论文部分内容阅读
目的探讨肿瘤抑制基因PTEN对人慢性粒细胞白血病K562细胞系的增殖、凋亡及对凋亡相关基因Bcl-2及Caspase-3/7及-9的影响。方法将携带有野生型PTEN及绿色荧光蛋白(GFP)的腺病毒(Ad-PTEN-GFP)及空载体腺病毒(Ad-GFP)转染人慢性粒细胞白血病K562细胞系。通过MTT检测细胞生长曲线,流式细胞仪分析转染效率、细胞凋亡率和细胞增殖指数,同时观察光镜、电镜下细胞形态,DNA ladder、荧光染色等方法检测细胞凋亡,荧光定量PCR(FQ-PCR)检测PTEN及Bcl-2 mRNA水平变化,Western Blotting检测PTEN及Bcl-2蛋白变化,应用试剂盒检测Caspase-3/7及-9蛋白活性。结果以感染复数为200的Ad-PTEN-GFP转染K562细胞后,最大生长抑制率为37.1%,早期和中晚期凋亡率均高于转染Ad-GFP组和未转染组(P<0.05),转染PTEN基因3d后Bcl-2 mRNA及蛋白表达水平分别是Ad-GFP组的0.27倍和0.58倍,Caspase-3/7及-9蛋白活性在转染PTEN基因后3d内呈时间依赖性升高。结论过表达PTEN基因可能通过抑制抗凋亡分子Bcl-2表达,增强Caspase-3/7及-9活性从而抑制K562细胞系增殖,促进细胞凋亡。
Objective To investigate the effects of tumor suppressor gene PTEN on proliferation, apoptosis, apoptosis related genes Bcl-2, Caspase-3/7 and -9 in human chronic myeloid leukemia K562 cell line. Methods Human K562 cell line was transfected with adenovirus (Ad-PTEN-GFP) and empty vector adenovirus (Ad-GFP) carrying wild-type PTEN and green fluorescent protein (GFP) The cell growth curve was detected by MTT, and the transfection efficiency, apoptosis rate and cell proliferation index were analyzed by flow cytometry. Meanwhile, the cell morphology was observed under light microscope and electron microscope, DNA ladder, fluorescence staining and other methods to detect apoptosis. (FQ-PCR) were used to detect the changes of PTEN and Bcl-2 mRNA levels. The protein expressions of PTEN and Bcl-2 were detected by Western Blotting. The activity of Caspase-3/7 and -9 protein was detected by kit. Results The maximum growth inhibition rate of K562 cells transfected with Ad-PTEN-GFP with a multiplicity of infection of 200 was 37.1%, and the apoptosis rate in early and middle stages was higher than that in Ad-PTEN-transfected and untransfected groups (P < 0.05). The expression of Bcl-2 mRNA and protein were 0.27 and 0.58 folds of that of Ad-GFP group after transfection for 3 days, respectively. The activity of Caspase-3/7 and -9 protein in PTEN gene transfered after 3 days Dependence increased. Conclusion Overexpression of PTEN gene may inhibit the proliferation of K562 cell line and promote cell apoptosis by inhibiting the expression of anti-apoptotic molecule Bcl-2, enhancing the activity of Caspase-3/7 and -9.