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目的:观察携带野生型PTEN基因的重组腺病毒(Ad-PTEN)体外转染子宫内膜癌细胞后的表达,并探讨其对肿瘤细胞产生抑制增殖、诱导凋亡的作用及机制。方法:细菌内同源重组方法构建Ad-PTEN,体外转染PTEN基因突变的子宫内膜腺癌RL95-2细胞中,X-gal染色检测转染效率。应用RT-PCR、Western印迹、细胞免疫组化检测Ad-PTEN在RL95-2细胞中的转录及表达;应用细胞计数、MTT实验,观察Ad-PTEN对RL95-2细胞生长的影响;应用光镜、透射电镜观察外源性PTEN基因表达对RL95-2细胞形态学及超微结构的影响;应用流式细胞分析仪(FCM)检测Ad-PTEN对RL95-2细胞周期分布的影响、凋亡诱导作用及半胱氨酸天冬氨酸特异蛋白酶casapase-3的激活。结果:外源性野生型PTEN基因经腺病毒介导成功转入RL95-2细胞,3种方法均检测出有PTENmRNA及PTEN蛋白的表达,当感染复数(MOI)为50时,体外转染效率达到100%。Ad-PTEN显著抑制RL95-2细胞生长并诱导其凋亡。此外,Ad-PTEN还能诱导RL95-2细胞周期G0/G1期阻滞以及caspase-3的激活。结论:重组腺病毒Ad-PTEN是高效的基因转移系统,能将PTEN目的基因转移到丧失该基因功能的子宫内膜癌RL95-2细胞中,对RL95-2细胞产生强有力的生长抑制及凋亡诱导作用,其机制可能包括细胞周期G0/G1阻滞及caspase-3蛋白酶的激活。
OBJECTIVE: To observe the expression of wild-type PTEN gene-transfected adenovirus (Ad-PTEN) after transfection into endometrial carcinoma cells in vitro and to explore its mechanism of inhibiting proliferation, inducing apoptosis of tumor cells. Methods: Ad-PTEN was constructed by homologous recombination in bacteria and transfected into endometrial adenocarcinoma RL95-2 cells with PTEN gene transfection in vitro. The transfection efficiency was detected by X-gal staining. The transcription and expression of Ad-PTEN in RL95-2 cells were detected by RT-PCR, Western blotting and immunohistochemistry. The cell growth and proliferation of RL95-2 cells were observed by cell counting and MTT assay. , The effect of exogenous PTEN gene expression on the morphology and ultrastructure of RL95-2 cells was observed by transmission electron microscopy. The effect of Ad-PTEN on the cycle distribution of RL95-2 cells and the induction of apoptosis were detected by flow cytometry (FCM) Activation of caspase-3 and cysteine aspartate specific protease. Results: The exogenous wild-type PTEN gene was successfully transfected into RL95-2 cells by adenovirus. The expression of PTEN mRNA and PTEN protein were detected by three methods. When the MOI was 50, the in vitro transfection efficiency Reached 100%. Ad-PTEN significantly inhibited RL95-2 cell growth and induced its apoptosis. In addition, Ad-PTEN also induced G0 / G1 phase arrest and caspase-3 activation in RL95-2 cell cycle. CONCLUSION: Recombinant adenovirus Ad-PTEN is an efficient gene transfer system that can transfer PTEN gene to RL95-2 cells with loss of function of the gene and exert strong growth inhibition on RL95-2 cells The mechanism of apoptosis may include cell cycle G0 / G1 arrest and caspase-3 protease activation.