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alterations of serum levels of bdnf-related mirnas in patients with depression

alterations of serum levels of bdnf-related mirnas in patients with depression
alterations of serum levels of bdnf-related mirnas in patients with depression

Alterations of Serum Levels of BDNF-Related miRNAs in Patients with Depression

You-Jie Li1.,Mei Xu2.,Zong-Hua Gao3,Ya-Qi Wang1,Zhen Yue1,Yan-Xia Zhang1,Xin-Xin Li1,

Can Zhang4,Shu-Yang Xie1*,Ping-Yu Wang1,5*

1Department of Biochemistry and Molecular Biology,Binzhou Medical University,YanTai,ShanDong,P.R.China,2Department of Psychiatry of Yantai Fushan District People’s Hospital,YanTai,ShanDong,P.R.China,3Department of Chemistry,Binzhou Medical University,YanTai,ShanDong,P.R.China,4Genetics and Aging Research Unit,Department of Neurology,Massachusetts General Hospital and Harvard Medical School,Charlestown,Massachusetts,United States of America,5Department of Epidemiology,Binzhou Medical University,YanTai,ShanDong,P.R.China

Abstract

Depression is a serious and potentially life-threatening mental disorder with unknown etiology.Emerging evidence shows that brain-derived neurotrophic factor(BDNF)and microRNAs(miRNAs)play critical roles in the etiology of depression.Here this study was aimed to identify and characterize the roles of BDNF and its putative regulatory miRNAs in depression.First, we identified that miR-182may be a putative miRNA that regulates BDNF levels by bioinformatic studies,and characterized the effects of miR-182on the BDNF levels using cell-based studies,side by side with miR-132(a known miRNA that regulates BDNF expression).We showed that treatment of miR-132and miR-182respectively decreased the BDNF protein levels in a human neuronal cell model,supporting the regulatory roles of miR-132and miR-182on the BDNF expression.Furthermore, we explored the roles of miR-132and miR-182on the BDNF levels in depression using human subjects by assessing their serum https://www.doczj.com/doc/fe562850.html,pared with the healthy controls,patients with depression showed lower serum BDNF levels(via the enzyme-linked immunosorbent assays)and higher serum miR-132and miR-182levels(via the real-time PCR).Finally,the Pearson’s(or Spearman’s)correlation coefficient was calculated to study whether there was a relationship among the Self-Rating Depression Scale score,the serum BDNF levels,and serum BDNF-related miRNA levels.Our results revealed that there was a significant negative correlation between the SDS scores and the serum BDNF levels,and a positive correlation between the SDS scores and miR-132levels.In addition,we found a reverse relationship between the serum BDNF levels and the miR-132/miR-182levels in depression.Collectively,we provided evidence supporting that miR-182is a putative BDNF-regulatory miRNA,and suggested that the serum BDNF and its related miRNAs may be utilized as important biomarkers in the diagnosis or as therapeutic targets of depression.

Citation:Li Y-J,Xu M,Gao Z-H,Wang Y-Q,Yue Z,et al.(2013)Alterations of Serum Levels of BDNF-Related miRNAs in Patients with Depression.PLoS ONE8(5): e63648.doi:10.1371/journal.pone.0063648

Editor:Kenji Hashimoto,Chiba University Center for Forensic Mental Health,Japan

Received November20,2012;Accepted April4,2013;Published May21,2013

Copyright:?2013Li et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License,which permits unrestricted use,distribution,and reproduction in any medium,provided the original author and source are credited.

Funding:This study was supported by the NCET-10-0919,’Taishan scholar’position and National Natural Science Foundation(No.30801324,81141114, 81200601),the Shandong Science and Technology Committee(ZR2009CQ033,ZR2009CL005)and the Foundation of ShanDong Educational Committee of China (No.J09LF11,J11LC01).The funders had no role in study design,data collection and analysis,decision to publish,or preparation of the manuscript.

Competing Interests:The authors have declared that no competing interests exist.

*E-mail:wpinggirl@https://www.doczj.com/doc/fe562850.html,(PW);shuyangxie@https://www.doczj.com/doc/fe562850.html,(SX)

.These authors contributed equally to this work.

Introduction

Depression is a serious and potentially life-threatening clinical mental disorder with unknown etiology.In some cases,it shows severe and persistent symptoms,and insidiously impacts on the life quality of the patients[1].Depression is sometimes associated with impaired recovery from a number of medical conditions,including stroke,hip fracture,and myocardial infarction[2–4].Gender has also been found to associate with depression,with women demonstrating higher levels of depressed mood compared with men[5,6].Though the molecular and cellular mechanisms of depression have still not been completely elucidated,it was suggested that nearly50%of the risk for depression is contributed to genetic factors[7].Furthermore,emerging evidence shows that changes in gene expression play crucial roles in the pathogenesis of depression[8,9].The bdnf gene is one of the most essential genes which are involved in the pathophysiology of several mental disorders,including depression.The BDNF protein is a neurotro-phin and plays essential roles in neuronal development and plasticity.Decreased BDNF protein has been suggested to affect the pathology of major depressive disorder(MDD)[10,11].Bdnf has also been suggested as a susceptibility gene for MDD and schizophrenia(SCZ),especially in a subgroup of patients with schizophrenia and a lifetime history of depressive symptoms. Moreover,a polymorphism in the bdnf gene is associated with depression-related traits in several independent studies[12–15]. Because it is difficult to assess the BDNF levels directly from patients’brain,increasing effort has focused on measuring the BDNF levels in the blood.The serum BDNF levels were found to be lower in depressed patients compared with control subjects [16].Interestingly,the serum BDNF levels were increased following the treatment of antidepressant medications[17], suggesting that the serum BDNF levels could be as a surrogate biomarker for determining both the depression status and the efficacy of antidepressant treatment.

