【作者】 刘奇峰；

【导师】 刘闺男；

【作者基本信息】 中国医科大学 ， 内科学， 2009， 博士

【摘要】 Egr-1与骨桥蛋白在大鼠血管平滑肌细胞中的相关性及其机制的研究前言动脉粥样硬化性疾病、血管重建术后再狭窄（restenosis,RS）、高血压等血管重塑相关性疾病已经愈来愈严重地威胁着人类的健康。目前认为,血管中膜平滑肌细胞（smooth muscle cell,SMC）向内膜下迁移与增殖是血管重塑的主要病理基础之一。研究表明,某些转录因子（transcription factors,TF）可以调节多个血管平滑肌细胞（vascular smooth muscle cell,VSMC）增殖相关基因的表达,从不同层面调控VSMC的迁移与增殖。同时,人们也逐渐注意到细胞外基质（extracellular matrix,ECM）在血管重塑过程中的重要地位。研究发现,ECM在各种外界刺激的作用下,可以作为细胞外信号,经跨膜信号传递系统到达核内,激活一系列调控VSMC增殖的TF,从而推动VSMC的分裂、迁移及增殖。TF与ECM在血管重塑的过程中密不可分,因此将二者有机地联系在一起,并对其相互作用关系进行深入探讨,将有助于全面了解血管重塑的细胞与分子机制。早期生长反应因子-1（eally growth response factor-1,Egr-1）作为一种锌指结构TF,参与多种基因的调控,与细胞增殖关系密切。大量研究表明,Egr-1在介导VSMC迁移、增殖过程中发挥着重要的作用,成为目前血管重塑机制研究中的焦点。骨桥蛋白（osteopontin,OPN）作为ECM中一种重要的功能性蛋白,被认为是血管损伤重塑过程中重要的始动因素,应用抗OPN抗体可以抑制VSMC的表型转化、迁移和增殖。尽管Egr-1和OPN在介导VSMC迁移、增殖过程中均起着重要的作用,但二者之间的关系尚不清楚。本课题拟在以往研究的基础上,对培养的大鼠主动脉VSMCs分别稳定转染Egr-1、OPN基因,并检测转染前后OPN与Egr-1的表达变化,从而分析二者的相关性。并应用染色质免疫沉淀（chromatin immunoprecipitation,ChIP）方法检测Egr-1与OPN启动子的结合情况,同时通过向OPN稳定转染细胞株中加入细胞外信号调节蛋白激酶（extracellular signal-regulated kinase,ERK）的磷酸化抑制剂,检测抑制ERK磷酸化后,OPN上调Egr-1的水平变化,从而对Egr-1与OPN相关性的内在机制进行深入探讨,并为抑制VSMC的迁移、增殖,控制血管壁的不适当重塑提供理论及实验基础。材料与方法1、大鼠VSMC的培养大鼠A10主动脉VSMC细胞株购自ATCC细胞库,用含10%胎牛血清（fetalbovine serum,FBS）的DMEM培养基,在37℃、5%CO₂、饱和湿度的培养箱中培养,用0.25%的胰酶消化、传代。2、质粒的提纯、扩增及稳定转染委托TaKaRa公司对pCMV-Egr-1/NEO、pCMV（-）/NEO质粒及pET-28-rOPN/NEO、pET-28（-）/NEO质粒进行提纯、扩增及鉴定,其中pET-28-rOPN/NEO和pET-28（-）/NEO质粒被连于CMV启动子上,以便在VSMC中表达。待培养的VSMCs生长到80%汇合度时,以100μg/ml～1mg/ml的新霉素（neomycin418,G418）浓度进行最低G418浓度筛选。当培养的VSMCs在6孔板内达到80%汇合度时,应用FuGENE6分别向VSMCs内转染pCMV-Egr-1/NEO、pCMV（-）/NEO质粒及pCMV-ET-28-rOPN/NEO、pCMV-ET-28（-）/NEO质粒。细胞转染24小时（hour,h）后,加入400μg/ml G418的培养液进行培养。2周后,采用有限稀释法将细胞传至96孔板中进行单克隆化培养（于含200μg/ml G418的条件培养液中）,待长满后进行扩大培养,并检测细胞中Egr-1、OPNmRNA的表达。3、应用ERK磷酸化抑制剂阻断ERK信号传导通路当OPN稳定转染的VSMCs生长至80%汇合度时,向细胞培养液中加入10μMERK磷酸化抑制剂PD98059,继续培养24h后,进行Western blot检测。4、反转录多聚酶链反应（reverse transcriptase polymerase chainreaction,RT-PCR）检测Egr-1、OPNmRNA用Trizol Reagent提取VSMCs的总RNA。用RNA PCR Kit Ver.3.0扩增目的片段。PCR产物电泳后,用溴化乙啶（ethidium bromide,EB）染色,BioImaging Systems系统成像,用NIH image软件分析产物条带灰度,以Egr-1、OPN产物条带灰度值与甘油醛-3-磷酸脱氢酶（glyceralde-3-phosphate dehydrogena,GAPDH）产物条带灰度值的比值分别作为Egr-1、OPNmRNA的相对表达量。5、Western blot检测Egr-1、OPN、ERK、P-ERK蛋白提取细胞总蛋白,上样量为80μg。转印到PVDF膜上后用5%脱脂奶粉封闭,用Egr-1兔抗大鼠多克隆抗体（1:300）、OPN兔抗大鼠多克隆抗体（1:500）、ERK兔抗大鼠多克隆抗体（1:200）、磷酸化细胞外调节蛋白激酶（phosphoextracellularsignal-regulated kinase,P-ERK）兔抗大鼠多克隆抗体（1:200）及兔抗大鼠α-tubulin多克隆抗体（1:500）4℃孵育过夜,分别用相应的二抗37℃孵育2h,ECL发光1分钟,胶片中的蛋白条带经BioImaging Systems采集后进行灰度值测定。目的蛋白灰度值与内对照α-tubulin比值为其相对蛋白定量。6、ChIP方法检测Egr-1与OPN启动子的结合分别取2×10⁶个对数生长期的稳定转染Egr-1的VSMCs及正常VSMCs,用甲醛交联,0.125M甘氨酸终止交联。超声剪切染色质脱氧核糖核酸（deoxyribonucleicacid,DNA）成500～1000个碱基对（basepair,bp）片段。分别取50μl全细胞抽提物作为实验和阴性对照样本。用ChIP缓冲液10倍稀释后各取其中10μl合并作为输入对照（input）,在实验样本中加入5g Egr-1兔抗大鼠多克隆抗体（1:300）,将实验样品及阴性对照样本4℃孵育过夜。