基于RNAi技术研究参芪扶正注射液联合IFN-α对肝癌细胞增殖能力的影响

Effect of shenqifuzheng injection combined with IFN-α on the proliferation of hepatoma cells based on RNAi technique

目的:研究参芪扶正注射液与干扰素α(IFN-α)联用时对肝癌细胞增殖能力的影响,并通过RNA干扰技术明确其对IFN-α协同增效的作用机制。方法:运用细胞增殖实验(MTT)技术检测IFN-α注射液(6000 IU)单独或与参芪扶正注射液(0.5 g/L)联合时对SMMC-7721细胞增殖能力的影响;用实时荧光定量聚合酶链反应(Real-time PCR)技术和蛋白质印迹法(Western-blot)分别检测IFN-α单独或与参芪扶正注射液联用时,实验组、阴性对照组(NaCl)和空白对照组中STAT1的RNA转录和蛋白质表达情况,确定参芪扶正注射液对STAT1转录和表达的影响;运用Western blot检验STAT1基因经RNA干扰后SMMC-7721的STAT1蛋白质表达,确定干扰效果;MTT实验检测IFN-α单独或联合参芪扶正注射液作用于STAT1基因"敲低"细胞株时,对细胞增殖能力的影响。结果:与单独使用IFN-α及参芪扶正注射液相比,小剂量参芪扶正注射液与IFN-α联用可以增强IFN-α对人肝癌细胞SMMC-7721增殖能力的抑制作用,并上调STAT1 mRNA和蛋白质的表达;SMMC-7721的STAT1基因"敲低"细胞株中STAT1蛋白表达明显降低;针对SMMC-7721的STAT1基因"敲低"细胞株,参芪扶正注射液协同IFN-α与单独IFN-α及阴性对照组相比,对细胞增殖能力的影响无显著性差异。结论:参芪扶正注射液通过上调人肝癌细胞SMMC-7721中STAT1的表达,是提升干扰素α对肝癌细胞增殖能力抑制作用的主要机制之一。

Objective： To study the effect of Shenqi Fuzheng Injection Combined with interferon alpha（IFN-alpha） on the proliferation of hepatocellular carcinoma cells, and to clarify the mechanism of its synergistic effect on IFN-alpha by RNA interference. Methods： By using cell proliferation assay（MTT） detection of interferon alpha（IFN-alpha） injection（6000 IU） alone or with Shenqifuzheng Injection（0.5 g/L） effects on the proliferation ability of SMMC-7721 cells combined; using real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） and Western blot（Western-blot） were detected respectively. IFN-alone or with Shenqi Fuzheng Injection Combined with, the experimental group, negative control group（NaCl） and STAT1 control group the expression of RNA mRNA and protein, to determine the effect of Shenqi Fuzheng Injection on the expression and transcription of STAT1; expression of STAT1 protein by Western blot test of STAT1 gene by RNA interference SMMC-7721 sure, the jamming effect is stable; IFN-alpha MTT assay alone or combined With Shenqifuzheng Injection on STAT1 gene knockdown cell lines, the The effect of cell proliferation.Results： Compared with the use of IFN-alpha and Shenqi Fuzheng injection alone, can inhibit the enhancement of IFN-alpha on the proliferation of human hepatocellular carcinoma SMMC-7721 cells combined with small dose of Shenqifuzheng injection and IFN-alpha, and STAT1 mRNA up-regulated expression and protein; STAT1 gene SMMC-7721 was knocked decreased expression of STAT1 protein and low cell lines; ＂SMMC-7721 for STAT1 gene knockdown cell lines, Shenqifuzheng injection compared with single IFN-collaborative IFN-alpha alpha and negative control group, no significant effect on cell proliferation. Conclusion： Shenqifuzheng injection through the expression of STAT1 in human hepatocellular carcinoma cells SMMC-7721, is one of the main mechanisms effectively enhance the inhibitory effect of interferon alpha on the proliferation of hepatoma cells.