Considerable effort has been focused on identifying and characterizing the factors that modulate BDNF levels,one of which that has been recently intensively studied is microRNA (miRNA).miRNAs are endogenous small non-coding RNAs that regulate various gene expression at post-transcriptional levels through targeting mRNAs for cleavage or translational repression [18].miRNAs are essential for different cellular processes, including metabolism,differentiation and apoptosis in both animals and plants[19–21].Increasing evidence suggests a critical role of miRNAs in the pathogenesis of neuropsychiatric disorders, including depression.For example,two polymorphisms,located in pre-miR-182and pre-miR-30e,were associated with the increased risk of the major depressive disorder[22,23].Another study showed that miR-124a,one of the most abundant miRNAs in the brain[24],may contribute to depression by suppressing the expression of glucocorticoid receptor,the activation of which interferes with the hippocampal neurogenesis[25].In addition,a study showed that either up-regulation of miR-16in raphe or down-regulation of miR-16in locus coeruleus induced behavioral responses using depressive mouse models[26].In summary,these studies clearly showed that miRNAs are involved in the etiology of depression by regulating their targeting genes.

Considering the important roles of BDNF and miRNAs in the pathogenesis of depression,our current study was aimed at identifying the novel miRNAs that regulate serum BDNF levels, and characterizing the relationship between miRNAs and depression.First,in a neuronal cell-based model,we used miR-132as a positive control and confirmed that it regulated the expression of BDNF as previous report[27].We also found that miR-182was a novel putative miRNA that regulated the BDNF expression through bioinformatic and cell-based functional studies. Next,we measured the serum levels of BDNF,miR-132and miR-182in human subjects with depression and controls.Our results demonstrated that serum BDNF levels were decreased in depressed patients compared with control subjects,consistent with previous studies.We also found that the levels of miR-132and miR-182were increased in patients compared with those in healthy controls.Our results demonstrate that the serum BDNF and its related miRNAs may be utilized as important biomarkers for the diagnosis or as the therapeutic targets of depression. Results

Clinical Characteristics of Patients and Controls

First,we characterized the demographic and clinical charac-teristics of all the patients and controls that took part in this study (Table1).Forty patients with depression and forty controls participated in the study.Notably,the patients in this study with melancholic features showed observable psychomotor retardation and all patients diagnosed with psychotic depression showed mood-congruent delusions,like delusions of guilt,delusions of poverty or nihilistic delusions.None of the patients with depression in the present study met criteria for atypical depression or suffered from hallucinations.

Lower Serum BDNF Levels in Patients with Depression We next studied the serum BDNF levels in patients with depression and controls by ELISA.Our results showed that the mean serum BDNF levels in the patient samples were 20.0565.98ng/ml,which were much lower than those in healthy controls25.2265.17ng/ml(Table1,Fig.1A).This finding was supported by a previous reported showing that the serum BDNF levels were decreased in patients compared with control subjects [16].Thus,these independent studies suggest that the serum BDNF levels could be utilized as a surrogate biomarker of depression.

Higher Levels of BDNF-related miRNAs in Patients with Depression

Next,we explored to identify and characterize putative miRNAs that regulate BDNF protein levels.We utilized an widely-utilized online miRNA analysis software,which provides a comprehensive analysis to identify the targeting genes of specific miRNAs(https://www.doczj.com/doc/fe562850.html,/microrna/getMirnaForm. do,or https://www.doczj.com/doc/fe562850.html,/index.html).We found that the BDNF mRNA39-untranslated regions(UTR)was targeted by miR-182through this computational analysis(Fig.1B).As a positive control,miR-132was also investigated in this study,which has been reported to regulate BDNF expression[27].

In our next set of experiments,we investigated the functional roles of miR-182in regulating the levels of BDNF using cell-based models.After chemical synthesis of miRNAs,the human neuroblastoma SH-SY5Y cells were transfected with miR-132or miR-182,and the BDNF expression was detected by western blotting analysis.Our results showed that the BDNF levels were decreased in miR-182-treated cells,as well as in miR-132-treated cultures,compared with control treatment(Fig.2A,B).Thus,our results supported the roles of both miR-182and miR-132in regulating the BDNF expression.

Furthermore,we investigated the serum levels of the novel putative BNDF-regulating miR-182,along with miR-132,in our characterized human subjects by real-time PCR.We found that the serum miR-132levels in patients with depression were 0.586103copies/ml(n=40),which were significantly higher than those in the serum of healthy controls(0.136103copies/ml, P,0.01,n=40,Table1,Fig.3A).In addition,the serum miR-182 levels in patients with depression were0.026103copies/ml (n=40),which were also significantly higher than those in the serum levels of healthy controls(P,0.01,Table1,n=40,Fig.3B). Our results demonstrated that miR-182was a novel putative BNDF-regulating miRNA,which may be involved in the pathogenesis of depression.

