信号转导分子smad4基因表达抑制体外培养人血管平滑肌细胞的增殖

Inhibition of signal transduction molecule smad4 on proliferation of vascular smooth muscle cells cultured in vitro

目的:探讨细胞内转化生长因子β信号通路中信号转导分子Smad4与平滑肌细胞的增殖关系。方法:实验于2003-08/10在哈尔滨医科大学医学遗传学研究室完成。①取体质量200g左右的健康雄性SD大鼠,常规胶原酶消化法原代培养大鼠血管平滑肌细胞。用体积分数为0.2和0.05(去血清培养)胎牛血清和的Dulbecco改良的Eagle培养基,37℃,体积分数0.05CO2进行培养,2.5g/L胰酶消化传代。血管平滑肌细胞经锥虫蓝染色,存活细胞数在98%以上。形态学上出现典型的“峰和谷”样生长状态,抗平滑肌特异α-肌动蛋白免疫组织化学染色阳性。取3～8代细胞用于实验。②观察项目:转染前后细胞增殖能力和细胞凋亡能力的检测应用流式细胞分析仪和脱氧核糖核酸末端转移酶介导的缺口末端标记法凋亡检测试剂盒完成。smad4的表达及部分与细胞周期调控相关基因(p16、细胞周期调控激酶4和细胞周期因子E)和细胞凋亡基因(半胱天冬酶2和半胱天冬酶3)表达变化应用免疫印迹法和荧光免疫组化方法检测。结果:①转染前后细胞增殖能力和细胞凋亡能力流式细胞分析仪和脱氧核糖核酸末端转移酶介导的缺口末端标记法测定结果:smad4基因过表达调控人血管平滑肌细胞增殖能力下降DNA合成前期细胞明显增加[(72.33±4.35)%],正常培养细胞DNA合成前期细胞数相对较少[(45.88±1.58)%]。②smad4的表达及部分与细胞周期调控相关基因免疫印迹法和荧光免疫组化方法检测结果:转染后细胞凋亡能力增加;细胞周期抑制基因p16表达增加,细胞周期调控激酶4和细胞周期因子E表达下降,凋亡相关基因半胱天冬酶2和半胱天冬酶3表达增加。结论:smad4基因对血管平滑肌细胞增殖能力的抑制作用主要通过抑制细胞增殖和诱导细胞凋亡的发生而实现。转化生长因子β信号传导通路对血管平滑肌细胞增殖的抑制作用除了能够有效阻滞细胞周期的演进外,还能够诱导细胞凋亡。

AIM: To investigate the relationship of transforming growth factor beta(TGF-β) and signal transduction molecule smad4 with proliferation of vascular smooth muscle cells (VSMCs). METHODS: The experiment was finished in Genetics Institute of Harbin Medical University from August to October 2003. ① Healthy male SD rats with body mass about 200 g were selected. Vascular smooth muscle cells were primary cultured with routine collagenase sigestion method. The blood serum of fetus cattle (cultured without blood serum) with the volume of 0.2 and 0.05 and Eagle medium improved by Dulbecco were adopted at 37 ℃. The volume of 0.05 CO2 were used for culture, and transferred by 2.5 g/L pancreatin digestion. Vascular smooth muscle cells were stained by trypan blue, and the stock rate of the cells was over 98%. In morphology the characteristic hill-and-valley appearance were exhibited.Anti-smooth muscle unique alpha actin immunohistochemistry staining was positive. The cells of 3-8 passages were used to study. ② Observation program: The proliferation and apoptosis of cells were detected with Flow Cytometry (FCM) analysis and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) technique in pre- and post-transferation. The changes of expression of smad4 and genes related with cell cycle (p16, CDK4 and cyclin E) and cell cycle genes (caspases 2 and caspases 3) were detected with Western blot and immunohischemistry. RESULTS: ① The results of proliferation and apoptosis of cells detected with FCM and TUNEL in pre- and post-transferation: smad4 gene overexpression, the ability of proliferation of cells was decreased. Compared with normal cells, overexpression of smad4 protein in cells resulted in G1 phase arrest (G1 phase cells was (72.33±4.35)% vs (45.88±1.58)%.② The results detected by Western blot and immunohischemistry: The ability of cell apoptosis after transferation was increased, and the inhibiting genes of cell cycle regulators p16 increased, but CDK4 and cyclin E were both decreased. Furthermore, the expression of associated genes of apoptosis-caspase-2 and caspase-3 both enhanced. CONCLUSION: The inhibition of smad4 gene on VSMCs is conducted through the inhibition of cell proliferation and the induction of apoptosis. The inhibition of TGF-β signal pathway on VSMCs proliferation not only effectively prevents the evolution of cell cycle, but induces cells apoptosis.