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目的探讨不同体外培养条件与血管平滑肌细胞(VSMCs)表型、增殖和细胞骨架蛋白表达之间的差异及生物学意义。方法体外培养大鼠主动脉血管平滑肌细胞,实验分为6组:2代对照组、2代血清饥饿组、4代对照组、4代血清饥饿组、6代对照组和6代血清饥饿组;用5-Brdu标记增殖细胞;RT-PCR检测表型基因SM22ɑmRNA的表达;免疫细胞化学检测细胞骨架蛋白SMɑ-actin、β-Tubulin和Desmin的表达。结果体外培养的VSMCs,随培养代数的增多,细胞骨架蛋白表达减少,电镜超微结构可见胞质中内质网、高尔基体等细胞器增多,有大量的分泌小泡;血清饥饿胞中线粒体增多并电子密度增高。血清培养条件下SMɑ-actin随培养代数的增加其表达减少,血清饥饿培养可使SMɑ-actin表达可逆性上调;β-Tubulin和Desmin随培养代数的增加表达减少更明显,而血清饥饿上调作用也不明显,至第6代基本不表达。结论血清培养和血清饥饿培养在不同代中对VSMCs表型转化、增殖和细胞骨架有着不同的影响,它们之间存在着内在的联系,在维持细胞形态、收缩功能和血管重构方面起了重要作用。β-Tubulin和Desmin表达消失可能具有重要生物学意义。
Objective To investigate the differences and biological significance of different phenotypes, proliferation and cytoskeletal protein expression in cultured rat vascular smooth muscle cells (VSMCs). Methods The rat aortic vascular smooth muscle cells were cultured in vitro. The experiment was divided into 6 groups: control group 2, serum starvation group 2, control group 4, serum starvation group 4, control group 6 and serum starvation group 6; The proliferating cells were labeled with 5-Brdu. The expression of phenotypic gene SM22ɑmRNA was detected by RT-PCR. The expressions of SMɑ-actin, β-Tubulin and Desmin were detected by immunocytochemistry. Results The VSMCs cultured in vitro showed decreased expression of cytoskeleton proteins with the increase of culture algebra. The ultrastructure of electron microscope showed that the number of organelles such as endoplasmic reticulum and Golgi bodies increased and there were a large number of secretory vesicles. The number of mitochondria increased in serum starvation cells Elevated electron density. The expression of SMɑ-actin decreased with the increase of culture algebra under serum culture conditions, and the expression of SMɑ-actin was up-regulated by serum-starvation culture. The expression ofβ-Tubulin and Desmin decreased with the increase of culture algebra, while the up-regulation of serum starvation Not obvious, to the sixth generation basically not expressed. Conclusions Serum culture and serum starvation culture have different effects on the phenotype transformation, proliferation and cytoskeleton of VSMCs in different generations, and they are intrinsically linked and play an important role in maintaining cell morphology, contractile function and vascular remodeling effect. The loss of β-Tubulin and Desmin expression may have important biological significance.