Relationship between the Serum Levels of BDNF and its Regulatory miRNAs

We have identified that the serum BDNF levels were decreased and the BDNF-related miRNAs including miR-182and miR-132 were increased in human subjects with depression compared with control subjects.Due to the reported role of miRNAs in negatively regulating their targeting gene expression,we reasoned that there might exist a direct negative relationship between the serum BDNF levels and its-related miRNAs.To test this hypothesis,the relationship between the serum levels of BDNF and its-related miRNAs was examined by calculating Spearman’s correlation coefficient.Our results showed a significant negative correlation (Spearman r s=20.307,P=0.006)between the serum BDNF levels and the miR-132levels in patients with depression(n=40) and controls(n=40,Fig.4A).However,there was no significant correlation(Spearman r s=0.098,P=0.385)between the serum BDNF levels and the miR-182levels in all patients(n=40)and controls(n=40,Fig.4B).

Relationship between SDS and the Serum Levels of BDNF-related miRNAs

Finally,we studied whether the serum levels of miRNAs may be applied in diagnosing depression.The relationship between the serum levels of miRNAs and the Self-Rating Depression Scale

(SDS)score was investigated by calculating Pearson’s (or Spear-man’s)correlation coefficient.A significant negative correlation was revealed (Pearson r =20.427,P =7.75E-05)between the serum BDNF levels and the SDS scores in all subjects (n =80),including 40patients with depression and 40controls (Fig.5A).In addition,there was a significant positive correlation (Spearman r s =0.347,P =0.002)between the serum miR-132levels and the SDS scores in all subjects (n =80),including 40patients with depression and 40controls (Fig.5B).A significant positive correlation (Spearman r s =0.242,P =0.030)was also found between the serum miR-182levels and the SDS scores in all subjects (n =80,Fig.5C).Thus,our results indicated that the serum levels of miRNAs were statistically correlated with the SDS scores,and suggested that their levels should be able to assist in the diagnosis of depression.

Discussion

In current study,we characterized the roles of BDNF and its related miRNAs in the pathogenesis of depression.BDNF is an important member of the neurotrophin family of growth factors,

which induces stress in both animal models [28]and human psychopathologies,including major depression,or posttraumatic stress disorder [10,29].We identified miR-132and miR-182as BDNF regulatory miRNAs by bioinformatics,and functionally validated their role in the negative regulation of the BDNF expression using a human neuronal cell model.Then we further explored the roles of BDNF and its related miRNAs in patients with depression and control subjects.Our results showed that the serum BDNF levels in patients with depression were significantly decreased compared with controls.Interestingly,the serum levels of miR-132and miR-182were increased in patients with depression,compared with their controls.A negative relationship between BDNF and miR-132was found in serum of both patients and controls.There was also a negative correlation between the BDNF levels and the SDS scores in depressed patients and healthy controls.Moreover,significant positive correlations were found not only between the serum miR-132level and SDS score,but also between the serum miR-182levels and the SDS scores in depressed patients and healthy controls.

Identifying biomarkers and applying them in the diagnosis of major depressive disorder have remained a goal of clinicians and scientists for many decades.Karege F.and colleagues [30]demonstrated that cortical BDNF levels were consistent with serum BDNF levels in rodents and they also showed that serum

Figure 1.BDNF serum levels and its related miRNAs.(A )BDNF concentrations in patients and controls.The serum levels of BDNF in the patients with depression (n =40;20.0565.98ng/ml)were significantly decreased compared with those of the healthy controls (n =40;25.2265.17ng/ml).(B )The targeting site on BDNF-mRNA-39UTR by miR-182,which was aligned with the mRNA-39UTR of human bdnf gene with the nucleotide position.Vertical lines indicate identity.doi:10.1371/journal.pone.0063648.g001Figure 2.miR-132/182regulating BDNF expression.(A,B )Detection of BDNF by western blotting.SH-SY5Y cells were treated with miR-182or miR-132,the results showed that BDNF expression in the miR-182-or miR-132-treated cultures was much lower than that of negative control miRNA cultures.*P ,0.05,miR-182-or miR-132-treated cultures vs.control cultures.Relative values for BDNF vs .Actin are indicated in Fig.2B.

doi:10.1371/journal.pone.0063648.g002

BDNF levels were lower in patients with depression than those in matched controls [16].Other reports have also indicated that serum BDNF levels were lower in drug-free major depressed subjects than in control subjects [31,32].Similarly,we found that serum BDNF levels were lower in patients with depression compared with controls.We also found that a negative correlation between BDNF level and SDS score in both patients and controls.Thus,the above studies suggest that serum BDNF may be a clinically useful biomarker for depression.

Despite the consistence from many findings showing lower serum BDNF levels in patients with depression,decreased BDNF levels have also been found in several other disease,like Huntington’s disease [33],Alzheimer’s disease [34],schizophrenia [35],and bipolar disorder [36].Thus,the apparent lack of diagnostic specificity in the BDNF levels is likely to be a major drawback,which limits the true clinical application of this measure.Therefore,serum BDNF-related miRNAs,combined with serum BDNF levels,were investigated in this study to increase the feasibility of applying BDNF-related miRNAs in the diagnosis of depression.Our study also warrant future studies of the levels of these BDNF-related miRNA in other mental disorders.

