摘要
目的经皮冠状动脉介入治疗冠心病有较好的近期疗效,但患者长期预后却受血管再狭窄的制约,血管平滑肌细胞(VSMC)的表型转化是导致血管狭窄的主要原因。文章通过构建敲低Tudor-SN稳定株,检测Tudor-SN基因对大鼠血管平滑肌细胞A7R5增殖、迁移、凋亡的影响,为研究血管平滑肌细胞表型转化和血管再狭窄发病机制提供细胞模型。方法通过制备敲低Tudor-SN基因的重组慢病毒,感染A7R5细胞,加入嘌呤霉素药物筛选出低表达Tudor-SN基因的稳定株;使用Western blot检测Tudor-SN敲低效果;在表型实验中,将A7R5细胞分为pLKO-Vector对照组和sh-Tudor-SN实验组,利用CCK-8实验、免疫荧光实验、细胞划痕实验和流式细胞术分别检测对照组和实验组A7R5细胞的增殖、迁移和凋亡的变化;使用PDGF诱导pLKO-Vector对照组和sh-Tudor-SN实验组细胞表型转化,检测Tudor-SN蛋白表达。结果成功构建了敲低Tudor-SN的shRNAs的重组慢病毒稳定株;表型实验结果显示,敲低Tudor-SN后,实验组从第3天起细胞增殖能力明显低于对照组,实验组迁移面积(1.6、1.7μm^(2))与对照组(2.7μm^(2))相比明显下降(P<0.05),细胞凋亡能力增加(P<0.05);给予PDGF刺激,Tudor-SN蛋白表达增加(P<0.05);与对照组相比,实验组Tudor-SN蛋白表达有所增加(P<0.05)。结论敲低Tudor-SN血管平滑肌细胞增殖及迁移能力降低,凋亡水平增加,并且敲低Tudor-SN降低了PDGF诱导的VSMC表型转化,表明Tudor-SN蛋白参与并促进了VSMC表型转化。
Objective Percutaneous coronary intervention(PCI)is the main clinical treatment for coronary heart disease,though this treatment have improved tremendously over the past decades,in-stent restenosis(ISR)still occurred in a proportion of patients and became the major limitation of PCI.The proliferation vascular smooth muscle cells(VSMCs)is the main cause of ISR.This work aimed to clarify the effect of knockdown Tudor-SN on proliferation,migration and apoptosis of rat VSMCs,and to provide a cell model for further study of VSMCs phenotypic switching and vascular restenosis.Methods A7R5 cells were infected with the lentiviruses and screened by adding puromycin drug to construct stable knock-down Tudor-SN gene cell lines.Western blot analysis were used to determine Tudor-SN expression level.In phenotypic experiments,A7R5 cells were divided into pLKO-Vector control group and sh-Tudor-SN experimental group.CCK-8 and immunofluorescence assays were applied to analyze the effect of Tudor-SN on the proliferation.Wound healing assay and flow cytometry were used to detect the effect of Tudor-SN for migration and apoptosis of A7R5 cells.PDGF was used to induce phenotypic switching of cells in pLKO-Vector control group and sh-Tudor-SN experimental group,and the expression of Tudor-SN protein was detected.Results Recombinant lentivirus stable strains with Tudor-SN knockdown shRNAs were successfully constructed.The results of the phenotype experiment showed that after knocking down Tudor-SN,the cell proliferation ability of the experimental group was significantly lower than that of the control group from the third day.Compared with the control group(2.7μm^(2)),the migration area of the experimental group(1.6μm^(2),1.7μm^(2))was significantly decreased,and the apoptosis capacity was increased(P<0.05).Given PDGF stimulation,the expression of Tudor-SN was increased.Compared with the control group,the expression of Tudor-SN was increased in the knockdown group after stimulation with PDGF.Conclusion Knocking down Tudor-SN could inh
作者
刘明霞
马锦征
苏超
杨洁
魏民新
LIU Ming-xia;MA Jin-zheng;SU Chao;YANG Jie;WEI Min-xin(Department of Biochemistry and Molecular Biology,School of Basic Medical Sciences,Tianjin Medical University,Tianjin 300070,China;Division of Cardiac Surgery,Cardiovascular Medical Center,The University of Hong Kong-Shenzhen Hospital,Shenzhen 518053,Guangdong,China)
出处
《医学研究生学报》
CAS
北大核心
2022年第7期687-693,共7页
Journal of Medical Postgraduates
基金
国家自然科学基金(82170435)
深圳市科技研发基金基础研究学科部局项目(JCYJ20180508152222104)。
