血性炎的病变特征。 2．掌握化脓性炎、纤维素性炎、炎性息肉的大体变化。 3．认识炎细胞和脓细胞。 二、实验内容 【病理组织观察】 （一）观察方法 1．肉眼观察炎症的基本病理变化：变质、渗出、增生。（1）变质性炎:器官体积增大或缩小（大片细胞坏死），不同程度的变性、坏死。（2）渗出性炎：①浆液性炎：局部形成炎性水肿，或体腔积液。②纤维素性炎：纤维素渗出部位不同，形态不一。常发生于粘膜、浆膜和肺。在粘膜形成假膜，在心包形成绒毛状物，称绒毛心。③化脓性炎：大量中性粒细胞渗出伴不同程度组织坏死，有黄绿色、浑浊、粘稠的脓液形成，可表现为表面化脓和积脓、蜂窝织炎、脓肿等，如阑尾蜂窝织炎。（3）增生性炎：①非特异性增生性炎常表现为器官体积增大或管腔壁增厚，组织变硬韧；粘膜可形成炎性息肉，突出于粘膜表面；在疏松组织可形成炎性假瘤。②肉芽肿性炎常有境界清楚的结节状病灶形成。 2．镜下观察①各种炎细胞的大小、形状、核的分叶数量以及胞浆、胞核的染色情况。 ②阑尾壁血管有何变化，各层结构是否完整，有无破坏和微脓肿形成，以何种炎细胞浸润为主要，阑尾腔内有无脓液存在。③组织有无增生、水肿，血管是否扩张，可见哪些炎细胞浸润。 （二）观察内容 肉眼观察 1．肾脓肿肾表面或切面可见一个或多个境界清楚的、圆形、灰黄色质化脓病灶。 2．细菌性痢疾结肠粘膜表面附有一层灰白色膜状物，有的标本呈糠皮样，粘膜充血、水肿、增厚。
3．绒毛心心包膜不光滑，失去正常光泽，表面有灰白色绒毛状渗出物附着，故名绒毛心。 4．慢性阑尾炎阑尾肿胀变粗，浆膜表面失去光泽，切面见见阑尾壁厚薄不一，局部有出血呈黑褐色，部分区域附有黄白色脓性渗出物。 5．炎性息肉皮肤或粘膜表面可见短小带蒂的凸起状肿物，即为息肉。 镜下观察 1．各种炎细胞观察各种炎细胞的大小、形状、核的分叶数量以及胞浆、胞核的染色情况。 2．急性化脓性阑尾炎阑尾壁充血、水肿、粘膜坏死，有脓性渗出物，病灶有炎症细胞浸润，以中性粒细胞为主。 （三）思考题 1．炎症的局部组织有哪些基本的病理变化？ 2．按照病理变化的不同，可将炎症分为几大类，各有哪些主要的病理变化？ 3．何为假膜性炎?是如何形成的？ 4．脓肿与蜂窝织炎如何区别？ 5．血道播散可导致哪几种后果？ （四）病案讨论 患者男性，32岁，臀部长“疮”，伴肿、痛及发热就诊。查体：局部红肿、隆起，大小约5cm×5cm，有波动感，体温38.9℃，Bp100/65mmHg，肝脾肿大，心肺（-）。血常规：WBC 21.0×109/L，N90％。局部手术切开，引流出大量黄白色浓稠液体。 讨论： 1．做出诊断，写出诊断依据。 2．简述其病理变化。 3．用病理知识解释临床表现。
炎症的实验观察教学案例简介 案例名称：炎症的实验观察 课程名称：显微形态学 授课教师：杨静 学时数：4 授课对象：临床医学等专业本科生 教学目的： 1.掌握炎症的基本病变，病理类型及各类型的病变特点。 2.熟悉炎症的发生、发展和结局。 内容简介： 炎症是十分常见而又重要的基本病理过程，体表的外伤感染和各器官的大部分常见病和多发病(如疖、痈、肺炎、肝炎、肾炎等）都属于炎症性疾病。具有血管系统的活体组织对损伤因子的防御性反应称为炎症。 本实验案例通过结合多媒体课件、教学录像、大体标本和组织切片的观察，掌握常见炎症性疾病（阑尾炎、胆囊炎、绒毛心、白喉、鼻息肉、肺脓肿等）的病理学改变，为临床实习打下坚实的基础。 特点： 本实验案例教学以学生为主体，通过自己观察病变，总结病变特点，结合教师讲授，使学生深刻理解炎症的病变特点及临床病理联系。