A large number of miRNAs are shown to be specifically expressed in neuronal and non-neuronal cells in the brain and enriched in the central nervous system,and their expressions are regulated at various biological circumstances [24].miR-124and miR-128are primarily expressed in neurons,whereas miR-23,miR-26,and miR-29are expressed in high amounts in astrocytes [37].During acute or chronic stress,the expression of miR-134,miR-183,miR-132,Let-7a-1,miR-9-1,and miR-124a-1is altered in a hippocampal region responsible for mood [38].miR-124and cAMP response element-binding protein (CREB)are found to be involved in a feedback loop contributing to neuronal plasticity [39],which shows that miR-124can control serotonin-induced synaptic facilitation through posttranscriptional suppression of CRE

B and,conversely,CREB can further regulate the expression of miR-124[40].Likewise,a similar mechanism has been identified between BDNF and miR-132,BDNF can dramatically up-regulate neuronal expression of miR-132[41],while

suppres-

Figure 3.Serum miR-132and miR-182levels detected by real-time PCR.(A,B )Serum miR-132or miR-182levels in patients with depression and their controls,respectively.Real-time PCR showed that serum miR-132(or miR-182)levels in depressed patients (n =40)were much higher than those in healthy controls (n =40,P ,0.01).

doi:10.1371/journal.pone.0063648.g003

Figure 4.Relationship between levels of serum BDNF and its related miRNAs.(A )A significant negative correlation (Spearman r s =20.307,P =0.006)between the serum BDNF and miR-132levels in depressed patients (n =40)and controls (n =40).(B )No significant correlation (Spearman r s =0.098,P =0.385)was found between the serum BDNF and miR-182levels in depressed patients (n =40)and controls (n =40).doi:10.1371/journal.pone.0063648.g004

sion of miR-132by 29-O-methyl oligoribonucleotide can increase BDNF transcript levels [27].Similarly,our results demonstrated that miR-132treatment in human neuronal cells significantly decreased the BDNF expression.Thus,these studies indicate that there may be a feedback loop between the expression of miR-132and BDNF.

Our study further studied the neurobiological role of miR-132.MiR-132is important for enhancing neuronal survival and its expression is up-regulated in transplanted neurons [42].miR-132expression in the murine prefrontal cortex can regulate neuro-developmental processes,but its dysregulation contributes to the neuro-developmental pathologies in schizophrenia [43].Davis et al .reported that spontaneous cortical levels of miR-132were lower at the end of the sleep-dominant light period than those at the end of the dark period in rats,suggesting that miR-132played a regulatory role in sleep [44].In this study,our results showed that the serum miR-132levels were increased in patients with depression than those in controls,which might contribute to the lower serum BDNF levels.

We also investigated the biological roles of miR-182in depression.miR-182has been shown to regulate cell growth and suggested as a tumor-suppressive gene.Dysregulation of miR-182can result in tumorigenesis,and lead to gastric adenocarcinoma through a mechanism targeted by CREB1[45].Over-expression of miR-182expression can regulate cell cycle and suppress proliferation of lung cancer cells in vitro [46].However,Bai et al .reported different roles of miR-182in non-sonic hedgehog medulloblastoma,and showed that over-expression of miR-182contributed to leptomeningeal metastatic dissemination in non-sonic hedgehog medulloblastoma and the knockdown of miR-182decreased cell migration in vitro [47].miR-182has also been reported to involve in circadian rhythms in major depression patients with insomnia [22].In this study,our results identified and characterized that miR-182was a novel putative BDNF regulatory miRNA in a human neuronal cell model by both bioinformatic and molecular biological strategies.We also found that the serum levels of miR-182were higher in patients with depression compared with those in healthy controls.The serum levels of miRNAs (miR-132and miR-182)were found to be increased and BDNF levels were reduced in depressed patients compared with healthy controls,which supported that miR-132and miR-182could negatively regulate BDNF expression.Therefore,we found

that a negative correlation between the serum BDNF levels and the miR-132levels in depressed patients and healthy controls,but we did not found a significant negative correlation between the serum BDNF levels and the miR-182levels in patients and controls.The relatively small number of available studies may lead to this limitation.

In summary,we characterized the roles of BDNF and its related miRNAs in the pathogenesis of depression and validated that miR-182was a novel miRNA that regulated the BDNF levels.Our results also demonstrated that the serum BDNF levels were decreased,while the levels of miR-132and miR-182were increased in depressed patients compared with healthy controls.Our results clearly indicate that BDNF and its related miRNAs play important roles in the pathogenesis of depression.Further studies in larger and different subjects are warranted for more conclusive results.

Materials and Methods Subjects

This study was performed at the outpatient department of Psychiatry of Yantai Fushan District People’s Hospital in China.The research protocol was approved by the Medical Ethics Committee of Binzhou Medical University.All experiments were performed in accordance with relevant guidelines and regulations of the Medical Ethics Committee of Binzhou Medical University.Prior to inclusion,eligible patients with depression,or their legal relatives in case of incapacity,provided written informed consent after study procedures were fully explained.

Chinese classification of mental disorders (CCMD-3)was performed for diagnosis,which was based on the Clinical descriptions and diagnostic guidelines of ICD-10.The diagnostic criteria also refer to the Research Criteria of ICD-10,and the DSM-IV [48].Forty patients with depression,aged 23–61years old,fulfilled the CCMD-3criteria for depressive disorder and were diagnosed with depression first time.They did not take anti-depression drugs before.A careful diagnostic assessment was performed to rule out schizophrenia,paranoid psychosis,schizo-affective disorder,bipolar disorder,organic brain syndrome,chronic alcohol or drug abuse,and clinically relevant somatic illness.Then,they were evaluated by Self-Rating Depression Scale (SDS).SDS is a 20-item self-reported measurement of

the

Figure 5.Relationship between serum levels of BDNF-related miRNAs and SDS score.(A )A significant negative correlation (Pearson r =20.427,P =7.75E-05)was found between serum BDNF levels and SDS score in depressed patients (n =40)and controls (n =40).(B )An obvious positive correlation (Spearman r s =0.347,P =0.002)was revealed between the serum miR-132levels and SDS score in patients with depression (n =40)and controls (N =40).(C )There was an obvious positive correlation (Spearman r s =0.242,P =0.030)between the serum miR-182levels and SDS score in depressed patients (n =40)and healthy controls (N =40).doi:10.1371/journal.pone.0063648.g005

