CAJ | 학술논문

目的建立稳定表达候选抑癌基因DMBT1(deleted in malignant brain tumours 1)的胆囊癌细胞系GBC-SD,研究其迁移能力并探讨分子机制.方法以脂质体为介导在体外建立稳定表达DMBTl的胆囊癌细胞系.细胞分组:(1)GBC-SD/DMBT1为转染DMBT1组;(2)GBC-SD/Vector为转染空载体组;(3)GBC-SD为未处理组.免疫组化、Western blot及RT-PCR检测DMBT1蛋白、mRNA表达,确证转染成功,划痕法与Transwell法检测细胞迁移能力,Western blot检测E-钙粘蛋白、CD44V6、CD15等黏附转移分子表达.统计学采用配对或独立样本t检验,P＜0.01为差异有统计学意义.结果稳定转染后,实验组DMBT1蛋白及mRNA表达较对照组分别提高3.2倍、2.7倍;划痕法显示实验组细胞在迁移距离、迁移速率、迁移面积均显著小于对照组(P＜0.01);Tnmswell法表明转染DMBT1后,迁移细胞数亦显著少于对照组(P＜0.01);免疫印迹表明转染实验组较对照组E-钙粘蛋白表达上调,CD15表达量减少(P＜0.01),而CD44V6、α与β连环素、整合素α5在转染前后表达量无明显差异.结论在体外成功建立稳定表达DMBT1的胆囊癌细胞系;DMBT1在体外过表达显著抑制GBC-SD的迁移,至少部分依赖于上调E-钙粘蛋白表达,下调CD15表达.
목적 건립은정표체후선억암기인DMBT1(deleted in malignant brain tumours 1)적담낭암세포계GBC-SD,연구기천이능력병탐토분자궤제.방법 이지질체위개도재체외건립은정표체DMBTl적담낭암세포계.세포분조:(1)GBC-SD/DMBT1위전염DMBT1조;(2)GBC-SD/Vector위전염공재체조;(3)GBC-SD위미처리조.면역조화、Western blot급RT-PCR검측DMBT1단백、mRNA표체,학증전염성공,화흔법여Transwell법검측세포천이능력,Western blot검측E-개점단백、CD44V6、CD15등점부전이분자표체.통계학채용배대혹독립양본t검험,P＜0.01위차이유통계학의의.결과 은정전염후,실험조DMBT1단백급mRNA표체교대조조분별제고3.2배、2.7배;화흔법현시실험조세포재천이거리、천이속솔、천이면적균현저소우대조조(P＜0.01);Tnmswell법표명전염DMBT1후,천이세포수역현저소우대조조(P＜0.01);면역인적표명전염실험조교대조조E-개점단백표체상조,CD15표체량감소(P＜0.01),이CD44V6、α여β련배소、정합소α5재전염전후표체량무명현차이.결론 재체외성공건립은정표체DMBT1적담낭암세포계;DMBT1재체외과표체현저억제GBC-SD적천이,지소부분의뢰우상조E-개점단백표체,하조CD15표체.
Objective To establish the gallbladder carcinoma cell line GBC-SD with stable expression of DMBT1 (deleted in malignant brain tumours 1) and explore effects on its chemotaxis and migration.Methods The full-length expression clone of DMBT1 was transfeeted with Lipofectamine 2000 into GBC-SD which was subsequently screened with G418 filtration.Semi-quantitative reverse transcriptionPCR,Western blot and immunocytocbemistry were employed to determine the level of DMBT1 mRNA and protein.Scratch wound model and transwell assay were applied to evaluate alterations of cell migration ability.Western blot was used to detect variances in the levels of proteins relevant to invasion and migration,namely CD15,CD44V6,E-cadherin,a and β catenin,integrin α5 and β1.Results RT-PCR,Western blotting and immunocytocbemistry found significant increase of DMBT1 mRNA and protein,GBC-SD/DMBT1 vs GBC-SD/vector,P ＜ 0.01.Scratch wound model witnessed markedly lessened parameters of migration distance,area,and rate in GBC-SD/DMBT1,P ＜0.01,when compared with that of GBC-SD/vector.Transwell assay confirmed great discrepancy in migration,between GBC-SD/DMBT1 and GBC-SD/vector.Western blot detected decreased level of CD15 and increased level of E-cadherin whereas no significant difference was found in α and β catenin,integrin α5 and β1,and CD44V6 expression,P ＜0.01,between GBC-SD/DMBT1 and GBC-SD/vector.Conclusion The cell model with stable expression of DMBT1 was successfully established in vitro; DMBT1 over-expression can significantly inhibit the migration of the gallbladder carcinoma cell line GBC-SD possibly because of an up-regulation of E-cadherin and down-regulatian of CD15.