论文部分内容阅读
目的观察 Gax 基因转染对缺氧性肺动脉平滑肌细胞(PASMCs)凋亡及其相关基因表达的影响。方法腺病毒介导 Gax 转染体外原代培养的 PASMCs。透射电镜观察细胞凋亡形态;原位细胞凋亡检测法观察 Ad-Gax 转染前后在常氧和缺氧处理2h、6h、12h、24h、48h 大鼠 PASMCs凋亡情况;免疫细胞化学法测 PASMCs Bcl-2、Bax 蛋白表达。结果透射电镜观察到 Ad-Gax 转染后PASMCs 产生凋亡现象。未转染缺氧刺激前后,均无或可见极少量的阳性细胞;Ad-Gax 转染后,如无缺氧刺激,也无或仅可见少量的阳性细胞,予缺氧刺激后,阳性细胞显著增多,尤其在转染后24～48h更明显。转染组常氧、缺氧2h、6h、12h、24h、48h 细胞凋亡百分率均显著高于未转染组(P<0.01)。Ad-Gax 转染前,与常氧时比较,缺氧刺激 PASMCs 后,Bax 蛋白表达略为升高但无统计学意义;而 Bcl-2蛋白表达显著升高,差异有统计学意义(P<0.05)。Ad-Gax 转染 PASMCs 后,缺氧刺激使 Bcl-2蛋白表达显著降低(P<0.01),Bax 蛋白表达显著增高(P<0.01)。转染组细胞凋亡率与 Bcl-2/Bax 比值呈负相关(r=-0.53,P<0.01)。结论 Ad-Gax 转染可诱导缺氧性 PASMCs 凋亡,其机制可能是通过上调 Bax 蛋白和下调 Bcl-2蛋白的表达,尤其是通过降低 Bcl-2/Bax 比值实现的。
Objective To observe the effect of Gax gene transfection on the apoptosis of hypoxic pulmonary artery smooth muscle cells (PASMCs) and its related gene expression. Methods Adenovirus-mediated Gax transfection in primary cultured PASMCs. The morphological changes of apoptotic cells were observed by transmission electron microscopy. The apoptosis of PASMCs was detected by in situ cell apoptosis assay before and after Ad-Gax transfection in normoxia and hypoxia for 2h, 6h, 12h, 24h and 48h. Immunocytochemistry PASMCs Bcl-2, Bax protein expression. Results The apoptosis of PASMCs was observed by transmission electron microscope after Ad-Gax transfection. No significant positive or negative cells were observed before or after untransfected hypoxia. After Ad-Gax transfection, no or only a few positive cells were observed after hypoxia stimulation. After hypoxia stimulation, the positive cells were significantly Increase, especially in 24 ~ 48h after transfection more obvious. The percentage of apoptotic cells in normoxia, hypoxia 2h, 6h, 12h, 24h, 48h were significantly higher than those in untransfected group (P <0.01). Before Ad-Gax transfection, compared with normoxia, the expression of Bax protein was slightly increased after hypoxia-stimulated PASMCs, but the expression of Bcl-2 protein was significantly increased, the difference was statistically significant (P <0.05 ). After Ad-Gax transfection, the expression of Bcl-2 protein was significantly decreased (P <0.01) and the expression of Bax protein was significantly increased (P <0.01) after hypoxia stimulation. The rate of apoptosis in the transfected group was negatively correlated with the ratio of Bcl-2 / Bax (r = -0.53, P <0.01). Conclusion Ad-Gax transfection can induce apoptosis of hypoxic PASMCs by up-regulating Bax protein and down-regulating the expression of Bcl-2 protein, especially by decreasing the ratio of Bcl-2 / Bax.