几次洗涤后,复合物从珠子上洗脱下来。用酚氯仿抽提纯化DNA,最后应用于PCR分析。7、统计学分析采用SPSS11.0统计软件进行数据分析。Egr-1与OPN的相关性用双变量相关分析法（Spearman相关分析）,不同组间的参数比较用One-way ANOVA分析,两两比较采用LSD方法,P＜0.05有统计学意义。实验结果1、RT-PCR结果显示:Egr-1稳定转染组的Egr-1mRNA较正常对照组和空载体组表达明显增强（p＜0.01）;OPN稳定转染组OPNmRNA与正常对照组和空载体组比较,表达也明显增强（p＜0.01）。2、RT-PCR及Western blot方法检测Egr-1和OPN稳定转染VSMCs后OPN和Egr-1的表达水平。结果显示:与正常对照组和空载体组比较,Egr-1转染组的OPNmRNA及蛋白表达均随着Egr-1表达的增强而明显上调（p＜0.01）;同样,OPN稳定转染组的Egr-1mRNA及蛋白表达较正常对照组和空载体组明显升高（p＜0.01）。3、ChIP结果显示:无论正常对照组还是Egr-1稳定转染组的Egr-1抗体免疫沉淀DNA中均可以扩增出含OPN启动子结合位点的基因调控区片段。4、Western blot结果显示:正常对照组与OPN转染组给予PD98059后,与未处理组比较,P-ERK/ERK表达均明显减少（p＜0.01）,同时,Egr-1表达明显下调（p＜0.01）。结论1、成功建立了Egr-1、OPN的稳定转染细胞株。2、Egr-1无论在转录水平还是在翻译水平均可影响OPN的表达,同样,OPN也可以影响Egr-1的表达,二者的表达呈正相关性。3、VSMC中,OPN启动子区域存在着Egr-1的结合位点,Egr-1参与了对OPN基因的转录调控。4、OPN可以通过ERK途径反馈上调Egr-1的表达。5、VSMC中,Egr-1与OPN之间形成一个作用级联放大的正反馈环。更多还原

【Abstract】 ObjectiveVascular remodeling diseases,such as atherosclerosis,restenosis（RS）following reconstructive vascular operation,and hypertension has become an increasingly serious threat to human health.Currently,many studies have demonstrated that migration and proliferation of the vascular smooth muscle cells（VSMCs）in the vascular tunica media is one pathological basis of vascular remodeling.Studies have found that some transcription factors can regulate the expressions of several VSMC proliferation-associated genes,and thereby regulate VSMC proliferation. At the same time,under the influence of various external stimuli,some extracellular matrix can be used as extracellular signal reaches the nucleus and then through an intracellular signal transduction system to induces the expression of a series of VSMC proliferation-associated transcription factors,and thereby promotes VSMC migration and proliferation.With careful attention to the reciprocal relationship between transcription factors and extracellular matrix,which conduce to finding out the mechanism underlying vascular remodeling.Early growth response factor-1（Egr-1）,a zinc finger transcription factor,regulates the expression of multiple proliferation-associated genes,and its expression level is closely correlated with cell proliferation.Studies found that Egr-1 could mediate the migration and proliferation of VSMCs.Osteopontin（OPN）is a functionally important protein in the extracellular matrix（ECM）,and anti-OPN antibodies can inhibit the phenotypic modulation,proliferation and migration of VSMCs.Although OPN and Egr-1 both play a significant role in mediating the process of migration and proliferation of the VSMCs,relatively little is known about the relationship between them.We used Egr-1 and OPN cDNA to transfect VSMCs and then detected the expression changes of Egr-1 and OPN and observed the relationship between Egr-1 and OPN before and