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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 study the effect of exogenous PTEN cDNA expression on the apoptosis of endometrial carcinoma cells. Methods The replication-deficient adenovirus Ad-PTEN containing PTEN cDNA was constructed by homologous recombination in bacteria. The expression of PTEN in transfected Ad-PTEN cells was detected by Western blotting. Protein expression. RL95-2 cells were randomly divided into 3 groups: (1) blank control group: medium only without adenovirus; (2) Ad-CMV group: Ad-CMV transfected with replication-defective empty vector adenovirus ) Ad-PTEN group: Ad-PTEN transfected with replication-deficient adenovirus, the first two groups were control group. The three methods of detection of cell membrane phosphatidylserine (PS) valgus, activation of caspase-3 and chromosomal DNA fragmentation were used to detect the expression of PTEN protein on RL95 -2 cell apoptosis. Results The PTEN cDNA mediated by Ad-PTEN was able to express continuously and effectively in RL95-2 cells. The number of PS everted cells in RL95-2 cells were (6.09 ± 1.01)% and (9.98 ± 2.17)% respectively at 24, 48, 72 and 96 h after transfection in Ad-PTEN group, (11.74 ± 2.65)% and (27.69 ± 8.67)%, respectively. There were significant differences between the two control groups at each time point (P <0.05) The percentage of activated caspase-3 cells in the cells were (2.6 ± 0.5)%, (18.0 ± 4.4)%, (21.8 ± 5.1)%, (33.7 ± 9.9) )%, Respectively, compared with the two control groups at each time point, the differences were statistically significant (P <0.05). At 48 h after Ad-PTEN transfection, a specific chromosomal DNA ladder appeared in RL95-2 cells. Conclusions Adenovirus-mediated PTEN cDNA can effectively express in RL95-2 cells and induce apoptosis, providing a theoretical basis for gene therapy of endometrial cancer.