symptoms of depression,which including statements about cognitive,psychomotor,somatic,and affective symptoms.Each item is scored from1to4.The raw score is converted into standardized score.A cut-off higher than53is defined presence of depression according to the Chinese version of this scale(Zhang M,Handbook of psychiatry scales.Science and Technology Publishing Company of Hunan).Forty healthy controls,who came to Yantai Fushan District People’s Hospital for physical examina-tion during the period from March1st,2010to September30th, 2010,were diagnosed without any mental or physical illness.All the controls provided written informed consent and agreed to participate in this study after study procedures were fully explained.

Serum BDNF Detection

Serum samples from the patients with depression and controls

were collected between8:00and9:00a.m.After centrifugation for 20min at2,650g,serum was stored at80u C.Serum BDNF was analyzed in duplicate as follows:The samples were incubated in a 96-well plate,followed by the addition of rabbit anti-human antibodies against BDNF(SC-546;Santa Cruz Biotechnology, Santa Cruz,CA)according to the manufacturer’s instructions. Then antibody binding was detected by incubation with goat anti-rabbit IgG/HRP-labeled secondary antibodies(1:2000,Beijing Zhong Shan-Golden Bridge Technology Co.,Ltd.,China)and their substrates in turn.The optical density of the reaction was estimated spectrophotometrically at450nm using an ELX800 ELISA reader(Bio-Tek Instrument Inc.,USA).

Real-time PCR

The mirVana TM miRNA isolation kit(Ambion,USA)was used to extract miRNAs from plasma according to the manufacturer’s instructions.Then,miRNAs were added ploy(A)using poly(A) polymerase(Ambion).The cDNAs were synthesized by RT primer 59-AACATGTACAGTCCATGGATGd(T)30N(A,G,C or T)-39, miR-132and miR-182were analyzed by real-time PCR.The miR-132forward primer:59-ACAGTCTACAGCCATGGTC-39. Reverse primer was59-AACATGTACAGTCCATGGATG-39. The miR-182forward primer was59-GGCAATGGTAGAACT-CACACT-39.Reverse primer was59-AACATGTACAGTC-CATGGATG-39.Real-time PCR analysis was performed by SuperTaq Polymerase(Takara,Japan).The expression of miRNAs was detected using the RG3000system(Corbett Research,Austrilia)with the Quantitect SYBRGreen Kit(Qiagen) as our previous study[49,50]:an initial denaturation at95u C for 4min,followed by40cycles of95u C denaturation for30s,52u C annealing for30s,and extension at72u C for30s.Fluorescence was detected at585nm at each extension step of72u C.Human5S rRNA was added into each sample and served as control.All transfections were performed in triplicate.

Cell Culture and Transfection

Human neuroblastoma cells(SH-SY5Y,obtained from Shang-hai Institute of Cell Biology,China)were grown in DMEM/F12 medium(Hyclone,USA)containing10%FCS(Hyclone,USA) and100U/ml of penicillin-streptomycin(Sigma,USA)at37u C with5%CO2.

MiR-132and miR-182were chemically synthesized in the form of small interfering RNA(siRNA)duplexes according to Park’s study[51](Table2).16106cells were transfected with0.5m g miRNA in 1.5m l of lipofectamine2000(Invitrogen,USA), according to the manufacturer’s instructions.All transfections were performed in triplicate.Western Blotting

Forty-eight hours after transfection,total protein lysates of SH-SY5Y cells were prepared and each40m g protein sample was loaded onto10%SDS-PAGE.After electrophoresis,the protein was transferred to PVDF membrane,which was blocked with7% non-fat milk in TBST[50mmol/L Tris-HCl(pH7.6), 150mmol/L NaCl,0.1%Tween-20]for1.5h at room temper-ature.The TBST buffer solution was added to wash the membrane3times before immunoblotting with polyclonal rabbit anti-human BDNF antibody(1:400,SC-546;Santa Cruz Biotech-nology,Santa Cruz,CA).After being incubation at4u C overnight and a wash with TBST,the membrane was incubated with a HRP-labeled goat anti-rabbit IgG(1:4000,Beijing Zhong Shan-Golden Bridge Technology Co.,Ltd,China)for1h at room temperature.The same membrane also was stripped and re-probed with Actin antibody(1:400,Beijing Zhong Shan-Golden Bridge Technology Co.,Ltd,China)as control.All experiments were performed in triplicate.

Statistics

Data were first tested for normal distribution and variance homogeneity with shapiro.test and F test,respectively.If normal distribution was verified,data were expressed as means6SD,or expressed as median and quartiles.Since BDNF and SDS score showed normal distribution,differences between these two groups were analyzed using Student’s t-test and Pearson’s correlation coefficient was calculated to assess the relationship among variables.Since miR-132,miR-182did not showed normal distribution,nonparametric tests were applied.Differences in continuous variables(miR-132,miR-182)between groups were analyzed by Wilcoxon rank sum test,and the correlations were calculated by the Spearman rank test.Statistical analyses were performed with R version2.15.0(Copyright(C)2012,ISBN3-900051-07-0).For all statistical analyses,P,0.05was considered statistically significant.

Acknowledgments

We are grateful to Hongjie Cui for her help with revision.

