摘要
目的检测过表达人核糖核酸酶抑制因子(ribonuclease inhibitor,RI)基因的质粒pIRES2-EGFP-RI对人膀胱癌T24细胞侵袭转移及上皮细胞间质转化(epithelial mesenchymal transition,EMT)现象的影响。方法将连有RI的质粒pIRES2-EGFP-RI稳定转染T24细胞后利用G418筛选出阳性克隆。根据转染的质粒不同分为3组:T24组(未转染组)、T24-0(转染pIRES2-EGFP质粒)和T24-RI组(转染pIRES2-EGFP-RI质粒)。RT-PCR检测T24细胞中RI的mRNA水平变化,细胞免疫荧光及Western blot检测RI蛋白的表达,利用HE染色观察其细胞形态的变化,Transwell方法检测细胞侵袭及转移能力的变化,Western blot检测侵袭转移及EMT相关蛋白Twist、MMP-9和Snail及E-cadherin的表达,构建移植瘤模型,利用HE染色观测肺组织切片癌细胞转移情况,组织免疫化学方法检测移植瘤中RI、Twist、Snail及E-cadherin的表达。结果 T24-RI组人RI的mRNA水平及蛋白表达显著升高(P<0.05);HE染色结果表明细胞铺展良好,核质比明显减小,细胞由间质形态向上皮形态转变;Transwell方法显示T24-RI组细胞的侵袭及转移能力较T24组、T24-0组明显下降;Western blot结果表明T24-RI组Twist、MMP-9和Snail较T24组、T24-0组显著降低(P<0.01),E-cadherin较T24组、T24-0组显著升高(P<0.01);HE染色肺组织切片显示T24-RI组较其他两组癌细胞转移降低,组织免疫化学结果表明T24-RI组RI、E-cadherin蛋白明显升高,而Twist、Snail显著降低。结论过表达人RI载体提高了RI蛋白的表达水平,并抑制了人膀胱癌T24细胞EMT的发生,从而抑制了膀胱癌T24细胞的侵袭、转移。
Objective To observe the effect on the invasion, migration and epithelial mesenchymal of over-expression of human ribonuclease inhibitor （RI） transition （EMT） in human bladder cancer T24 cells. Methods The vectors of plRES2-EGFP （T24-0 group） and pIRES2-EGFP-RI （T24-RI group） were used to transfect human bladder cancer T24 cells, respectively, and the positive clones were screened by G418. The T24 ceils without transfection were served as control （T24 group）. The expression of RI gene at mRNA and protein levels was evaluated by RT-PCR, immunofluorescence and Western blot analysis. HE staining was used for detect the changes of cell morphology. The protein levels of twist, matrix metalloproteinase 9 （MMP-9）, snail and E-eadherin were determined by Western blot analysis. Tumor cells at 2 × 10^6 of the three groups were respectively injected subcutaneously into the back of BALB/c nude mice to establish the xenograft models. HE staining was performed to observe the EMT and metastasis of the T24 cells in lung tissues, and immunohisto- chemical assay was applied to detect the expression of RI, E-cadherin, twist and snail in the tumors. Results Compared with the T24 group and T24-0 group, the expression of RI was significantly higher in the T24-RI group （P 〈 0.05 ）. The cells spread well, the nucleo-cytoplasmic ratio decreased, and EMT was observed. The cell invasion and migration abilities significantly reduced in the T24-RI group compared with the 3＂24 group and T24-0 group. The expression of twist, MMP-9 and snail significantly decreased, but the expression of E-cadher- in significantly increased in the T24-RI group compared with the T24 group and T24-0 group （P 〈0.01 ）. Cellinvasion and metastasis reduced in the lung tissues of BALB/c nude mice receiving an injection of pIRES2- EGFP-RI-transfected T24 cells. The expression of RI and E-cadherin significantly increased, but the expression of twist and snail significantly decreased in the T24-RI group compared with the T24 group
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2012年第23期2378-2383,共6页
Journal of Third Military Medical University
基金
国家自然科学基金(81071719
81172424)
重庆市自然科学基金(CSTC2009BB5273)~~
