【作者】 陈建；

【导师】 夏豪；

【作者基本信息】 武汉大学 ， 心血管内科， 2022， 硕士

【摘要】 研究背景心脑血管系统疾病是威胁人民生命健康的临床常见病、多发病。近年来,随着中国人口老龄化趋势以及饮食、生活方式的改变,冠心病、心肌梗死等疾病的发病率呈日益上升趋势。目前,经皮冠状动脉支架植入介入治疗是临床治疗冠状动脉性心脏病最主要的方式,然而由于支架植入导致的内皮损伤以及血管平滑肌细胞(vascular smooth muscle cells,VSMCs)的增殖和迁移进而造成的术后血管再狭窄(restenosis,RS)始终是困扰医生与病人的一个问题。随着临床诊疗技术的不断发展,支架植入经历了从裸金属支架(bare metal stent,BMS)到药物洗脱支架(drug-eluting stent,DES)的更新换代,然而血管再狭窄的病理机制依旧没有完全揭开,对其发病机制以及治疗的研究对临床诊疗以及人民健康具有重要意义。Sema4d是一种Ⅰ型跨膜蛋白,其广泛分布于神经,骨骼肌,内皮,肿瘤等各个组织和器官,并参与细胞增殖、迁移、粘附、免疫应答、心血管发育等多种生理病理过程。有研究表明,在心力衰竭、冠心病等疾病中,其血清表达水平明显升高,且可通过plexin B1受体激活级联信号,对增殖相关的MAPK信号通路进行激活。由于血管平滑肌的增殖迁移、表型转换是血管再狭窄的重要病理机制,我们推测sema4d可能参与了血管再狭窄的发生过程,并通过本研究探究sema4d在此过程中发挥的作用。研究目的探究sema4d在病理性血管内膜增生中的作用以及相关信号通路机制方法第一部分:细胞实验部分,通过组织块贴壁法体外培养原代大鼠VSMCs,通过免疫荧光检测α-SMA进行细胞鉴定,并使用血小板衍生生长因子-BB(PDGF-BB)诱导其增殖与迁移,通过免疫荧光、western blot等方法观察诱导前后sema4d的表达水平改变;另外,通过腺病毒(Ad-sh Sema4d)降低sema4d的表达,将细胞分为对照组、PDGF-BB刺激组、PDGF-BB+Ad-GFP组、PDGF-BB+Ad-sh Sema4d四组,通过CCK-8、PCNA水平检测细胞增殖;通过transwell、伤口愈合实验、MMP-9水平检测细胞迁移;通过α-SMA、OPN表达水平检测细胞表型转换;通过western blot检测ERK1/2、p-ERK1/2水平探究sema4d对MAPK信号通路的影响。第二部分:动物实验部分,通过球囊导管建立大鼠颈总动脉损伤动物模型,并通过对血管损伤区域局部孵育Ad-sh Sema4d腺病毒的方式实施干预。动物实验分组为sham组、模型组、模型+Ad-GFP组、模型+Ad-sh Sema4d组。术后14天处死大鼠,对损伤血管处取材并进行石蜡包埋切片或提取组织蛋白,通过HE染色探究Sema4d对大鼠颈总动脉内膜增生的影响,通过组织免疫荧光与免疫组化探究Sema4d以及KI67、MMP-9、SM-22α、OPN的表达水平。结果第一部分:细胞实验部分,免疫荧光细胞鉴定显示:组织块贴壁法提取的SD大鼠颈总动脉VSMCs免疫荧光α-SMA染色阳性率大于95%;免疫荧光及western blot显示,使用PDGF-BB(20ng/ml)诱导后sema4d的表达水平明显升高(P<0.05);CCK-8细胞增殖实验显示,PDGF-BB刺激组(B组)、PDGFBB+Ad-GFP组(C组)细胞增殖活性显著高于对照组(A组)(P<0.05),而PDGF-BB+Ad-sh Sema4d组(D组)增殖活力则较PDGF-BB刺激组(B组)、PDGF-BB+Ad-GFP组(C组)有所降低(P<0.05),B、C两组增殖水平无显著差异(P>0.05);western blot检测PCNA表达水平显示,B组、C组PCNA表达水平较A组升高(P<0.05),D组较B组、C组PCNA表达明显降低(P<0.05),B、C两组无显著差异;Transwell结果显示:B组、C组穿出小室细胞数量显著多于A组(P<0.05),D组穿出小室细胞数量则较B组、C组明显降低(P<0.05);伤口愈合实验结果显示,与A组相比,B组、C组细胞迁移率明显提高(P<0.05)、而D组细胞迁移速率则相较B组、C组明显降低(P<0.05);western blot检测MMP-9表达水平显示,B、C两组MMP-9表达较A组明显增高(P<0.05),D组MMP-9表达较B组、C组则明显降低(P<0.05);western blot检测α-SMA结果显示,B组、C组α-SMA表达水平较A组明显降低(P<0.05),D组较B组、C组表达则明显升高(P<0.05);western blot检测OPN结果显示,B组、C组OPN表达较A组明显升高、D组较B组、C组明显降低;western blot检测ERK1/2、p-ERK1/2结果显示,四组总ERK1/2水平无显著差异,而p-ERK1/2水平,B组、C组较A组明显升高(P<0.05)、D组较B组、C组明显降低(P<0.05)。第二部分:动物实验部分:血管切片HE染色显示,与sham组(A组)相比,模型组(B组)以及模型+Ad-GFP组(C组)的血管内膜增生面积和内膜/中膜比值显著增加(P<0.05),模型+Ad-sh Sema4d组(D组)的血管内膜增生面积和内膜/中膜比值则较B组、C组明显降低(P<0.05);血管切片免疫组化显示,模型组的Sema4d表达明显高于sham组,且sema4d表达分布集中在增生内膜区域;免疫荧光检测切片KI67、MMP-9、OPN表达显示,B组、C组的KI67、MMP-9、OPN荧光强度均相应高于A组,D组KI67、MMP-9、OPN荧光强度则较B组、C组均相应降低(P<0.05);免疫荧光检测切片SM-22α表达显示,B组、C组SM-22α表达水平较A组明显降低(P<0.05)、D组则较B组、C组表达明显增高(P<0.05)。结论Sema4d在PDGF-BB诱导大鼠颈动脉血管平滑肌细胞模型与增生血管内膜中的表达水平升高;下调Sema4d的表达对PDGF-BB诱导的血管平滑肌细胞的增殖和迁移以及表型转化具有抑制作用,并对血管损伤后血管内膜病理性增生具有明显抑制作用,其机制可能与Sema4d促进MAPK通路的激活有关。更多还原

【Abstract】 BackgroundCardiovascular and cerebrovascular diseases are common and frequentlyoccurring diseases that threaten people’s life and health.In recent years,with the aging trend of China’s population and the change of diet and lifestyle,the incidence of coronary heart disease,myocardial infarction and other diseases is increasing.At present,percutaneous coronary stent implantation is the main clinical treatment for coronary heart disease,however,postoperative restenosis(RS)caused by endothelial injury caused by stent implantation and proliferation and migration of vascular smooth muscle cells(VSMCs)has always been a problem for doctors and patients.With the continuous development of clinical diagnosis and treatment technology,stent implantation has experienced the replacement from bare metal stent(BMS)to drugeluting stent(DES),but the pathological mechanism of vascular restenosis is still not fully revealed,and the study of its pathogenesis and treatment is of great