培养血管平滑肌细胞收缩蛋白表达与表现型转变及细胞增殖的关系

CONTRACTILE PROTEIN EXPRESSION,PHENOTYPIC MODULATION AND PROLIFERATION OF VASCULAR SMOOTH MUSCLE CELLS IN PRIMARY CULTURE

目的 探讨体外血管平滑肌细胞 (SMC)的失分化与其表现型转变及增殖的关系。 方法 采用电镜形态定量及蛋白印迹 (Westerblot)方法检测家兔主动脉SMC在原代培养过程中肌丝体密度及收缩蛋白表达的动态变化 ,并分析这些变化与细胞增殖状态的关系。 结果 根据对细胞生长曲线的分析 ,原代培养SMC的增殖可分为 3个阶段 ,即潜伏期 (第 1～ 6d)、对数增殖期 (第 6～ 12d之间 )和增殖后静止期 (第 12d以后 )。电镜定量显示 ,胞质中肌丝的体积密度均值由培养前的 (32 14± 1 4 4 ) %下降至潜伏期末的 (18 36± 2 0 1) % (P <0 0 5 ) ;然后逐渐增高 ,到细胞进入增殖后静止期第 3d恢复至培养前水平 [(32 5 0± 1 2 1) % ;与培养前相比 ,P >0 0 5 ]。Westernblot分析显示 ,培养过程中平滑肌特异型收缩蛋白 ,如α SM肌动蛋白、SM肌球蛋白重链随培养时间逐渐降低 ,其中作为血管SMC成熟标志的SM肌球蛋白重链亚型 2在培养第 9d后消失 ;而非肌细胞型收缩蛋白 ,β NM肌动蛋白则随培养时间逐渐增高。上述蛋白含量的变化不随细胞增殖状态及肌丝的消涨而波动。 结论 体外培养血管SMC的表现型转变和失分化是两个相对独立的过程。前者与SMC的增殖状态密切相关 ,可能是肌丝系统为适应细胞分裂而发生的可逆性重组 ;而?

Objective To determine the relationships between dedifferentiation,phenotypic modulation and proliferation of cultured vascular smooth muscle cells (SMC). Methods Dynamic changes in cytoplasmic volume fraction of myofilaments and expressions of contractile proteins in primary cultured rabbit aortic SMC were examined with morphometric electron microscopy and Western blot analysis.The data were correlated with the growth phases of the cell. Results The growth of SMC could be divided into three phases according to the growth curve:the initial quiescent phase (day 1 to day 6),the logarithmic replication phase(between day 6 and day 12)and the post-proliferation quiescent phase(after day 12).The average cytoplasmic volume fraction of myofilaments decreased from(32.13±1.44)%at day 0 to(18.36±2.01)%(P<0.05)at the end of the initial quiescent phase,and then gradually backed to the original level three days after SMCs entering the post-proliferation quiescent phase[(32.50±1.21)%;P>0.05,compared with the value at day 0].In the course of the culture,the contractile proteins of smooth muscle type,such as α-SM actin,SM-MHC were gradually decreased,with SM-2,a SM-MHC isoform that is a marker for mature SMC,quickly disappeared at day 9.In contrast,the contractile protein of non-muscle type,β-NM actin increased with culture time.The changes in contents of these structural proteins were independent on cell growth phases and fluctuations of myofilaments.Conclusion The phenotypic modulation and the dedifferentiation of SMCs in primary culture are two separate events.The former is a revertible change representing the disorganization and reorganization of SMC myofilaments during the cell mitosis,and the latter is an irreversible slow-going course independent of SMC proliferation.