摘要
目的:探讨应用短发夹RNA(short hairpin RNA,shRNA)干扰技术沉默磷脂酶Cε(phospholipase C epsilon,PLCε)基因对人膀胱癌移植瘤生长及凋亡相关蛋白Bcl-2和Bax表达的影响。方法:人膀胱癌BIU-87细胞种植于裸鼠皮下,待移植瘤体积达40 mm3左右时,在肿瘤部位分别直接注射建构有PLCε特异性shRNA的重组表达质粒pGenesil-PLCε、阴性对照重组pGenesil-NP质粒以及0.9%氯化钠溶液;观察肿瘤体积的变化情况,30 d后处死全部裸鼠,取瘤组织,称瘤质量;瘤组织制备病理切片,HE染色观察细胞形态的变化;RT-PCR鉴定瘤组织中PLCεmRNA的表达;Western印迹法检测瘤组织中PLCε、Bcl-2、Bax、p-Akt1/Akt1和p-Bad/Bad蛋白的表达。结果:pGenesil-PLCε组小鼠移植瘤生长明显缓慢,瘤组织体积和质量均明显小于对照组;HE染色结果显示,瘤组织中出现细胞核固缩和裂碎等凋亡征象;RT-PCR检测结果显示,PLCεmRNA表达明显降低(P<0.05);Western印迹法结果提示,PLCε和Bcl-2蛋白表达量明显降低,Bax蛋白表达增加,与2个对照组比较,差异均有统计学意义(P<0.05);而Akt1、Bad蛋白的表达在实验组以及对照组中差异均无统计学意义,p-Akt1和p-Bad在移植瘤中未见表达。结论:沉默PLCε基因可明显抑制PLCε的表达,并下调Bcl-2蛋白以及上调Bax蛋白的表达,促进膀胱癌细胞凋亡而抑制肿瘤生长。
Objective：To explore the effect of silencing phospholipase C epsilon（PLCε） gene expression with short hairpin RNA（shRNA） on Bcl-2/Bax protein expression in xenografted human bladder cancer cells in nude mice.Methods：Mice were subcutaneously implanted 2×106 BIU-87 cells.Once the volume of tumor reached 40 mm3,pGenesil-PLCε or pGenesil-NP vector or 0.9% NaCl solution was injected directly into the tumor bodies,respectively.The tumor volume was recorded.The nude mice were killed 30 d later.The tumor body was isolated and weighed.The pathological changes of carcinoma were observed after HE staining.The expression of PLCε mRNA was determined by RT-PCR.The protein expression of PLCε,Bcl-2,Bax,p-Akt1/Akt1,and p-Bad/Bad were detected by Western blotting.Results：The tumor grew slower in pGenesil-PLCε group.The size and weight of tumor bodies in pGenesil-PLCε group were significantly lower than those in control groups（P〈0.05）.In pGenesil-PLCε group the tumor cells showed apoptotic changes such as karyopyknosis and nuclear fragmentation under light microscope.The expression of PLCε mRNA was obviously decreased compared with the control groups（P〈0.05）.The protein expressions of Bcl-2 and PLCε were markedly decreased（P〈0.05） but Bax protein expression was increased compared with two control groups（P〈0.05）.Knocking down of PLCε did not influence the protein expression of Akt1 and Bad.The p-Akt1 and p-Bad protein had no expression in xenografted tumor.Conclusion：Silencing PLCε gene with the shRNA greatly decreased PLCε gene expression,down-regulated Bcl-2 protein expression,up-regulated Bax protein expression,induced cell apoptosis,and finally inhibited the growth of human bladder cancer.
出处
《肿瘤》
CAS
CSCD
北大核心
2010年第5期376-380,共5页
Tumor