Author Contributions

Conceived and designed the experiments:YL SX PW.Performed the experiments:YL MX ZG YW ZY YZ XL SX PW.Analyzed the data:PW SX.Contributed reagents/materials/analysis tools:SX PW.Wrote the paper:SX PW CZ.

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专家访谈-抑郁症

了解抑郁症 许志平 一、抑郁症有哪些主要症状? 1、情绪症状:持续的心境低落及愉快感丧失,可以表现闷闷不乐直至生不如死感,悲观绝望,过分的内疚和自责;兴趣和精力减退。常常伴有焦虑情绪。 2、认知症状:曲解的认知行为模式:消极的看待自我、世界和未来;注意力涣散和记忆减退,可能会误诊为“痴呆”。有时会伴有幻觉和妄想。 3、躯体不适症状:是抑郁症患者常见的主诉,如性质不一的躯体疼痛感,各器官系统的不适:乏力、心慌、胸闷、气短及腹部不适等,但不适感通常是模糊的,部位、性质及程度变化不定,缺乏特异性,躯体及辅助检查正常。 4、生物学症状:睡眠、食欲及性欲的改变。 二、如何初步判断是否患了抑郁症? 1、上述症状至少持续存在2周以上; 2、上述症状导致患者出现显著的精神痛苦或者功能损害。 3、要明确可能导致抑郁的躯体疾病、活性物质及药物。如甲状腺功能减退、脑血管病、多发性硬化及脑退行性病等。 三、抑郁症常见吗? 是一种常见病,据国际上流行病学调查,抑郁症终生患病率从7.8%到17.1%不等。平均发病年龄为20-30岁,成年女性患抑郁症的比例是男性2倍,老年人群的患病率将近25%。内科住院患者中有22%-33%诊断患有抑郁症及相关心里障碍。 四、抑郁症的危险因素或者病因有那些呢? 抑郁症的发生与生物、心理和社会因素有关,在有的病例中某些因素是重要的甚至是决定性的因素。 1、与遗传素质密切相关:调查发现大约将近或者超过50%的患者有抑郁症家族 史,亲属同病率远高于一般人群,血缘关系越近患病风险越高,目前认为是多基因遗传方式。 2、性别因素:成年女性患抑郁症的比例是男性2倍,性别差异可能与以下因素 有关,女性生命过程中要经历数次体内性激素的显著变化,女性生活更为艰难,遭遇负性生活事件更多,缺乏有效的应对策略。 3、与儿童期的不良经历密切相关:儿童期父母离世,缺乏父母关爱,受到虐待, 父母过分严厉,成长环境封闭,缺乏社会交往等。 4、性格因素:一些性格特征可能与抑郁症发生相关,如追求完美,道德感过强, 谨小慎微,过分看重工作成效而不顾乐趣和人际交往,容易紧张和忧虑,社交场合过分担心被指责和拒绝等。 5、心理社会环境因素:婚姻状况不良,社会经济地位低,重大突发的或者持续 的负性生活事件,负性生活事件为丧失和受屈辱性质,都是抑郁症的重要危险因素。 6、其他因素。越来越多的神经生化、神经内分泌,影像学及分子遗传学等研究 证据表明,抑郁症可能是一种生物学疾病。 五、抑郁症的治疗手段有哪些呢? 综合性的治疗手段,包括心理治疗、药物治疗及物理治疗等,大多数患者的治疗是有效的。 六、社会上对抑郁症的几个认识误区。

识图与构造机考题库(学生复习用)

《建筑识图与构造》题库 一、单项选择题:(共30题,每题2分,共60分) .国标中规定施工图中水平方向定位轴线的编号应是( C ) A、大写拉丁字母 B、英文字母 C、阿拉伯字母 D、罗马字母 .附加定位轴线2/4是指( B ) A、4号轴线之前附加的第二根定位轴线 B、4轴线之后附加的第二根定位轴线 C、2号轴线之后的第4根定位轴线 D、2号轴线之前附加的第4根定位轴线 .索引符号图中的分子表示的是( C ) A、详图所在图纸编号 B、被索引的详图所在图纸编号 C、详图编号 D、详图在第几页上 .有一图纸量得某线段长度为5.34cm,当图纸比例为1:30时,该线段实际长度是( C)米。 A、160.2 B、17.8 C、1.062 D、16.02 .门窗图例中平面图上和剖面图上的开启方向是指( A ) A、朝下,朝左为外开 B、朝上,朝右为外开 C、朝下,朝右为外开 D、朝上,朝左为外开 .房屋施工图中所注的尺寸单位都是以( C )为单位。 A、以米为单位 B、以毫米为单位 C、除标高及总平面图上以米为单位外,其余一律以毫米为单位 D、除标高以米为单位外,其余一律以毫米为单位 . 图标中规定定位轴线的编号圆圈一般用( A ) A、8mm

B、10mm C、6mm D、14mm . 总平面图中用的风玫瑰图中所画的实线表示( A ) A、常年所剖主导风风向 B、夏季所剖主导风风向 C、一年所剖主导风风向 D、春季所剖主导风风向 . 建施中剖面图的剖切符号应标注在( A ) A、底层平面图中 B、二层平面图中 C、顶层平面图中 D、中间层平面图中 . 楼梯平面图中标明的“上”或“下”的长箭头是以哪为起点( C ) A、都以室首层地坪为起点 B、都以室外地坪为起点 C、都以该层楼地面为起点 D、都以该层休息平台为起点 . 楼梯平面图中上下楼的长箭头端部标注的数字是指( B ) A、一个梯段的步级数 B、该层至上一层共有的步级数 C、该层至顶层的步级数 D、该层至休息平台的步级数 . 与建筑物长度方向一致的墙,叫( A ) A、纵墙 B、横墙 C、山墙 D、墙 . 施工图中标注的相对标高零点±0.000是指( C ) A、附近黄海平均海平面 B、建筑物室外地坪 C、该建筑物室首层地面 D、建筑物室外平台 . 计算室使用面积的依据是( C ) A、轴线到轴线间尺寸 B、墙外皮到墙外皮

