稳定转染PTEN基因后对卵巢上皮性癌细胞磷酸酰肌醇3激酶／蛋白激酶B信号传导通路的影响

Effects of PTEN over-expression on phosphatidyl inositol 3-kinase/protein kinase B signal pathway in ovarian epithelial cancer cells

目的 研究外源性PTEN基因稳定转染卵巢上皮性癌（卵巢癌）细胞后,对卵巢癌细胞磷酸酰肌醇3激酶（PI3K）/蛋白激酶B（Akt）信号传导通路的影响.方法 构建表达野生型PTEN基因的重组载体pcDNA3.1A-PTEN质粒,并转染卵巢癌细胞株HO-8910细胞（重组载体组）,以转染空载体pcDNA3.1A质粒（空载体组）作为阴性对照,以未转染质粒（空白组）作为空白对照.采用逆转录（RT）PCR技术检测3组细胞中PTEN、Akt1、Akt2、PI3K mRNA的表达水平,蛋白印迹法检测3组细胞中PTEN蛋白的表达强度,四甲基偶氮唑蓝（MTT）比色法检测3组细胞的生长情况[以吸光度（A）值表示,A值越大表示生长速度越快].结果 表达野生型PTEN基因的重组载体pcDNA3.1A-PTEN质粒,经测序证实构建成功.重组载体组细胞中PTEN mRNA的表达水平为（17372±23）copy/ml,高于空载体、空白组[分别为（39±1）、（78±4）copy/ml],差异均有统计学意义（P＜0.05）;重组载体组细胞中PTEN蛋白的表达强度也明显高于空载体、空白组.重组载体组细胞中Akt1、Akt2、PI3KmRNA的表达水平分别为（28±2）、（7±1）、（61±2）copy/ml,低于空载体[分别为（115±5）、（18±2）、（84±2）copy/ml]、空白组[分别为（77±4）、（17±2）、（1349±7）copy/m1],差异均有统计学意义（P＜0.05）.培养第5天,重组载体组细胞生长速度（A值）为90 158±47,低于空载体、空白组（分别为148 251±65、250 115±62）,差异均有统计学意义（P＜0.05）.结论 转染表达野生型PTEN基因的重组载体能有效提高卵巢癌HO-8910细胞中PTEN mRNA和蛋白的表达,抑制细胞生长,并通过显著降低Akt1、Akt2、PI3K mRNA的表达水平,明显抑制PI3K/Akt信号传导通路.

Objective To evaluate the effect of exogenous wild PTEN gene stable transfected into human ovarian cancer cell line HO-8910 on phosphatidyl inositol 3-kinase（ PI3K）/protein kinase B （Akt）siganal pathway and cells proliferation. Methods Wild-type PTEN recombinant eukaryotic expression plasmid was constructed and then was transfected into HO-8910 cells by lipofectamine 2000. The expression of PTEN, Akt1, Akt2, PI3K mRNA and protein of PTEN were tested by reverse transcription（ RT）-PCR and Western blot. The proliferation of HO-8910 after wild PTEN gene transfected was measured by methyl thiazolyl tetrazolium（MTT）. Results Wild-type PTEN gene was successfully transfected into HO-8910 cells. The results of RT-PCR and western bolt showed that there were the significant expression high level of PTEN mRNA and protein after infected by wild-PTEN plasmid than those in the control[ （ 17 372 ±23）vs.（39±1 ）vs. （78 ±4）copies/ml,P 〈0. 05 ]. While the expression of mRNA of Akt1, Akt2 and PI3K were decreased clearly than those in the control [ （28 ± 2 ） vs. （ 115 ± 5 ）, （7 ± 1 ） vs. （ 18 ± 2）, （61 ± 2 ） vs.（84 ± 2）copies/ml , all P 〈 0. 05 ]. The proliferation rate of HO-8910 cells was obviously slower than those in the control （90 158 ±47 vs. 148 251 ±65 vs. 250 115 ±62, P〈0.05）. Conclusion Transfection of PTEN may increase the expression of PTEN and inhibit the proliferation of HO-8910 cells, in which PI3K/ Akt siganal pathway is inhibit significantly.