家兔气管插管及颈部迷走神经分离术实验案例简介 案例名称：家兔气管插管及颈部迷走神经分离术 课程名称：机能实验学 授课教师：于利 学时数：4 授课对象：临床医学等专业本科生 教学目的： 1.掌握家兔麻醉、颈部手术实验操作方法。 2.学习气管插管及颈迷走神经分离实验方法。 内容简介： 家兔是常用于机能学实验的实验动物之一，可以用来进行呼吸系统、循环系统、泌尿系统、消化系统等生理、病理、药理学实验。熟练地掌握家兔的麻醉和实验手术技能是保证实验顺利完成的前提，本实验为学生提供一次学习掌握家兔实验手术方法的机会；其次，家兔气管插管和颈迷走神经分离，是进行家兔呼吸系统实验的前期准备，其成功与否直接关系到呼吸系统实验顺利进行。 本次实验课目的在于教授学生学习掌握家兔的呼吸系统实验的手术操作方法及步骤。 特点： 本实验教学案例以实验手术操作方法的讲解分析，现场实验手术操作示教，学生亲身实践相结合，使学生能够熟练掌握家兔麻醉、颈部手术及气管插管、颈迷走神经分离实验手术技能。
四川卫生康复职业学院教案
实验设备：显微镜（学生两人一台）、电视投影仪、组织切片、大体标本 实验内容： 一、大体标本：注意观察形态、大小、颜色、质地、数目、有无包膜 1.葡萄胎：大小不一的水泡状，由滋养细胞高度增生所致。 2.畸胎瘤：位于卵巢，可见毛发、骨骼等，提示高分化。 3.结肠息肉状腺瘤：呈外生性生长，为息肉状突起，由蒂与肠黏膜相连。 4.脂肪瘤：黄色、软腻，分叶状，包膜清楚。 5.子宫肌瘤（单发）：灰白色、鸡蛋大小，包膜完整，切面呈编织状。 6.子宫多发性肌瘤：数个大小不一的灰白色肿块，包膜完整。 7.小肠间质细胞瘤：小肠的一段，小肠壁上的肿块呈外生性生长，边界清楚，有完整包膜，切面呈灰 白色、致密、编织状。 8.卵巢多房性粘液性囊腺瘤：呈囊状，边界清，内容物呈胶冻状。 9.脂肪肉瘤：假包膜、体积大，呈淡黄色，切面呈多彩状。 10.结肠多发性息肉状腺瘤：结肠可见大量灰白色的息肉，以蒂相连，为癌前病变。 11.结肠多发性息肉状腺瘤恶变：结肠的一段，可见大量粟粒大小灰白色息肉，局部可见灰白色肿块， 侵入肠壁，切面可见多彩状。 12.骨肉瘤：干骺端可见一外生性生长的肿块，骨膜掀起，内部浸润。 13.阴茎癌：阴茎表面可见灰白色菜花状肿块。 14.肺癌：可见灰白色巨大肿块，将边缘肺组织压迫、萎缩。 15.结肠癌（溃疡性）：癌变处呈溃疡状，凹凸不平，向下浸润，呈灰白色，渗入肠全层。 二、病理切片： 1.高分化结肠腺癌：分清黏膜层、黏膜下层、肌层、浆膜层，分清肿瘤的实质与间质。 2.肺高分化鳞癌：重点观察癌巢中角化珠的形态结构。 实验方法： 1.第一节课由教师先结合电视投影讲解切片，然后学生观察切片，教师巡视、解答学生在观察中遇到 的问题 2.第二节课先由学生自行观察大体标本15分钟，再由教师对其问题进行解答、讲解。 板书设计 实验四肿瘤 实验目的：肿瘤的特征及生长方式，良恶性肿瘤的区别 实验内容： 一、大体标本 1.葡萄胎 2.畸胎瘤 3.结肠息肉状腺瘤 4.脂肪瘤 5.子宫肌瘤（单发） 6.子宫多发性肌瘤 7.小肠间质细胞瘤 8.卵巢多房性粘液性囊腺瘤 9.脂肪肉瘤 10.结肠多发性息肉状腺瘤 11.结肠多发性息肉状腺瘤恶变 12.骨肉瘤 13.阴茎癌 14.肺癌 15.结肠癌（溃疡性） 二、病理切片： 1.高分化结肠腺癌
实验报告 专业班级：康复治疗技术2 班实验小组：第四组姓名：卢锦锟实验日期：2015 年11 月10 日（一）实验项目：家兔动脉血压调节 （二）实验目的： 1、掌握神经体液因素及受体阻断或兴奋药物对家兔心血管活动的影响机制。 2、掌握动脉血压作为心血管功能活动的综合指标及其相对恒定的调节原理和重要意义。 3、掌握家兔实验的基本方法和技术（静脉麻醉、静脉输液、动脉插管、分离神经等）。 4、掌握压力生物信号采集与处理系统的使用。 （三）基本原理：（要求对写出关键点）动脉血压是心血管功能活动的综合指标。正常心血管的活动在神经、体液因素的调节下保持相对稳定，动脉血压相对恒定。动脉血压的相对恒定对于保持各组织、器官正常的血液供应和物质代谢是极其重要的。