应用载体介导的RNAi技术抑制人胃癌和腹膜间皮细胞中TGF-β_1的表达  

RNAi-mediated gene silencing of transforming growth factor-β_1 expression in human gastric cancer cell line SGC-7901 and peritoneal mesothelial cells

作　　者：吴涛[1] 徐惠绵[1] 吴晓华[2] 姜成钢[1] 于淼[1] 

机构地区：[1]中国医科大学附属第一医院肿瘤科,辽宁省沈阳市110001 [2]秦皇岛市第一医院普外科,河北省秦皇岛市066000

出　　处：《世界华人消化杂志》2005年第21期2530-2534,共5页World Chinese Journal of Digestology

基　　金：国家重点基础研究发展规划973项目资助;No.G1998051203;辽宁省自然科学基金资助;No.2001225002-3~~

摘　　要：目的:应用载体介导的RNAi技术特异地干扰TGF-β1在人胃癌细胞株SGC-7901和腹膜间皮细胞中的表达.方法:使用线性化的pcPURβiCassette质粒构建针对TGF-β1基因的siRNA表达载体;hU6载体启动子下游插入含TGF-β1基因特异性序列的62mer寡核苷酸片段;采用胰蛋白酶-EDTA消化法,从人的腹膜组织中分离腹膜间皮细胞.采用形态学和链霉菌抗生物素蛋白-过氧化物酶连接法对培养细胞进行鉴定.将TGF-β1-siRNA载体转染人的上述两种细胞,以半定量RT-PCR和Western印迹方法检测转染后两种细胞中TGF-β1的表达水平的变化.结果:电泳、DNA测序证实合成的siRNA基因序列正确并已准确克隆入pcPURβiCassette载体.免疫组化染色结果显示腹膜间皮细胞角蛋白,波形蛋白表达阳性,而白细胞共同抗原(CD45),第Ⅷ因子相关抗原阴性.与各自未处理组比较,两种细胞TGF-β1-siRNA载体组的mRNA和蛋白均明显下降,并以蛋白下降更为明显.人胃癌细胞株SGC-7901TGF-β1蛋白下降约65.8%,人腹膜间皮细胞TGF-β1蛋白下降约61.8%.结论:载体介导的RNAi技术可成功地干扰TGF-β1在人胃癌细胞株SGC-7901和腹膜间皮细胞中的表达,为今后胃癌腹膜转移的基因靶向治疗奠定了基础.AIM： To silence the expression of transforming growth factor-β1 （TGF-β1） gene in human gastric cancer cell line SGC-7901 and peritoneal mesothelial cells using vector based RNA interference （RNAi） technique. METHODS： A vector, which was used to transcribe functional short interfering RNA （siRNA）, was constructed, and 62 met oligonucleotide fragment was inserted into the downstream of the hU6 promoter. Mesothelial cells were isolated from human omenta by trypsin- EDTA disaggregation, and identified by morphology and streptomyces anti-biotin protein-peroxidase method. The plasmids containing TGF-β1 target sequences were transfected into the two kinds of cells menti-oned above, and then the expression level of TGF-β1 was detected by semiquantitive RT-PCR and Western blot technique. RESULTS： Electrophoresis and DNA sequencing confirmed that the TGF-β1-specific siRNA was synthesized and cloned into the expression vector pcPUR β iCassette successfully. Immunohistochemistry showed positive staining for cytokeratin and vimentin, but negative staining for leukocyte common antigen and factor Ⅷ-related antigen in cultured mesothelial cells. As compared with their respective untreated group, the TGF-β1 mRNA and protein expression in the two kinds of transfected cells were remarkably decreased, especially TGF-β1 protein. TGF-β1 protein was reduced by a percent of 65.8% and 61.8% in human gastric cancer cell line SGC-7901 and mesothelial cells, respectively. CONCLUSION： TGF-β1 expression can be notably inhibited in human gastric cancer cell line SGC-7901 and peritoneal mesothelial cells using plasmid-based TGF-β1-RNAi technique.

关 键 词：载体介导 RNAI TGF—β1 胃癌 腹膜间皮 

分 类 号：R735.2[医药卫生—肿瘤]