CAJ | 학술논문

目的观察黑色素瘤分化相关基因-7/白细胞介素-24(MDA-7/IL-24)基因对肝癌细胞株MHCC97-H侵袭转移及上皮-间充质转化(EMT)特性的影响.方法构建携带MDA-7/IL-24基因的腺病毒载体,转染肝癌细胞株MHCC97-H,噻唑蓝(MTT)、免疫荧光检测转染效率.采用Transwell侵袭实验和迁移实验分别检测未转染细胞(空白对照组)、转染阴性对照组(Ad.vec组)和实验组(转染Ad.MDA-7细胞组)侵袭转移能力.镜下观察转染前后细胞形态的变化,免疫荧光检测转染前后细胞上皮表型蛋白和间质表型蛋白的定位和表达,实时定量聚合酶链反应(Real-timePCR)检测转染后细胞MDA-7/IL-24基因的表达,Western blot检测E-钙黏蛋白(E-cad)、N-钙黏蛋白(N-cad)、波形蛋白(Vim)蛋白的表达.结果 MDA-7/IL-24可以有效转染肝癌细胞株MHCC97-H,稳定表达MDA-7/IL-24 mRNA(空白组:2.042±0.915比Ad.MDA-7组:37.512±2.354,P＜0.05)和蛋白.转染MDA-7/IL-24的肝癌细胞株MHCC97-H较空白对照组和阴性对照组侵袭迁移能力明显受到抑制(实验组迁移率:空白组迁移率=1.00：0.46,实验组侵袭率:空白组侵袭率=1.00：0.61,P＜0.01).镜下观察肝癌细胞株MHCC97-H转染MDA-7/IL-24后,由成纤维细胞纺锤体样、排列分散,转变为细胞排列紧密如圆形或椭圆形铺路石样.免疫荧光可见肝癌细胞株MHCC97-H转染后细胞膜表达的E-cad荧光由胞质转移至细胞周边浓聚,而Vim的荧光强度较前下降.Western blot进行蛋白定量分析,转染后MHCC97-H细胞上皮表型标志性蛋白E-cad表达量增高,间质表型标志性蛋白Twist、Vim表达降低,差异具有统计学意义(P＜0.05).结论再表达MDA-7/IL-24基因的MHCC97-H细胞可以增加上皮表型蛋白表达,减少间质表型蛋白,改变细胞骨架构型,逆转肝癌细胞EMT过程,使肿瘤细胞侵袭能力减弱.
목적 관찰흑색소류분화상관기인-7/백세포개소-24(MDA-7/IL-24)기인대간암세포주MHCC97-H침습전이급상피-간충질전화(EMT)특성적영향.방법 구건휴대MDA-7/IL-24기인적선병독재체,전염간암세포주MHCC97-H,새서람(MTT)、면역형광검측전염효솔.채용Transwell침습실험화천이실험분별검측미전염세포(공백대조조)、전염음성대조조(Ad.vec조)화실험조(전염Ad.MDA-7세포조)침습전이능력.경하관찰전염전후세포형태적변화,면역형광검측전염전후세포상피표형단백화간질표형단백적정위화표체,실시정량취합매련반응(Real-timePCR)검측전염후세포MDA-7/IL-24기인적표체,Western blot검측E-개점단백(E-cad)、N-개점단백(N-cad)、파형단백(Vim)단백적표체.결과 MDA-7/IL-24가이유효전염간암세포주MHCC97-H,은정표체MDA-7/IL-24 mRNA(공백조:2.042±0.915비Ad.MDA-7조:37.512±2.354,P＜0.05)화단백.전염MDA-7/IL-24적간암세포주MHCC97-H교공백대조조화음성대조조침습천이능력명현수도억제(실험조천이솔:공백조천이솔=1.00：0.46,실험조침습솔:공백조침습솔=1.00：0.61,P＜0.01).경하관찰간암세포주MHCC97-H전염MDA-7/IL-24후,유성섬유세포방추체양、배렬분산,전변위세포배렬긴밀여원형혹타원형포로석양.면역형광가견간암세포주MHCC97-H전염후세포막표체적E-cad형광유포질전이지세포주변농취,이Vim적형광강도교전하강.Western blot진행단백정량분석,전염후MHCC97-H세포상피표형표지성단백E-cad표체량증고,간질표형표지성단백Twist、Vim표체강저,차이구유통계학의의(P＜0.05).결론 재표체MDA-7/IL-24기인적MHCC97-H세포가이증가상피표형단백표체,감소간질표형단백,개변세포골가구형,역전간암세포EMT과정,사종류세포침습능력감약.
Objective To investigate the effecof the Melanomdifferentiation associated gene-7/ Interleukin-24 (MDA-7/IL-24) on the migration and invasion of human HepatocellulacarcinomcellMHCC97-H and the change of epithelial to mesenchymal transition (EMT).MethodConstructed Adenoviruvectotaken MDA-7/IL-24 gene transfected hunman HepatocellulacarcinomcellMHCC97-H.The expression of MDA-7/IL-24 wadetected by real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting.The migration and invasion of Non-transfected (blank control group), Ad.vegroup (negative control group) and Ad.MDA-7 (experimental group) transfected MHCC97-H cellwadetected by Transwell invasion assay and Transwell migration assay.The changein morphology of cellwere observed by optical microscopy.The localization and expression of the epithelial markerand the mesenchymal markerwere assayed by immunofluorescence and Western blotting.ResultThe stable MDA-7/IL-24 mRN(Ad.vegroup MDA-7 mRN2.042 ± 0.915/Ad.MDA-7 group MDA-7 mRN37.512±2.354) and protein expression of MDA-7 wadetected in the celltransfected with Ad.MDA-7.the migration rate of Ad.vegroup to the Ad.MDA-7 group were 1.00：0.46, (P ＜ 0.01).The invasion rate of Ad.vegroup to the Ad.MDA-7 group were 1.00：0.61,(P ＜0.01).The migration and invasion warestrained in the experimental group compared with control groups, (P ＜0.01).Transfection of Ad.MDA-7 induced morphologichange of MHCC97-H cellfrom fibroblastishape with cell scattering to paving stone structure with cell-cell adhesion.In Ad.MDA-7 transfected cells, the localization of E-cadherin waaltered from the cytoplasm to cell-cell contactand the level of Vimentin decreased.Moreover, the expression of the epithelial marker, E-cadherin and β-catenin, were increased;the expression of the mesenchymal marker, twisand Vimentin, were reduced.Conclusion MHCC97-H reexpression of MDA-7/IL-24 induced the increasing expression of epithelial markeand the decrease expression of mesenchymal markereversed HCEMthen suppressed the HCcellmigration and invasion.