通过实验改变神经、体液因素或施加药物，观察动脉血压的变化，间接反映各因素对心血管功能活动的调节或影响。 实验仪器与试剂：bl-420 生物信号采集与处理系统、血压换能器、刺激电极、哺乳类动物手术器械、注射器（5ml、1ml）等；3%戊巴比妥钠、0.3%肝素、1:10000盐酸肾上腺素、1:10000 去甲肾上腺素、1:1000 异丙肾上腺素、0.01%多巴胺、1%酚妥拉明、0.01%普萘洛尔、0.001%乙酰胆碱、0.01%阿托 品 2、静脉注射0.01%重洒石酸去甲肾上腺素：去甲肾上腺素与血管平滑肌上的a和B 2受体结合，使血管收缩，管径变小，外周阻力增加，从而使平均动脉压升高。此外，去甲肾上腺素还可以使心率增加，心收缩力变大，因此血压升高。 3、静脉注射0.005%盐酸异丙肾上腺素：异丙肾上腺素能与骨骼肌血管B 2受体结合，骨 骼肌血管（在全身血管中比例较大）持续舒张抵消了皮肤粘膜血管的收缩作用，因而出现后降压作用。 4、静脉注射0.01%盐酸肾上腺素：肾上腺素能与心肌B 1受体结合激动心肌心肌收缩力 增强，因此心率加快，传导加速，心排出量增多而导致收缩压升高。肾上腺素能激动腹腔内脏血管a 1受体，使动腹腔内脏血管收缩，血压升高。 5、静脉注射1%酚妥拉明：a受体阻断剂，a受体的作用：a受体为传出神经系统的受体，根据其作用特性与分布不同分为两个亚型: a 1 、a 2。 a 1 受体主要分布在血管平滑肌（如皮肤、粘膜血管，以及部分内脏血管），激动时引起血管收缩; a 1 受体也分布于瞳孔开大肌，激动时瞳孔开大肌收缩，瞳孔扩大。 a2 受体主要分布在去甲肾上腺素能神经的突触前膜上，受体激动时可使去甲肾上腺素释放减少，对其产生负反馈调节作用。 （四）实验主要设备和仪器、药品和用品（要求分类、简洁、清晰表述） 1、仪器设备：婴儿秤、兔手术台、哺乳动物手术器械一套，bl-420 生物机能实验系统，动脉夹、压力换能器等； 2、试剂用品：20%氨基甲酸酯（5ml/kg ）、1%去甲肾上腺素、1%肝素、生理盐水、1g/l 异丙肾上腺素、1g/l 酚妥拉明、5ml、10ml、20ml 注射器各一支、三通管、插管、小烧杯等； （五）实验的步骤与方法（列出主要步骤、方法要点） 1 、动物手术： （1）、麻醉、固定：家兔称重后，按5ml/kg 从耳缘静脉缓慢注射20%氨基甲酸酯。将家兔麻醉后，使其仰卧固定在兔手术台上。 （2）颈部手术：颈部备皮，沿颈前正中切开皮肤5~6cm,分离气管并做气管插管。分离两侧的迷走神经，穿线备用。手术完毕后，用温生理盐水纱布覆盖手术野。分离左侧颈总动
实验题目：糖皮质激素对炎症的治疗 班级：12级临七3班姓名：廖梦宇学号：2012021320 一、实验原理： 二甲苯涂于小鼠耳部，可致局部组织炎症，释放某些炎症物质，造成耳部急性渗出性炎性水肿。糖皮质激素可明显抑制各种致炎因素引起的炎症，从而改善红、肿、热、痛等症状。通过测定小鼠耳片的重量，观察炎症的发生及糖皮质激素的抗炎作用。 二、实验目的： 观察糖皮质激素对二甲苯所致小鼠耳部水肿及毛细血管渗透的影响。 三、实验步骤： 1.分组：取6只小鼠，随机分为2组。分别称重并标记编号。观察一般活动。 2.给药：1#, 2#, 3# 小鼠给地塞米松，0.2ml/10g 4#, 5#, 6# 小鼠给生理盐水，0.2ml/10g 给药途径：腹腔注射 3.致炎：30分钟后，两组小鼠于左耳前后两面均匀涂二甲苯0.03ml，记录时间。另 侧做对照。 4.耳片：致炎后30分钟，小鼠颈椎脱臼处死，沿耳廓基线剪下两耳，用打孔器分别 在两耳同一部位打下耳片，称重，记录。 5.计算：肿胀程度=(左耳片重量-右耳片重量)/右耳片重量*100% 6.统计学检验 四、实验结果： 编号给药左耳片重量右耳片重量肿胀程度（%）
