中华妇产科杂志
中華婦產科雜誌
중화부산과잡지
CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
2010年
9期
682-685
,共4页
翟永宁%徐玲玲%沈悦%XIA Hong%沈宇飞
翟永寧%徐玲玲%瀋悅%XIA Hong%瀋宇飛
적영저%서령령%침열%XIA Hong%침우비
卵巢肿瘤%PTEN磷酸水解酶%1-磷脂酰肌醇3-激酶%原癌基因蛋白质c-akt%信号传导
卵巢腫瘤%PTEN燐痠水解酶%1-燐脂酰肌醇3-激酶%原癌基因蛋白質c-akt%信號傳導
란소종류%PTEN린산수해매%1-린지선기순3-격매%원암기인단백질c-akt%신호전도
Ovarian neoplasms%PTEN phosphohydrolase%1-Phosphatidylinositol 3-kinase%Proto-oncogene proteins c-akt%Signal transduction
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.
目的 研究外源性PTEN基因穩定轉染卵巢上皮性癌(卵巢癌)細胞後,對卵巢癌細胞燐痠酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)信號傳導通路的影響.方法 構建錶達野生型PTEN基因的重組載體pcDNA3.1A-PTEN質粒,併轉染卵巢癌細胞株HO-8910細胞(重組載體組),以轉染空載體pcDNA3.1A質粒(空載體組)作為陰性對照,以未轉染質粒(空白組)作為空白對照.採用逆轉錄(RT)PCR技術檢測3組細胞中PTEN、Akt1、Akt2、PI3K mRNA的錶達水平,蛋白印跡法檢測3組細胞中PTEN蛋白的錶達彊度,四甲基偶氮唑藍(MTT)比色法檢測3組細胞的生長情況[以吸光度(A)值錶示,A值越大錶示生長速度越快].結果 錶達野生型PTEN基因的重組載體pcDNA3.1A-PTEN質粒,經測序證實構建成功.重組載體組細胞中PTEN mRNA的錶達水平為(17372±23)copy/ml,高于空載體、空白組[分彆為(39±1)、(78±4)copy/ml],差異均有統計學意義(P＜0.05);重組載體組細胞中PTEN蛋白的錶達彊度也明顯高于空載體、空白組.重組載體組細胞中Akt1、Akt2、PI3KmRNA的錶達水平分彆為(28±2)、(7±1)、(61±2)copy/ml,低于空載體[分彆為(115±5)、(18±2)、(84±2)copy/ml]、空白組[分彆為(77±4)、(17±2)、(1349±7)copy/m1],差異均有統計學意義(P＜0.05).培養第5天,重組載體組細胞生長速度(A值)為90 158±47,低于空載體、空白組(分彆為148 251±65、250 115±62),差異均有統計學意義(P＜0.05).結論 轉染錶達野生型PTEN基因的重組載體能有效提高卵巢癌HO-8910細胞中PTEN mRNA和蛋白的錶達,抑製細胞生長,併通過顯著降低Akt1、Akt2、PI3K mRNA的錶達水平,明顯抑製PI3K/Akt信號傳導通路.
목적 연구외원성PTEN기인은정전염란소상피성암(란소암)세포후,대란소암세포린산선기순3격매(PI3K)/단백격매B(Akt)신호전도통로적영향.방법 구건표체야생형PTEN기인적중조재체pcDNA3.1A-PTEN질립,병전염란소암세포주HO-8910세포(중조재체조),이전염공재체pcDNA3.1A질립(공재체조)작위음성대조,이미전염질립(공백조)작위공백대조.채용역전록(RT)PCR기술검측3조세포중PTEN、Akt1、Akt2、PI3K mRNA적표체수평,단백인적법검측3조세포중PTEN단백적표체강도,사갑기우담서람(MTT)비색법검측3조세포적생장정황[이흡광도(A)치표시,A치월대표시생장속도월쾌].결과 표체야생형PTEN기인적중조재체pcDNA3.1A-PTEN질립,경측서증실구건성공.중조재체조세포중PTEN mRNA적표체수평위(17372±23)copy/ml,고우공재체、공백조[분별위(39±1)、(78±4)copy/ml],차이균유통계학의의(P＜0.05);중조재체조세포중PTEN단백적표체강도야명현고우공재체、공백조.중조재체조세포중Akt1、Akt2、PI3KmRNA적표체수평분별위(28±2)、(7±1)、(61±2)copy/ml,저우공재체[분별위(115±5)、(18±2)、(84±2)copy/ml]、공백조[분별위(77±4)、(17±2)、(1349±7)copy/m1],차이균유통계학의의(P＜0.05).배양제5천,중조재체조세포생장속도(A치)위90 158±47,저우공재체、공백조(분별위148 251±65、250 115±62),차이균유통계학의의(P＜0.05).결론 전염표체야생형PTEN기인적중조재체능유효제고란소암HO-8910세포중PTEN mRNA화단백적표체,억제세포생장,병통과현저강저Akt1、Akt2、PI3K mRNA적표체수평,명현억제PI3K/Akt신호전도통로.
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.
