携带野生型PTEN基因的重组腺病毒治疗子宫内膜癌的体外研究

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PTEN mRNA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.

Inhibitory effects of recombinant adnovirus carrying wild type PTEN gene on endometrial carcinoma cells: an in vitro study

Objective:To observe the expression of Ad-PTEN in human endometrial carcinoma cell line RL95-2 after in vitro infection and investigate the mechanism by which Ad-PTEN inhibits tumor cell proliferation and induces apoptosis. Methods: Ad-PTEN was constructed through a bacterial homologous recombinant system; the expression of Ad-PTEN in RL95-2 cells was determined by RT-PCR, Western blotting and immunohistochemical staining. The efficiency of adenovirus mediated gene transfer of Ad-PTEN was determined by X-gal staining. The inhibitive effect of Ad-PTEN on RL95-2 cells proliferation was determined by cell growth analysis and MTT assay; the morphologic and ultrastructural changes of RL95-2 cells transfected with Ad-PTEN were observed by the light and electron microscopy; and Flow cytometry was used to study the cell cycle and apoptosis. Results: The expression of Ad-PTEN mRNA and protein in RL95-2 cells was confirmed by RT-PCR, Western blot and cell immunohistochemical staining. The efficiency of adenovirus mediated Ad-PTEN gene transfer was 100% when the multiplicities of infection (MOI) was 50. Exogenous PTEN gene significantly suppressed the growth of RL95-2 cells and iduced the apoptosis. Ad-PTEN could also induce cell cycle arrest (G_(0)/G_(1) ) and activated caspase-3 . Conclusion: The constructed Ad-PTEN transfection system is highly efficient in introducing wild type PTEN gene into human endometrial carcinoma RL95-2 cells. Ad-PTEN can strongly inhibit cell proliferation and induce apoptosis in RL95-2 cells, which may be associated with cell cycle arrest (G0/G_(1)) and the activation of caspase-3.