2 地塞米松0.0095 0.0071 33.8 3 地塞米松0.0136 0.0053 156.6 4 生理盐水0.0170 0.006 5 161.5 5 生理盐水0.0115 0.0074 55.4 6 生理盐水0.0125 0.0078 60.3 五、讨论： 根据实验结果，生理盐水没有明显的药理作用，小鼠因耳部涂抹二甲苯而产生炎症反应，出现“红、肿、热、痛”的现象；地塞米松属于糖皮质激素的一种，有显著的抗炎作用。本实验中，由于操作失误，1号小鼠在涂抹二甲苯时用力过大致死，实验数据丢失。
实验报告 专业班级：康复治疗技术2班实验小组：第四组姓名：卢锦锟实验日期：2015年11月10日 （一）实验项目：家兔动脉血压调节 （二）实验目的： 1、掌握神经体液因素及受体阻断或兴奋药物对家兔心血管活动的影响机制。 2、掌握动脉血压作为心血管功能活动的综合指标及其相对恒定的调节原理和重要意义。 3、掌握家兔实验的基本方法和技术（静脉麻醉、静脉输液、动脉插管、分离神经等）。 4、掌握压力生物信号采集与处理系统的使用。 （三）基本原理：（要求对写出关键点） 动脉血压是心血管功能活动的综合指标。正常心血管的活动在神经、体液因素的调节下保持相对稳定，动脉血压相对恒定。动脉血压的相对恒定对于保持各组织、器官正常的血液供应和物质代谢是极其重要的。通过实验改变神经、体液因素或施加药物，观察动脉血压的变化，间接反映各因素对心血管功能活动的调节或影响。 实验仪器与试剂：bl-420生物信号采集与处理系统、血压换能器、刺激电极、哺乳类动物手术器械、注射器（5ml、1ml）等；3%戊巴比妥钠、0.3%肝素、1:10000盐酸肾上腺素、1:10000去甲肾上腺素、1:1000异丙肾上腺素、0.01%多巴胺、1%酚妥拉明、0.01%普萘洛尔、0.001%乙酰胆碱、0.01%阿托品 2、静脉注射0.01%重洒石酸去甲肾上腺素：去甲肾上腺素与血管平滑肌上的α和β2受体结合，使血管收缩，管径变小，外周阻力增加，从而使平均动脉压升高。此外，去甲肾上腺素还可以使心率增加，心收缩力变大，因此血压升高。 3、静脉注射0.005%盐酸异丙肾上腺素：异丙肾上腺素能与骨骼肌血管β2受体结合，骨骼肌血管（在全身血管中比例较大）持续舒张抵消了皮肤粘膜血管的收缩作用，因而出现后降压作用。 4、静脉注射0.01%盐酸肾上腺素：肾上腺素能与心肌β1受体结合激动心肌心肌收缩力增强，因此心率加快，传导加速，心排出量增多而导致收缩压升高。肾上腺素能激动腹腔内脏血管α1受体，使动腹腔内脏血管收缩，血压升高。 5、静脉注射1%酚妥拉明：α受体阻断剂，α受体的作用：α受体为传出神经系统的受体，根据其作用特性与分布不同分为两个亚型:α1、α2。 α1受体主要分布在血管平滑肌(如皮肤、粘膜血管，以及部分内脏血管)，激动时引起血管收缩;α1受体也分布于瞳孔开大肌，激动时瞳孔开大肌收缩，瞳孔扩大。 α2受体主要分布在去甲肾上腺素能神经的突触前膜上，受体激动时可使去甲肾上腺素释放减少，对其产生负反馈调节作用。 （四）实验主要设备和仪器、药品和用品（要求分类、简洁、清晰表述） 1、仪器设备：婴儿秤、兔手术台、哺乳动物手术器械一套，bl-420生物机能实验系统，动脉夹、压力换能器等； 2、试剂用品：20%氨基甲酸酯（5ml/kg）、1%去甲肾上腺素、1%肝素、生理盐水、1g/l 异丙肾上腺素、1g/l酚妥拉明、5ml、10ml、20ml注射器各一支、三通管、插管、小烧杯等； （五）实验的步骤与方法（列出主要步骤、方法要点） 1、动物手术： （1）、麻醉、固定：家兔称重后，按5ml/kg从耳缘静脉缓慢注射20%氨基甲酸酯。将家兔麻醉后，使其仰卧固定在兔手术台上。 （2）颈部手术：颈部备皮，沿颈前正中切开皮肤5~6cm，分离气管并做气管插管。分离两侧的迷走神经，穿线备用。手术完毕后，用温生理盐水纱布覆盖手术野。分离左侧颈总动