【摘要】：目的:探討攜帶凋亡素基因(apopptin)的溶瘤腺病毒ATV感染對(duì)人宮頸癌HeLa細(xì)胞自噬和凋亡的影響。方法:分別用攜帶凋亡素基因的溶瘤腺病毒ATV與對(duì)照病毒Ad-MOCK(均為前期工作構(gòu)建)感染HeLa細(xì)胞后,應(yīng)用WST-1法檢測(cè)ATV感染對(duì)HeLa細(xì)胞增殖的影響,流式細(xì)胞術(shù)檢測(cè)ATV感染對(duì)HeLa細(xì)胞凋亡和細(xì)胞周期的影響,Western blotting法檢測(cè)ATV感染對(duì)HeLa細(xì)胞自噬相關(guān)蛋白LC3、P62與mTOR表達(dá)的影響,MDC染色法檢測(cè)ATV感染對(duì)HeLa細(xì)胞自噬水平的影響。結(jié)果:ATV感染后抑制HeLa細(xì)胞增殖,并具有一定的時(shí)間效應(yīng)關(guān)系;以ATV 100 MOI感染72 h后抑制率達(dá)到59.26%,其抑制能力明顯強(qiáng)于同時(shí)間段對(duì)照組(P0.01)。ATV感染48 h,ATV組HeLa細(xì)胞凋亡率顯著高于對(duì)照組[(38.995±4.009)%vs(14.680±1.174)%,P0.01]。感染后的HeLa細(xì)胞,隨著ATV作用時(shí)間的延長(zhǎng),S期出現(xiàn)阻滯,48 h阻滯最強(qiáng),顯著高于對(duì)照組[(58.490±2.447)%vs(43.235±4.419)%,P0.05]。與對(duì)照組相比,ATV感染HeLa后,LC3表達(dá)量6、12 h逐漸增高,12 h達(dá)到最大值(P0.01),24 h表達(dá)量最低(P0.01);P62蛋白在24 h出現(xiàn)最高表達(dá)水平(P0.01);而mTOR隨著作用時(shí)間延長(zhǎng)逐漸降低(48 h時(shí),P0.01)。熒光顯微鏡下可見(jiàn)HeLa細(xì)胞核周區(qū)域MDC陽(yáng)性染色,隨著ATV作用時(shí)間延長(zhǎng),自噬小體明顯增多;與對(duì)照組相比,6、12h自噬小體數(shù)量顯著增多[(28.000±2.828)vs(8.500±2.121),(37.000±4.243)vs(14.000±1.414);均P0.01],24 h相對(duì)減少[(12.000±2.828)vs(17.000±1.414),P0.01]。結(jié)論:攜帶凋亡素基因的ATV溶瘤腺病毒可促進(jìn)人宮頸癌HeLa細(xì)胞的凋亡和自噬水平,進(jìn)而特異性殺傷腫瘤細(xì)胞。
[Abstract]:Aim: to investigate the effect of adenovirus ATV infection with apoptin gene (apopptin) on autophagy and apoptosis of human cervical cancer HeLa cells. Methods: HeLa cells were infected with adenovirus ATV carrying apoptin gene and control virus Ad-MOCK, respectively. The effect of ATV infection on the proliferation of HeLa cells was detected by WST-1 assay. The effect of ATV infection on apoptosis and cell cycle of HeLa cells was detected by flow cytometry. The effect of ATV infection on the expression of LC3,P62 and mTOR in HeLa cells was detected by Western blotting. The effect of ATV infection on the autophagy level of HeLa cells was detected by MDC staining. Results the proliferation of HeLa cells was inhibited by the infection of 1: ATV and had a time-effect relationship. The inhibitory rate of ATV 100 MOI infection for 72 h was 59.26, which was significantly higher than that in the control group (P0.01). The apoptosis rate of HeLa cells in the 48 h ATV group was significantly higher than that in the control group [(38.995 鹵4.009) vs (14.680 鹵1.174) P0.01]. With the prolongation of ATV, HeLa cells had the strongest arrest at 48 h in S phase, which was significantly higher than that in control group [(58.490 鹵2.447) vs (43.235 鹵4.419) P 0.05]. Compared with the control group, the expression level of LC3 increased gradually from 6h to 12h after ATV infection and reached the lowest (P0.01) at 24 h (P0.01) and the highest expression level of P62 protein (P0.01) at 24 h (P0.01), while mTOR decreased gradually (P0.01 at 48h). The positive staining of MDC in the perinuclear region of HeLa was observed under fluorescence microscope, and the number of autophagy bodies increased significantly with the prolongation of ATV action time, and the number of autophagy bodies increased significantly at 612h compared with the control group [(28.000 鹵2.828) vs (8.500 鹵2.121), (37.000 鹵4.243) vs (14.000 鹵1.414); P0.01] at 24 h, the number of autophagy bodies decreased relatively [(12.000 鹵2.828) vs (17.000 鹵1.414) P0.01]. Conclusion: ATV adenovirus carrying apoptin gene can promote apoptosis and autophagy of human cervical cancer HeLa cells and then specifically kill tumor cells.
【作者單位】： 長(zhǎng)春中醫(yī)藥大學(xué)藥學(xué)院;軍事科學(xué)院軍事獸醫(yī)研究所分子病毒學(xué)與免疫學(xué)實(shí)驗(yàn)室;吉林醫(yī)藥學(xué)院檢驗(yàn)學(xué)院;吉林省腫瘤醫(yī)院頭頸外一科;長(zhǎng)春中醫(yī)藥大學(xué)基礎(chǔ)醫(yī)學(xué)院;
【基金】：國(guó)家重點(diǎn)研發(fā)計(jì)劃(973計(jì)劃)資助項(xiàng)目(No.2016YSC1200900) 國(guó)家“重大新藥創(chuàng)制”科技重大專項(xiàng)資助(No.2014ZX09304314) 吉林省重大科技攻關(guān)項(xiàng)目資助(No.20150201002YY) 吉林省產(chǎn)業(yè)技術(shù)創(chuàng)新戰(zhàn)略聯(lián)盟項(xiàng)目資助(No.20140309006YY) 長(zhǎng)春市重大科技攻關(guān)項(xiàng)目資助(No.16ss11) 吉林省科技發(fā)展計(jì)劃項(xiàng)目資助(No.20150101124JC)~~
【分類號(hào)】：R737.33

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 郭海寧;張萍;韓繼成;靖杰;曹亮;肖朋朋;解長(zhǎng)占;李一權(quán);馬海彬;南福龍;崔卓棟;李霄;田明堯;魯會(huì)軍;金寧一;;轉(zhuǎn)瓶培養(yǎng)與生物反應(yīng)器微載體培養(yǎng)重組腺病毒的比較[J];中國(guó)病原生物學(xué)雜志;2016年01期

2 楊冬梅;方先龍;楊s，

本文編號(hào)：2252997