缺氧肺动脉平滑肌细胞中PKCα/c-fos、Bax/Bcl-2表达变化及其对增殖和凋亡的影响

Effects of PKCα/c-fos,Bax/Bcl-2 expression on the proliferation and apoptosis of rat pulmonary arterial smooth muscle cells in hypoxia

目的:研究缺氧大鼠肺动脉平滑肌细胞(PASMCs)PKCα/c-fos、Bax/Bcl-2表达的变化及对其增殖和凋亡的影响。方法:将原代培养的大鼠PASMCs分为常氧组(5%CO_2、21%O_2、74%N_2)和低氧组(5%CO_2,2%O_2,93%N2)24 h,48 h。通过氮蓝四唑盐(MTT)比色法测定PASMCs的增殖水平,以流式细胞仪Annexin V-FITC/PI染色法检测细胞凋亡率,并通过用RT-PCR和Western blot法检测PKCα/c-fos、Bax/Bcl-2基因和蛋白表达的变化。实验至少重复3次,每组设置至少3个复孔。结果:低氧24 h和48 h组PASMCs增殖明显,与常氧组相比差异具有统计学意义(P<0.01),而细胞凋亡率无显著变化。相对于常氧组,低氧24 h和48 h组PASMCs PKCα、c-fos表达增强,Bax表达变化不明显,而Bcl-2表达增加。结论:低氧培养的PASMCs增殖明显,凋亡无明显变化。同时PKCα、cfos表达增强,而抗凋亡基因Bcl-2表达增加。

Objective： To explore the effects of PKCα/c-fos, Bax/Bcl-2 on the proliferation and apoptosis of rat pulmonary arterial smooth musclecells（PASMCs） in hypoxia. Methods： The PASMCs of rats had been isolated and cultured, and then were cuhured under normoxia （ 5 % CO2,21% 02,74 % N2 ） and hypoxia（5 % CO2,2 % 02,93 % N2 ） condition for 24 h and 48 h, respectively. The proliferation of PASMCs was tested by methyhhiazolyhetrazolium （MTT）. The changes of PASMCs apoptosis were detected by Annexin V-FITC/PI staining combined flow cytometry. RT-PCR and Western blot analysis were performed to detect the gene and protein levels of PKCα/c-fos, Bax/Bcl-2, respectively. All experiments were repeated three times with at least triplicate samples. Results： The proliferation of PASMCs in hypexia group were stronger than that of normoxia group after 24 h and 48 h （P〈0.01）, while the apoptosis rate did not change significantly. Meanwhile, the higher levels of PKCα, c-los, Bcl-2 mRNA and proteins after hypoxia for 24 h and 48 h were detected by RT-PCR and Western blot analysis, while the expression levels of Bax had no significant difference under normoxic and hypoxic conditions. Conclusion： Elevated proliferation and expression of PKCα, c-fos, Bcl-2 was observed in rat PASMCs in hypoxia, and while the apoptosis rate had no significant change.