after transfection.Meanwhile,we performed a chromatin immunoprecipitation（CHIP）assay to examine whether Egr-1 could bind to the OPN promoter,and In order to clarify the mechanism of OPN-mediated changes in Egr-1 expression,extracellular signal-regulated kinase（ERK）inhibitor was added to OPN-transfected cells.These results of the interaction between Egr-1 and OPN will likely provide an important theoretical and experimental basis needed to control the inappropriate remodeling of vessel walls.Materials and Methods1.Rat A10 aortic VSMCs were culturedRat A10 aortic VSMCs were purchased from ATCC.Cells were cultured in Dulbecco’s Modified Eagle Medium（DMEM）with 10%fetal bovine serum（FBS）at 37℃in a humid atmosphere of 5%CO₂.2.Vectors and stable transfectionpCMV-Egr-1/NEO and pCMV（-）/NEO were kindly provided by Dr.Lorraine E. Chalifour,Division of Experimental Medicine,McGill University,Montreal,Canada. pET-28-rOPN/NEO and pET-28（-）/NEO were kindly provided by Dr.Harvey A Goldberg,University of Western Ontario,London,Canada.The plasmids were purified, amplified,identified and the ET-28-rOPN and ET-28（-）cassette were placed under control of a CMV promoter to drive OPN expression by TaKaRa.To establish stable cell lines,cells were cultured at 80%confluence in 6-well dishes and then transfected with either pCMV-Egr-1/NEO or pCMV（-）/NEO or pCMV-ET-28-rOPN/NEO or pCMV-ET-28（-）/NEO,which both express Geneticin 418（G418）resistance.All transfections were performed using FuGENE6 according to the manufacturer’s instructions.One day later,cells were subcultured and grown in the presence of 400μg/ml G418.After 2 weeks,single G418-resistant colonies were obtained by serial dilution in 96-well dishes.Colonies were maintained in a medium containing 200μg/ml of G418 and analyzed individually for expression of Egr-1 or OPN.3.ERK inhibitor to inhibit the ERK pathwayCells expressing OPN were cultured at 80%confluence and then treated with 10μMPD98059,the ERK pathway inhibitor.24h after PD98059 treatment,we analyzed Egr-1 expression via Western blot.4.Reverse transcriptase polymerase chain reaction（RT-PCR）for Egr-1、OPNmRNATotal RNA was extracted from VSMCs using Trizol Reagent according to the manufacturer’s instructions.RT-PCR analysis was performed using TaKaRa RNA PCR Kit（AMV）Ver.3.0 according to the manufacturer’s instructions.Products were resolved by 1%agarose gel and bands visualized by ethidium bromide staining. Densitometric analysis of bands was performed using BioImaging Systems.5.Western blot analysis for Egr-1、OPN、ERK、P-ERK proteinProtein lysate（80μg）from cells was resolved by 12%sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE）and transferred to PVDF membranes. The filters were blocked with TBST buffer