腺病毒介导的PTEN cDNA对子宫内膜癌细胞凋亡的影响

Adenoviral-mediated expression of PTEN cDNA induces apoptosis in endometrial carcinoma cells

目的研究外源性PTEN cDNA的表达对子宫内膜癌细胞凋亡的影响。方法用细菌内同源重组法构建含野生型抑癌基因PTEN cDNA的复制缺陷型腺病毒Ad-PTEN,蛋白印迹法鉴定转染Ad-PTEN后,子宫内膜癌细胞系RL95-2细胞中PTEN蛋白的表达。将RL95-2细胞随机分成3组: (1)空白对照组:仅用培养基而不加腺病毒;(2)Ad-CMV组:转染复制缺陷型空载体腺病毒Ad-CMV; (3)Ad-PTEN组:转染复制缺陷型腺病毒Ad-PTEN,前两组均为对照组。采用细胞膜磷脂酰丝氨酸(PS)外翻检测、活化半胱氨酸天冬氨酸蛋白酶3(caspase-3)测定及染色体DNA片段化检测这3种方法,从不同角度检测PTEN蛋白的表达对RL95-2细胞凋亡的影响。结果经Ad-PTEN介导的PTEN cDNA能够在RL95-2细胞中持续有效地表达。Ad-PTEN组转染后24、48、72、96 h,RL95-2细胞中细胞膜PS外翻细胞分别占(6．09±1．01)％、(9．98±2．17)％、(11．74±2．65)％、(27．69±8．67)％,各个时间点分别与两个对照组比较,差异均有统计学意义(P<0．05);RL95-2细胞中活化caspase-3细胞分别占(2．6±0．5)％、(18．0±4．4)％、(21．8±5．1)％、(33．7±9．9)％,各个时间点分别与两个对照组比较,差异均有统计学意义(P<0．05)。Ad-PTEN转染后48 h,RL95-2细胞中出现特异性的染色体DNA梯状条带。结论腺病毒介导的PTEN cDNA能够有效地在RL95-2细胞中表达,并诱导其凋亡,为子宫内膜癌的基因治疗提供了理论基础。

Objective To investigate whether the human endometrial carcinoma cells, RL95-2 infected with recombinant Ad-PTEN can steadily produce PTEN protein and enter apoptosis. Methods The recombinant adenovirus containing PTEN cDNA was constructed using the method of homologous recombination in bacteria. The viral titer was examined by plaque assay and the expression of PTEN protein was detected by western blot assay. The apoptosis of RL95-2 cells was evaluated as following： flipping of membrane phosphatidylserine （PS） and identification of activating caspase-3 positive cells was determined by flow cytometer （FCM）, and furthermore genomic DNA fragmentation was detected by agarose electrophoresis. Results The recombinant adenovirus encoding PTEN cDNA was successfully constructed, and viral titers of Ad-PTEN were 5 × 10^9 pfu/ml. After infected by Ad-PTEN, the expression of PTEN protein was steady in human RL95-2 cells. After infected by Ad-PTEN for 24,45,72 and 96 h,the relative cell number of membrane PS flipping were （6.09 ± 1.01 ）% , （9. 98 ± 2. 17 ）% , （ 11.74 ± 2. 65 ）% , （27.69 ± 8. 67 ）% , which significantly increased than control group（ P 〈 0. 05 ）, the relative cell number of activated caspase-3 positive were （2. 6 ± 0. 5 ） % , （ 18.0 ± 4. 4） % , （ 21.8 ± 5.1 ） % , （33.7 ± 9.9） %, respectively, which significantly increased than control group（ P 〈 0. 05 ） , and genomic DNA fragmentation was verified also. Conclusions The recombinant Ad-PTEN vector is constructed successfully and the expression of specific PTEN is steady in RL95-2 cell line. The expression of PTEN induces RL95-2 cells to apoptosis. PTEN gene may be a novel therapeutic target in endometrial carcinoma.