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目的:探讨PTEN对体外培养的人胃黏膜上皮细胞系GES-1增殖凋亡的影响及其磷酸化通路研究。方法:体外培养GES-1细胞,构建针对PTEN基因的shRNA慢病毒载体,感染GES-1细胞系,干扰PTEN的表达,实验分为空白对照组、慢病毒阴性对照组和干扰PTEN实验组;荧光显微镜、实时荧光定量PCR和免疫印迹法检测其感染效率;CCK8测定GES-1细胞的增殖,流式细胞术测定细胞的周期和凋亡;实时荧光定量PCR检测GES-1的PTEN、Akt、ERK的mRNA表达水平;Western blot检测GES-1的Akt和磷酸化Akt(p-Akt),ERK1/2及磷酸化ERK1/2(p-ERK1/2)表达。结果:干扰PTEN的慢病毒成功感染GES-1,降低了PTEN基因的转录和翻译水平。与空白对照组相比,PTEN表达降低后使G0/G1期的细胞比率减低,而G2/M和S期细胞比率增高,同时凋亡细胞减少,促进GES-1的增殖而抑制其凋亡。p-Akt和p-ERK1/2表达显著上调(P<0.05),而对GES-1的Akt和ERK的mRNA及总蛋白表达没有影响(P>0.05)。结论:抑癌基因PTEN可以通过促进Akt和ERK的磷酸化,调节GES-1细胞下游的相关凋亡和信号通路,进而调节GES-1的增殖和凋亡。
AIM: To investigate the effects of PTEN on proliferation and apoptosis of human gastric epithelial cell line GES-1 cultured in vitro and its phosphorylation pathway. METHODS: GES-1 cells were cultured in vitro and shRNA lentiviral vector targeting PTEN gene was constructed and infected with GES-1 cell line to interfere PTEN expression. The experiment was divided into blank control group, lentivirus negative control group and PTEN experimental group. Fluorescence The infection efficiency of GES-1 cells was detected by microscope, real-time fluorescence quantitative PCR and Western blotting. The proliferation and proliferation of GES-1 cells were detected by CCK8. Cell cycle and apoptosis were detected by flow cytometry. The expression of PTEN, Akt and ERK The expressions of Akt, p-Akt, ERK1 / 2 and p-ERK1 / 2 in GES-1 were detected by Western blot. Results: The lentivirus that interfered with PTEN successfully infected GES-1, which reduced the transcription and translation of PTEN gene. Compared with the blank control group, the decrease of PTEN expression decreased the cell ratio of G0 / G1 phase, while the ratio of G2 / M and S phase cells increased, while the number of apoptotic cells decreased, and promoted the proliferation of GES-1 and inhibited the apoptosis. The expressions of p-Akt and p-ERK1 / 2 were significantly upregulated (P <0.05), but had no effect on the mRNA and protein expression of Akt and ERK in GES-1 (P> 0.05). Conclusion: PTEN can regulate the phosphorylation of Akt and ERK, regulate the apoptosis and signal pathway downstream of GES-1 cells, and then regulate the proliferation and apoptosis of GES-1.