不同方法对血管平滑肌细胞表型特性影响的实验研究  

The research of different methods on the phenotype characteristics of vascular smooth muscle cells

作　　者：尚松安[1] 马占龙[1] 陈相汛[1] 安艳丽[1] 丁洁[1] 滕皋军[1] 

机构地区：[1]江苏省分子影像与功能影像重点实验室,东南大学附属中大医院放射科,南京市210009

出　　处：《中国心血管病研究》2013年第10期794-797,I0001,I0002,共6页Chinese Journal of Cardiovascular Research

基　　金：国家自然科学基金面上项目（项目编号：81071193）

摘　　要：目的 通过两种分离方法体外获取血管平滑肌细胞（VSMCs）,比较细胞表型差异及生物学特性,为血管性疾病研究提供精确实验基础.方法 分别采用组织块法及酶消化法获取SD大鼠胸主动脉VSMCs,分为组织块组与酶消化组.倒置显微镜、结晶紫染色观察形态学改变并定量分析；MTT比色法及TransweU细胞迁移实验分别检测分析细胞增殖与迁移活性；流式细胞周期测定细胞周期分布；细胞免疫荧光染色鉴定VSMCs并测定分析收缩型标记蛋白α-平滑肌肌动蛋白（SMA）以及合成型标记蛋白平滑肌胚胎型肌球蛋白重链（SMemb）、原肌球蛋白-4（TPM-4）表达量的变化.结果 两组细胞SMA细胞免疫荧光染色阳性率≥95％,细胞呈现谷峰状结构生长.组织块组与酶消化组两组细胞长径径值分别为（95.10±16.23）μm和（114.67±15.92）μm.与酶消化组相比,组织块组细胞增殖及迁移活性分别增加23.04％和1.54倍（P＜0.05）,S＋G2期所占比例增加49.68％（P＜0.05）,SMA蛋白表达量下降（P＜0.05）,SMemb及TPM-4蛋白表达均增加（P＜0.05）.结论 组织块法获取细胞多以合成表型为主,酶消化法则主要呈收缩表型.伴随细胞形态学、增殖及迁移活性、表型特异性蛋白表达等不同改变,两种细胞获取方法可为不同目的研究提供精确的体外模型.Objective To obtain the vascular smooth muscle cells （VSMCs） with two isolation meth- ods and compare the cells biological characteristics and phenotypic differences, providing a precise experi- mental basis for the research of vascular disease. Methods The primary and subculture of VSMCs from SD rats were done by enzymatic dispersion and tissue explants, respectively. The VSMCs were observed using inverted microscope and crystal violet staining for morphological observation and quantitative analysis. The third passage cells viability and migration were detected by MTY assay and transwell assay, respectively. Im- munofluorescence staining for contractile marker a-smooth muscle actin （SMA） and synthetic markers non- muscle MHC isoform-B （SMemb）, Tropomyosin-4 （TPM-4） were performed for VSMCs identification and quantitative analysis. Results The post-confluent cells exhibited the ＂hill and valley＂ growth pattern of the two groups of cultured VSMCs. More than 95% of cells were both positively stained by immunofluorescence staining for SMA. Compared with the enzymatic dispersion group, the cells of the tissue explants group had a significant morphological alteration, with the polygonal shape （P〈O.05）, the viability and mobility of VSMCs increased 23.04% and 1.54 times, respectively （P〈0.05）. The expression of both SMemb and TPM-4 proteins increased while of SMA protein decreased, significantly （P〈O.05）. Conclusion Tissue explants for VSMCs is given priority to synthetic phenotype, while the enzyme digestion is mainly contractive phenotype, along with the cell morphology, viability and migration, and phenotypic specificity proteins expression change differently, the two methods can provide accurate models in vitro for different research purpose.

关 键 词：血管平滑肌细胞 表型转化 细胞培养 

分 类 号：Q95-93[生物学—动物学] R543[医药卫生—心血管疾病]