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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 investigate the relationship between loss of differentiation and phenotypic changes and proliferation of vascular smooth muscle cells (SMCs) in vitro. Methods The dynamic changes of myofibrillar density and contractile protein expression in the aortic SMC of rabbit aorta were detected by electron microscopy and Western blotting (Westerblot). The relationship between these changes and cell proliferation status was analyzed. Results According to the cell growth curve analysis, the proliferation of primary culture SMC can be divided into three stages, namely, the incubation period (1st ~ 6d), logarithmic proliferation period (6 ~ 12d) and quiescent period 12d later). Electron microscopy showed that the mean bulk density of myofilaments in the cytoplasm decreased from (32 14 ± 1 44)% before incubation to (18 36 ± 2 0 1)% (P 0 05) at the end of incubation period Increased to the level of pre-culture [(32 5 0 ± 1 2 1)%] in the third day after the cells entered the quiescent stage of proliferation, compared with that before the culture (P 0 05). Western blot analysis showed that the smooth muscle-specific contractile proteins such as α SM actin and SM myosin heavy chain decreased gradually with the culture time during culturing. The SM myosin heavy chain subtype 2, which is the marker of vascular SMC maturation, Disappeared on the 9th day; while the non-myocyte contractile protein, β NM actin gradually increased with the culture time. Changes in the above protein content does not fluctuate with the proliferation of cells and the disappearance of myofilaments. Conclusion The phenotypic changes and dedifferentiation of cultured vascular SMC are two relatively independent processes. The former is closely related to the proliferative state of SMC, which may be the reversible recombination of myofilament system to adapt to cell division;