最新精神分裂症病例

[临床类型] 疗反应和预后有一定指导意义。临床上常见的类型有偏执型、单纯型、青春型的紧张型。此外,尚有其他类型。 一、偏执型(Paranoid type):又称妄想型。本型最多见。发病年龄多在中年(25~35),起病缓慢或亚急性起病,症状以妄想为主,关系和被害妄想多见,次为夸大、自罪、影响、钟情和嫉妒妄想等。妄想可单独存在,也可伴有以幻听为主的幻觉。情感障碍表面上可不明显,智力通常不受影响。患者的注意和意志往往增强,尤以有被害妄想者为著,警惕、多疑且敏感。在幻觉妄想影响下,患者开始时保持沉默,以冷静眼光观察周围动静,以后疑惑心情逐渐加重,可发生积极的反抗,如反复向有关单位控诉或请求保护,严重时甚至发生伤人或杀人。患者也可能感到已成为“众矢之的”,自己已无力反抗的心境下,不得已采取消极的自伤或自杀行为。因而此型患者容易引起社会治安问题。病程经过缓慢,发病数年后,在相当长时期内工作能力尚能保持,人格变化轻微。患者若隐瞒自己表现或者说强调理由时,往往不易早期发现,以致诊断因难。

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混凝土的结构识图题库

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抑郁症案例分析

抑郁症案例分析 案例介绍: 徐某,男,19岁,某中学高二年级的学生。出身于知识分子世家。在新中国成立后的历次政治运动中,该生的家属大部分受到冲击,其中受迫害致死的3人,徐某的父亲则在“文革”中因受冲击而患精神分裂症。 徐某本人从小就有受歧视感。上小学时,他曾听邻居说过:“这孩子可怜,爸爸是精神病。”听到这种议论,他当即便产生了自卑的想法,觉得“世上哪有我这样不幸的人”。进入初中后,他认为父亲有病是家丑,不敢让父亲出席学校召开的家长会。由于徐某学习成绩不佳,初一时经常受到老师惩罚,并被老师封为班里的“第二号呆子”。为此,他非常恨老师,同时自卑感进一步加剧。初二时,他得了慢性肾炎,只好休学一年。重新复学后,学习成绩仍不见起色,身体状况也一直不好。他渐渐地感到,自己离班级、离同学越来越远。随着年龄增长,看到同学们各有所乐,徐某的无用感、无助感进一步强化,但他同时又认为,周围的同学、老师都具有小市民习气,他们不可能理解自己的行为。虽然有腰酸、背痛、头晕、头疼及乏力等症状,他也不愿到医院进行检查。 从初三开始,徐某便产生了想死的念头,并具体设想了如何去死,但缺乏勇气。进入高中后,他的状况依旧,一切均无好转。他对社会、对家庭、对人生、对自己都失去了信心,悲观丧气,提不起精神上学,也不想上学,觉得自己是社会中多余的成员,还是死了为好。下面是他自己的一段诉说:“我从小受歧视,养成内向的性格。我自卑又自尊,很留意别人对我的评价。我什么都不行,包括身体、成绩、家庭。同学们对社会充满希望,我却很悲观。世上小人太多,话说多了就会遭到歧视。我不敢反驳别人,见人低着头走,我要保护自己。我身体不好,痛苦多,随它去,死了算了。达尔文说‘适者生存’,我不适就让社会淘汰吧。我是过一天算一天,还上什么学,死了好!但我母亲对我好,我不能死在她前面(哽咽),我为母亲而活着。” 案例分析: 徐某认为自己什么都不行,很留意别人对自己的评价,自我评价过低。他有腰酸、背痛、头晕、头疼及乏力等症状,产生了精神疲乏感。此外,他反复出现想死的念头,并具体设想了如何去死,而且他对一切都没有兴趣,也不愿意上学。所有这些行为表现都符合抑郁症的诊断标准,而且已经达到了抑郁症的诊断标准。因此,徐某患的是抑郁症。 徐某的抑郁症主要是由于长期的社会不良因素的刺激。特殊的政治背景对徐某幼小的心灵的影响,家庭气氛的长期压抑,老师的责罚与刺伤,使他心中产生了阴影,对社会形成悲观的看法。在其人格的发展中,自卑、自暴自弃等性格上的弱点,对徐某的抑郁倾向的形成也有不可低估的影响。 在抑郁症的心理治疗中,认知—行为治疗、短程动力治疗和人际治疗疗效比较理想。对于徐某,可以从以下四个方面着手。(1)耐心倾听,使其充分发泄压抑多年的苦闷。当然,耐心倾听的前提是要充分得到他的信任感。(2)引导他正确认识社会、家庭和自己。我们可以举出大量的例子,说明他的遭遇并不是唯一,也不是最惨的。通过认知调整,使他已有的一些模糊认识和不合理的观念得到澄清。(3)通过智力测验和人格测验发现他的长处,鼓励他振作起来,从而多方面感受人生的价值。(4)及时与家长和学校取得联系,尽量改善他的外部心理环境。让他的母亲在平时的生活中,多关心他,创造一个关心体贴的家庭氛围。同时让他的老师多给他创造可以展示自己才能的机会,不让他有孤立的感觉。