containing 5%skim milk,incubated with primary antibodies(anti-Egr-1（1:300）,anti-OPN（1:500）,anti-ERK（1:200）, anti-P-ERK（1:200）,andα-tubulin（1:500）overnight at 4℃Samples were then incubated with HRP-IgG secondary antibody and enhanced chemiluminescence（ECL） was used to visualize the bands.Band quantification was performed using Quantity One.Experiments were performed in triplicate.6.Chromatin immunoprecipitation（ChIP）analysis to observe the Egr-1 binds to the OPN gene promoter. ChIP assays were performed using the ChIP assay kit according to the manufacturer’s instructions.The procedure included DNA-protein cross-linking inchromatin,shearing DNA into smaller fragments,immunoprecipitation with anti-Egr-1 antibody,and PCR identification of associated DNA sequences.Input was used as the positive control.7.Statistical analysesAll values were expressed as mean±SD.SPSS11.0 software was used for all statistical analysis.The relationship between Egr-1 and OPN was analyzed using bivariate correlation analytical method（Spearman’s correlation analysis）.Data were analyzed using one-way analysis of variance followed by a least significant difference test（LSD）for multiple comparisons.Differences were considered significant If p＜0.05.Results1.RT-PCR results showed that Egr-1 mRNA was more highly expressed in VSMCs which were stably transfected with Egr-1 than in control cells and vector-transfected cells（p＜0.01）,similarly,OPN mRNA was more highly expressed in cells stably transfected with OPN cDNA than controls group and vector-transfected group（p＜0.01）.2.We analyzed Egr-1 and OPN expression via RT-PCR and Western blot.The results showed that OPN mRNA and protein expression levels in the Egr-1-transfected VSMCs were significantly increased（p＜0.01）,compared to untransfected cells and vector-transfected cells.Similarly,Egr-1 mRNA and protein levels were increased in the OPN-transfected VSMCs（p＜0.01）.3.ChIP results showed that the OPN promoter sequence can be amplified in both control and Egr-1-transfected cells.4.Western blot results showed that levels of phospho-ERK were decreased relative to ERK in the PD98059 treated group compared to controls（p＜0.01）. Furthermore,we found that Egr-1 expression was reduced in PD98059 treated cells compared to control-treated cells（p＜0.01）.Conclusion1.Egr-1 cDNA or OPN cDNA was successfully transfected into VSMC_S.2.Egr-1 can affect the expression of OPN,Similarly,OPN can also affect the expression of Egr-1,and Egr-1 and OPN expression were positively correlated.3.Egr-1 can bind to the OPN promoter,and is likely to directly regulate its transcription.4.OPN acts through the ERK pathway to upregulate Egr-1.5.Egr-1 and OPN factors are likely to operate in a positive feedback loop in VSMC.更多还原