中文摘要：

目的构建整合素连接激酶（Integrin linked kinase,ILK）基因siRNA干扰质粒,探讨ILK基因沉默对膀胱癌BIU-87细胞增殖能力及糖原合成酶激酶3（Glycogen synthase kinase 3,GSK3）、β-环连蛋白（β-catenin）表达的影响。方法构建针对人ILK基因的2个特异性siRNA表达质粒和1个无同源性的阴性对照质粒,在脂质体介导下转染膀胱癌BIU-87细胞,通过RT-PCR及Western blot检测ILK基因mRNA和蛋白的表达水平,筛选出1个干扰效果较强的质粒用于后续试验,Western blot检测细胞GSK3和β-catenin蛋白的表达,Am-blue法检测细胞的增殖活力,流式细胞术检测细胞周期的分布。结果经酶切和测序证明重组干扰质粒构建正确;稳定转染pGenesil-1 siRNA1和pGenesil-1 siRNA2质粒的BIU-87细胞ILK基因mRNA和蛋白表达水平较BIU-87细胞组（未转染组）分别下降了69%、52%和70%、55%,选择pGenesil-1 siRNA1为最有效干扰序列;BIU-87siRNA1组GSK3的表达水平较阴性对照组和BIU-87细胞组分别升高了41.87%和30.12%,β-catenin的表达水平较阴性对照组和BIU-87细胞组分别降低了38.67%和48.05%;BIU-87 siRNA1组细胞增殖活力明显下降;BIU-87 siRNA1组G0-G1期细胞比例明显增加,S期减少。结论已成功构建ILK基因siRNA表达质粒,其可明显抑制膀胱癌BIU-87细胞的增殖,机制可能为ILK基因通过介导GSK3、β-catenin信号分子促进膀胱癌BIU-87细胞的增殖。

英文摘要：

Objective To construct the siRNA plasmid for integrin linked kinase（ILK） gene and investigate the effect of ILK gene silencing on proliferation of bladder cancer BIU-87 cells as well as expressions of glycogen synthase kinase 3（GSK3） and β-catenin.Methods Two specific siRNA plasmids for human ILK gene and one non-homologous negative control plasmid were constructed and transfected to BIU-87 cells in mediation of liposome.The expressions of ILK gene at mRNA and protein levels were determined by RT-PCR and Western blot respectively,based on which a plasmid with highly interferential efficiency was selected for further test.The BIU-87 cells were determined for expressions of GSK3 and β-catenin by Western blot,for proliferative activity by Am-blue method,and the distribution of cell cycle by flow cytometry.Results Both restriction analysis and sequencing proved that recombinant interfering plasmid was constructed correctly.Compared with those in BIU-87 cells untransfected,the expression levels of ILK mRNA in BIU-87 cells stably transfected with plasmids pGenesil-1 siRNA1 and pGenesil-1 siRNA2 decreased by 69% and 52%,while those of ILK protein decreased by 70% and 55% respectively.Plasmid pGenesil-1 siRNA1 was selected as the most effective sequence.Compared with those in negative control and BIU-87 groups,the expression levels of GSK3 in BIU-87 siRNA group increased by 41.87% and 30.12%,while those of β-catenin decreased by 38.67% and 48.05% respectively,the cell proliferative activity decreased significantly,the percentages of cells at G0-G1 stages increased significantly while those at S stage decreased.Conclusion The siRNA plasmid for ILK gene was constructed successfully,which inhibited the proliferation of BIU-87 cells significantly by a possible mechanism associated with signal pathway molecules GSK3 and β-catenin.