摘要目的 观察TMPRSS4基因沉默对人胰腺癌SW1990细胞体外生长增殖和侵袭的影响.方法 体外合成4个靶向TMPRSS4基因和阴性对照的真核表达载体,瞬时转染到SW1990细胞,实时定量PCR法检测转染细胞的TMPRSS4 mRNA表达.以干扰效率最高的真核表达载体转染SW1990细胞,G418筛选出稳定的TMPRSS4基因沉默的细胞株,蛋白质印迹法检测稳定细胞株TMPRSS4蛋白抑制效率,CCK-8法检测细胞生长抑制率,Transwell小室检测细胞侵袭能力.结果 成功构建了稳定下调TMPRSS4表达的细胞株SW1990/psi-TMPRSS4,细胞转染效率为82.9%.与亲本SW1990细胞比较,TMPRSS4 mRNA和蛋白水平分别下调了80.1%、60%.SW1990/psi-TMPRSS4组穿膜细胞数为(118.6±13.4)个,显著低于阴性对照组的(157.4±12.9)个和亲本细胞组的(157.0±9.5)个(P值均<0.01).SW1990/psi-TMPRSS4组细胞的侵袭抑制率为24.5%.但各组细胞增殖无明显变化.结论 成功筛选出稳定下调TMPRSS4表达的细胞株.下调TMPRSS4表达能有效抑制胰腺癌SW1990细胞的侵袭能力,但对细胞增殖无影响.

abstractsObjective To study the influence of the small interfering RNA (siRNA) interference TMPRSS4 expression on human pancreatic cancer SW1990 cell's proliferation and invasion. Methods The four eukaryotic expression vector of TMPRSS4 gene were synthesized in vitro and were transfected transiently into human pancreatic cancer SW1990 cells. TMPRSS4 mRNA expression of transfected cells was detected by real-time RT-PCR. The most efficient eukaryotic expression vector was used to be transfected into SW1990 cells. By using G418, cell strain that can silence TMPRSS4 gene stably was screened. The TMPRSS4 mRNA expression of the stable cell strain was detected by real time PCR TMPRSS4 protein expression was detected by western blot. The proliferation ability of transfected SW1990 cells was detected by CCK-8 method. By Transwell, the invasion change of SW1990 cell was detected. Results A stable cell strain, SW1990/psi TMPRSS4, was successfully constructed, in which the expression level of TMPRSS4 could be reduced stably by RNA interference. Cell transfection efficiency was 82.9%. Compared with the control group, the TMPRSS4 mRNA and protein levels were reduced by 80.1% and 60% ,and number of penetrating cells was 118.6 ±13.4 in SW1990/psi TMPRSS4 group, which was significantly lower than those in the negative control group (157.4 ± 12.9) and control group (157.0±9.5, P <0.01). Cells invasion inhibitory rate was 24.5% in SW1990/psi TMPRSS4 group. The cell proliferation was not significantly different among all the groups. Conclusions A stable cell strain is screened successfully in which the expression level of TMPRSS4 can be reduced stably. The down-regulation of TMPRSS4 gene expression level can inhibit the invasion of SW1990 cells, but has no effect on cell proliferation.