大鼠血管平滑肌细胞体外培养的表型转换及其鉴定  

The Phenotype Transformation of Rat's Vascular Smooth Muscle Cell During in Vitro Culture and Its Characterization

作　　者：王生兰[1] 苏娟[1] 徐一洲[1] 谢静[1] 王树人[1] 

机构地区：[1]四川大学华西基础医学与法医学院病理生理学教研室

出　　处：《中国动脉硬化杂志》2008年第4期268-272,共5页Chinese Journal of Arteriosclerosis

基　　金：高等学校博士学科点专项科研基金(20050610050)

摘　　要：目的探讨体外传代培养对血管平滑肌细胞表型转换的影响,确认体外培养的血管平滑肌细胞不同表型的最佳指标。方法选择以未经培养传代的血管平滑肌细胞和体外培养的第4代血管平滑肌细胞作对比研究,采用免疫组织化学法检测血管平滑肌细胞α肌动蛋白,逆转录聚合酶链反应检测血管平滑肌细胞肌动蛋白22α、基质γ-羧基谷氨酸蛋白和骨桥蛋白各目的基因mRNA相对表达水平,透射电镜观察血管平滑肌细胞的形态学特征,确定其表型转换特征及各表型指标的区分效能。结果电镜观察显示,未经培养传代的血管平滑肌细胞胞质中肌丝分布丰富、均匀,并可见到收缩表型血管平滑肌细胞的典型结构—密斑,内质网、高尔基复合体等细胞器相对较少;而体外培养至第4代时血管平滑肌细胞胞质中肌丝明显减少,内质网、高尔基复合体明显增多。原代血管平滑肌细胞α肌动蛋白免疫组织化学染色强且广泛,而体外培养至第4代时α肌动蛋白染色明显变浅、稀疏。第4代血管平滑肌细胞的骨桥蛋白mRNA表达量较原代血管平滑肌细胞显著增高(P<0.01),肌动蛋白22α mRNA在血管平滑肌细胞中的表达量较原始血管平滑肌细胞降低,但差异不大,基质γ-羧基谷氨酸蛋白mRNA表达量较未经培养传代的血管平滑肌细胞升高(P<0.05)。结论血管平滑肌细胞体外培养至第4代,即可从典型的收缩表型转化为合成表型,提示血管平滑肌细胞是一种很容易去分化的细胞。电镜形态观察α肌动蛋白和骨桥蛋白在两型血管平滑肌细胞中的差异,可作为血管平滑肌细胞表型区分的有效标志。而肌动蛋白22α和基质γ-羧基谷氨酸蛋白的表达在两型血管平滑肌细胞中虽有一定的差异,但重叠较大。Aim To investigate the effect of culture of vascular smooth muscle cell （VSMC}in vitro on its phenotype transformation and to survey the best parameters for characterizing the phenotype. Methods The primary VSMC and the fourth passage of VSMC in vitro culture were compared by their morphological characteristics under electron microscope, and the α- SM actin expression measured by immunohistochemical staining, the osteopontin {OPN}, SM22a, matrix Gla protein {MGP} mRNA transcription levels measured by RT-PCR were all comparatively studied. Results The myofilament was abundant, the dense patch--a typical structure of contractile phenotype of VSMC well-distributed, while the endoplasmic reticulum, Golgi＇ s complex were less-distributed in primary VSMC. The VSMC in fourth passage, however, showed scattered distribution of myofil- ament in cytosol, but much more endoplasmic reticulum, Golgi＇ s complex, and mitochondria under electron microscope observa- tion. The immunohistochemical staining showed much strongem-SM actin expression in primary VSMC compared with the fourth passnge （ P 〈 0.05）. OPNm RNA level increased very significantly （P〈0.01） in fourth passage of VSMC compared with primary ones. The transcription level of SM22α mRNA in fourth passage of VSMC decreased compared with primary VSMC, but the difference was not significant （ P 〉 0.05 ）. MGP mRNA transcription levels increased in fourth passage of VSMC （ P 〈 0.05 ）. Conclusions Four passages of VSMC in vitro can transform the typical contractile phenotype into the typical synthetic phe- notype, which indicated that the VSMC had the trait of easy-dedifferentiation, this may suggest some implication in VSMC re- search. Electron microscope observation, αSM actin, OPN showed significant difference between contractile phenotype and syn- thetic phenotype, which may represent a good marker for distinguishing the two different phenotypes. The SM22α and the matrix Gla had some difference, but overlaps were also significant betwee

关 键 词：病理学与病理生理学 血管平滑肌细胞 表型转换 体外培养 Α肌动蛋白 骨桥蛋白 

分 类 号：R363[医药卫生—病理学]