significance to clinical diagnosis and treatment as well as people’s health.Sema4d is a type I transmembrane protein,which is widely distributed in various tissues and organs such as nerve,skeletal muscle,endothelium and tumor,and is involved in a variety of physiological and pathological processes such as cell proliferation,migration,adhesion,immune response and cardiovascular development.Studies have shown that in heart failure,coronary heart disease and other diseases,its serum expression level is significantly increased,and its receptor,plexin B1,can activate the cascade signal to activate the proliferation-related MAPK signaling pathway.Since the proliferation,migration and phenotypic transformation of vascular smooth muscle are important pathological mechanisms of vascular restenosis,we speculated that Sema4d might be involved in the occurrence of vascular restenosis,and explored the role of Sema4d in this process through this study.ObjectiveTo explore the role of Sema4d in pathological intimal hyperplasia and related signaling pathways.MethodPart one: Cell experiment,the primary rat VSMCs were cultured by tissue block adhesion method in vitro,and the cells were identified by immunofluorescence assayα-SMA,and the concentration of platelet-derived growth factor BB(PDGF-BB).The proliferation and migration of Sema4d were induced,and the expression of Sema4d before and after induction was observed by immunofluorescence and western blot.In addition,Sema4d expression was reduced by adenovirus(AD-sh Sema4d),and cells were divided into control group,PDGF-BB stimulation group,PDGF-BB+ AD-GFP group,and PDGF-BB+ AD-sh Sema4d group.Cell proliferation was detected by CCK-8 and PCNA levels.Cell migration was detected by Transwell,wound healing assay and MMP-9 level.Phenotypic transformation was detected by expression levels of α-SMA and OPN.Western blot was used to detect ERK1/2 and P-ERK1/2 levels to explore the effect of Sema4d on MAPK signaling pathway.Part two: Animal experiment,the rat animal model of common carotid artery injury was established by balloon catheter,and the intervention was carried out by local incubation of AD-sh Sema4d adenovirus in the vascular injury area.The animals were divided into sham group,model group,model + AD-GFP group and model +AD-sh Sema4d group.The rats were sacrificed 14 days after the operation.Samples were taken from the injured blood vessels and paraffin-embedded sections or histopathological proteins were extracted.The effect of Sema4d on intimal hyperplasia of rat common carotid artery was investigated by HE staining,and the expression levels of Sema4d,KI67,MMP-9,SM-22α and OPN were investigated by tissue immunofluorescence or immunohistochemistry.ResultPart one:In cell experiment,immunofluorescence cell identification showed that the positive rate of immunofluorescence α-SMA staining of the common carotid artery VSMCs extracted by tissue block adhesion method was more than 95%.Immunofluorescence and Western blot showed that sema4 D expression level was significantly increased after PDGF-BB(ng/ m L)induction(P < 0.05).CCK-8 cell proliferation assay showed that the proliferation activity of PDGF-BB stimulation group(group B)and PDGF-BB+ AD-GFP group(group C)was significantly higher than that of control group(group A)(P < 0.05).The proliferation activity of PDGFBB+AD-sh Sema4d group(group D)was lower than that of PDGF-BB stimulation group(group B)and PDGF-BB+ AD-GFP group(group C)(P < 0.05),and there was no significant difference in proliferation level between group B and C(P > 0.05).Western blot showed that the expression level of PCNA in group B and