CAJ | 학술논문

目的：探讨依维莫司（ EVE）对人宫颈癌SiHa细胞增殖、凋亡及上皮间质转化（ EMT）的影响。方法常规培养SiHa细胞达对数生长期后，采用终浓度为1、10、100μmol／L EVE处理SiHa细胞，采用四甲基偶氮唑盐比色法测定对照组及不同浓度EVE处理24、48、72和96 h后的细胞增殖情况，分别采用Annexin V?FITC／PI双染联合流式细胞术和实时荧光定量聚合酶链反应（ qRT?PCR）检测不同浓度EVE处理96 h的早期和晚期凋亡率及凋亡相关基因（ Bax、Bad和Bcl?2）的表达情况，Western blotting和免疫荧光法检测各组处理96 h后的EMT相关标记分子包括上皮型钙粘附素（ E?cad）、纤维连接蛋白（ FN）和波形蛋白（ Vim）的水平。结果 EVE在1～100μmol／L范围内对SiHa细胞有增殖抑制作用，且增殖抑制率呈剂量和时间依赖性，除1μmol／L作用24 h外，其余浓度及作用时间的增殖抑制率均高于对照组（ P＜0?05）；与对照组相比，EVE处理96 h后的早期、晚期凋亡率及Bax、Bad的mRNA水平均升高，而Bcl?2 mRNA水平降低，差异均有统计学意义（ P＜0?05），且呈剂量依赖性；EVE处理后的E?cad水平均高于对照组，而FN和Vim水平均低于对照组（ P＜0?05）。结论采用EVE阻断mTOR通路对宫颈癌SiHa细胞有毒性作用，不仅抑制其增殖且诱导凋亡，可能与抑制其EMT过程有关。
목적：탐토의유막사（ EVE）대인궁경암SiHa세포증식、조망급상피간질전화（ EMT）적영향。방법상규배양SiHa세포체대수생장기후，채용종농도위1、10、100μmol／L EVE처리SiHa세포，채용사갑기우담서염비색법측정대조조급불동농도EVE처리24、48、72화96 h후적세포증식정황，분별채용Annexin V?FITC／PI쌍염연합류식세포술화실시형광정량취합매련반응（ qRT?PCR）검측불동농도EVE처리96 h적조기화만기조망솔급조망상관기인（ Bax、Bad화Bcl?2）적표체정황，Western blotting화면역형광법검측각조처리96 h후적EMT상관표기분자포괄상피형개점부소（ E?cad）、섬유련접단백（ FN）화파형단백（ Vim）적수평。결과 EVE재1～100μmol／L범위내대SiHa세포유증식억제작용，차증식억제솔정제량화시간의뢰성，제1μmol／L작용24 h외，기여농도급작용시간적증식억제솔균고우대조조（ P＜0?05）；여대조조상비，EVE처리96 h후적조기、만기조망솔급Bax、Bad적mRNA수평균승고，이Bcl?2 mRNA수평강저，차이균유통계학의의（ P＜0?05），차정제량의뢰성；EVE처리후적E?cad수평균고우대조조，이FN화Vim수평균저우대조조（ P＜0?05）。결론채용EVE조단mTOR통로대궁경암SiHa세포유독성작용，불부억제기증식차유도조망，가능여억제기EMT과정유관。
Objective To investigate the effect of an mTOR pathway inhibitor everolimus( EVE) on the proliferation, apopto?sis and epithelial mesenchymal transition( EMT) of human cervical cancer SiHa cells. Methods After being cultured in normal cul?ture, the SiHa cells were treated with EVE at final concentrations of 1, 10 and 100 μmol/L. The cell proliferation was measured by methyl thiazolyl tetrazolium in the control group and different concentrations of EVE at 24, 48, 72 and 96 h after exposure. The early?and late?apoptosis rate was measured by Annexin V/PI double staining via flow cytometry, and the apoptosis related gene expression ( Bax, Bad and Bcl?2) was assessed by real?time fluorescence quantitative polymerase chain reaction. Western blotting and immunoflu?orescence assay were used to detect the levels of EMT related marker molecules, including E?cadherin( E?cad) , Fibronectin( FN) and Vimentin(Vim) at 96 h after EVE exposure. Results EVE had inhibitory effect on the proliferation of SiHa cells in the range of 1?100μmol/L in a dose?and time?dependent manner. Except for 1μmol/L at 24 h, the proliferation inhibition rates of the remaining concen?tration and action times were higher than that of the control group( P<0?05) . Compared with the control group, EVE treatment for 96 h could dose?dependently increase the apoptosis rate and mRNA levels of Bax and Bad but decrease Bcl?2 level(P<0?05). Compared with the control group, the level of E?cad was increased but the levels of Vim and FN were decreased after treatment with different con?centrations EVE for 96 h( P<0?05) . Conclusion Blocking mTOR pathway by EVE has a toxic effect on cervical cancer SiHa cells, which not only inhibit the proliferation and induce apoptosis, but may be related to the inhibition of EMT process.