建筑制图与识图练习题与答案

1、以下建筑平面图纸中需要标注指北针的是()。 B、底层平面图 2、原位标注代表该部位的特殊数值,施工时应以原位标注取值优先。 C、平法 3、两个或两个以上的点、线、面具有同一的投影时,则称它们的投影()。 D、重合 4、建筑详图的比例范围为()。 D 、1:1-1:50 5、楼梯详图中需要标注的不只是各种尺寸,还有步数、平台标高、墙段轴线、梯段上下 关系。 C、结构设计 6、建筑用地范围内所有建筑物占地的面积与用地总面积之比叫做()。 A、建筑系数 7、将原状图放大后加以充实,清晰详尽地标注尺寸、写出说明,并将装饰纹样进行具象补 充以便参照的图样是()。 B、局部放大图 8、建筑给排水施工图中应画成粗实线的是()。 C、排水和重力流管线 9、基础详图通常为用()剖切平面沿垂直于定位轴线方向切开基础所得到的断面图。 B、铅垂 10 、柱平法施工图中,柱在不同标准层截面多次变化时,可采用(),其余情况宜用截面注写方式。 C、列表注写方式 11 、用于绘制晒图底版的图纸是()。 C、硫酸纸 12 、装饰平面布置图的剖切形式和位置与建筑平面图()。 B、完全一样 13 、框架结构的楼层结构布置图中被现浇板遮盖住的梁不可见轮廓线应画成()。 A、细虚线 14 、房屋建筑的平面图通常采用的比例为()。 B 、 1:100 15 、图纸的大小称为()。 A、图幅 16 、主要表示承重结构的布置情况,构件类型、大小以及构造作法等的图纸是()。 B、结构施工图

17 、反映电气系统基本组成、主要电气设备、元件之间的连接情况以及规格、型号、参数 等的图纸称为()。 C、电气系统图 18 、建筑总平面图观察基地的地貌特征主要依靠阅读()。 D、等高线 19 、观察以下图例,阅读正确的是()。 D、自然土壤、砖、钢筋混凝土 20 、建筑平面图中对墙、柱进行轴线编号的作用是()。 D、定位 21 、建筑剖面图是假想用一个或多个垂直于外墙轴线的(),将房屋剖开,所得的投影图。 B、铅垂剖切面 22 、建筑物宽度方向的墙叫做()。 B、横墙 23 、建筑施工图特指建筑设计工种在()阶段的图纸。 D、施工 24 、建筑工程图纸中的标高数据采用的单位是()。 D、米 25 、详细表现出装饰面连接处的构造,注有详细的尺寸和收口、封边的施工方法的图样是()。 C、构造节点图 26 、仿宋字的字号表示字的()。 C、高度 27 、能够满足设备材料采购、非标准设备制作和施工的需要的是()。 C、施工图 28 、图纸中书写的汉字字高应不小于()mm。 C 、3.5 29 、消防设计专篇中应对总平面设计中的()和消防登高面作出要求。 D、防火间距、消防车道 30 、绘图铅笔尾部的“ B或”“ H等”字样表示铅笔的()。 C、硬度 31 、以下部分在建筑剖面图中用粗线表达的是()。 B、剖到的墙体 32 、以下不属于建筑图纸作用的是()。

马克思主义哲学知识体系结构图

马克思主义哲学知识体 系结构图 公司标准化编码 [QQX96QT-XQQB89Q8-NQQJ6Q8-MQM9N]

马克思主义哲学理论结构图 【整体结构图】 物质及其存在形式辩证唯物主义物质范畴辩证唯物论物质世界与人的实践存在形式:运动、时间、空间 世界物质统一性与实事求是 普遍联系 基本特征 永恒发展 对立统一规律:揭示事物发展的动力和源泉 唯物辩证法基本规律质量互变规律:揭示事物发展的形式和状态 马否定之否定规律:揭示事物发展的方向和道路 克原因与结果 思现象与本质

主基本范畴内容与形式 义可能与现实 哲偶然与必然 学 认识是主体对客体的能动反映 认识的本质 认识与实践 第一次飞跃:从感性认识到理性认识 辩证唯物主义认识的过程第二次飞跃:从理性认识到实践 认识论认识的循环性和上升性 认识的真理性 真理观检验真理的标准 真理与谬误 思维方法:分析与综合、归纳与演绎、抽象与具体、历史与逻辑 社会存在

社会本质和社会的实践本质 基本结构社会结构 社会基本矛盾:社会发展的根本动力 历史唯物论社会发展规律科学技术:第一生产力 和历史创造者人民群众:历史创造者 社会历史进程 社会发展和人的本质和价值 人的发展共产主义社会 【第一章结构图】 哲学是理论化系统化的世界观 哲学哲学与世界观的关系 哲学与具体科学的关系 哲学和哲学朴素唯物主义的基本问题唯物主义形而上学唯物主义

第一性问题辩证唯物主 义和历 史唯物主义 哲学基本问题主观唯心主义 (思维和存在唯心主义 的关系问题)客观唯心主义 可知论 马克第二性问题 思主不可知论 义哲历史根源和阶级基础 学是马哲产生的自然科学和社会科学前提 科学马克思主义历史必然性直接理论来源 的世哲学的基本主观条件 界观特征科学性 和方马哲的本质革命性 法论特征实践性

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