C was higher than that in group A(P < 0.05),and the expression level of PCNA in group D was significantly lower than that in group B and C(P < 0.05),but there was no significant difference between groups B and C.Transwell assay results showed that the number of cells coming from the chamber in group B and C was significantly higher than that in group A.Compared with groups B and C,the number of cells in group D was significantly lower(P < 0.05).The results of wound healing experiment showed that compared with group A,cell mobility of group B and C was significantly increased(multiple?).The cell migration rate of group D was significantly lower than that of group B and group C(P < 0.05).Western blot showed that the expression of MMP-9in group B and C was significantly higher than that in group A(P < 0.05),and the expression of MMP-9 in group D was significantly lower than that in group B and C(P < 0.05).Western blot analysis of α-SMA showed that the expression of α-SMA in group B and C was significantly lower than that in group A(P < 0.05),and the expression of α-SMA in group D was significantly higher than that in group B and C(P < 0.05).Western blot analysis of OPN showed that the expression of OPN in group B and C was significantly higher than that in group A,and that in group D was significantly lower than that in group B and C.Western blot analysis of ERK1/2 and P-ERK1/2 showed that there was no significant difference in the total ERK1/2 level of the four groups,while the p-ERK1/2 level of group B and C was significantly higher than that of group A(P < 0.05),and group D was significantly lower than that of group B and C(P < 0.05).Part two : Animal experiments: HE staining of vascular sections showed that compared with sham group(group A),the intima hyperplasia area and intima/media ratio of model group(group B)and model + AD-GFP group(group C)were significantly increased(P < 0.05).The intima hyperplasia area and intima/media ratio of model + AD-Shsema4 D group(GROUP D)were significantly lower than those of group B and group C(P < 0.05).Immunohistochemistry of vascular sections showed that the expression of Sema4d in model group was significantly higher than that in Sham group,and the expression of Sema4d was concentrated in intima hyperplasia region.Immunofluorescence analysis of KI67,MMP-9 and OPN expression showed that the fluorescence intensity of KI67,MMP-9 and OPN in group B and group C was higher than that in group A,and the fluorescence intensity of KI67,MMP-9 and OPN in group D was lower than that in group B and group C(P < 0.05).Immunofluorescence assay showed that the expression of SM-22α in group B and C was significantly lower than that in group A(P < 0.05),and the expression of SM-22αin group D was significantly higher than that in group B and C(P < 0.05).Western blot analysis of Sema4d,ERK1/2 and P-ERK1/2 expressions of vascular tissue proteins showed that the Sema4d expression of group B and C was significantly higher than that of group A(P < 0.05),and the Sema4d expression of group D was significantly lower than that of group B and C(P < 0.05).There was no significant difference between group B and group C.ConclusionSema4d expression was increased in PDGF-BB induced rat carotid vascular smooth muscle cell model and Intimal hyperplastic vessels.Down-regulated Sema4d expression can inhibit the proliferation,migration and phenotypic transformation of PDGF-BB-induced vascular smooth muscle cells,and significantly inhibit the pathological hyperplasia of intima after vascular injury,the mechanism may be related to the promotion of MAPK pathway activation by Sema4d.更多还原