摘要
目的 建立血管平滑肌细胞 (VSMC)的收缩及合成表型 ,探讨蛋白激酶Cα(PKCα)亚型对其转化的影响。方法 用酶消化法及传代培养分别获 2种VSMC表型细胞 ,细胞特性通过免疫细胞化学及透射电镜鉴定。用竞争性逆转录聚合酶链反应 (竞争RT PCR) ,观察 2种表型细胞的PKCα表达。结果 酶消化法分离的VSMC较传代培养的VSMC显示缬蛋白(desmin)表达呈强阳性。2种表型细胞经超微结构观察分别显示典型的收缩表型及合成表型变化。PKCα表达在酶消化法分离的VSMC中明显高于传代培养的VSMC(t=3 .5 68;P <0 .0 5 )。结论 PKCα亚型在收缩表型的表达量明显高于合成表型 ,提示该亚型对维持VSMC的收缩表型起了一定作用。研究中选用竞争RT PCR方法克服了以往使用 β actin的局限性。
Objective The purpose of this study was to investigate the expression of protein kinase Cα(PKCα) in two phenotypic states of vascular smooth muscle cell (VSMC) by establishing contractile and synthetic phenotype.Methods VSMC enzymatically isolated from rat aorta were of contractile phenotype while the passaged cells were of synthetic phenotype. The cell phenotypes were identified by transmission electron microscopy and immunocytochemical staining. The gene expressions of PKCα were determined by competitive reverse transcriptase PCR (RT PCR).Results The freshly dispersed VSMC were positively stained by desmin antibodies compared with passaged VSMC. The two type of cells showed typical morphology of contractile and synthetic phenotypes, respectively. The expression of PKCα in freshly dispersed VSMC was significantly higher than that of VSMC of synthetic phenotype ( t =3.568; P <0.05). Conclusion The present data demonstrate an association between PKCα expression and phenotypic transition. It is proposed that PKCα plays a distinct role in sustaining contractile phenotype of VSMC. The competitive RT PCR used in this study overcomes the limitations of β actin method.
出处
《中华心血管病杂志》
CAS
CSCD
北大核心
1999年第6期453-455,共3页
Chinese Journal of Cardiology
基金
国家自然科学基金! ( 3 96 70 3 5 6 )
上海市科委资助!( 96 0 15 )
中法先进研究计划!(PRA97 0 5 )
