抑制胰腺癌细胞中诱骗受体3表达对T淋巴细胞功能的影响

Small interfering RNA inhibit decoy receptor 3 gene in pancreatic cancer cell and its effect on the function of T lymphocyte in vitro

目的 观察胰腺癌Panc-1细胞中诱骗受体3（DcR3）基因沉默对T淋巴细胞功能的影响.方法 构建针对DcR3基因的小干扰RNA真核表达载体,脂质体LipofectamineTM 2000介导DcR3干扰质粒转染人胰腺癌Panc-1细胞,转染48 h后采用反转录-聚合酶链反应（RT-PCR）和酶联免疫吸附试验（ELISA）检测DcR3的表达变化;与淋巴细胞混合培养72 h后采用流式细胞术检测T细胞亚群的变化及Th1、Th2细胞的偏移,ELISA法检测干扰素（IFN）-γ和IL-4水平.结果 与空载体转染组比较,转染靶向DcR3的小干扰RNA（DcR3-siRNA） 48 h后的Panc-1细胞中DcR3 mRNA和蛋白表达下降（P＜0.05）;沉默DcR3的胰腺癌Panc-1细胞与淋巴细胞混合培养后CD3＋ CD4＋T细胞增多（P＜0.05）,而CD8＋T细胞无明显变化（P＞0.05）;Th1细胞增多[（7.87±1.07）％比（5.57±0.70）％,P＜0.05],Th2细胞细胞减少[（0.91±0.15）％比（2.83±0.21）％,P＜0.05];细胞上清液中Th1型细胞因子的IFN-γ浓度升高[（20.88±1.32） μg/L比（15.61±1.26） μg/L,P＜0.01],而Th2型细胞因子的IL-4水平下降[（3.27±0.34） μg/L比（5.31±0.37） μg/L,P＜0.01].结论 在胰腺癌细胞中靶向沉默DcR3的表达,可以有效恢复T淋巴细胞的免疫调节功能,纠正失衡的Th1/Th2细胞因子网络.

Objective To investigate the effect of T lymphocyte function by silencing decoy receptor 3 （DcR3） in human Pancreatic cancer cell line Panc-1.Methods Small interfering RNA （siRNA）eukaryotic expression vector targeting DcR3 gene was transfected into Panc-1 cells by Lipofectamine 2000.The expression of DcR3 was detected by using reverse transcriptase-polymerase chain reaction （RT-PCR） and enzyme linked immunosorbent assay （ELISA） after transfection 48 h.After mixed lymphocyte tumor cell cultured for 72 h,lymphocyte subsets were examined by flow cytometry analysis and the level of interferon （IFN）-γ,interleukin （IL）-4 was determined by ELISA.Results In the DcR3-siRNA transfection group,the expression of DcR3 was decreased in Panc-1 cells remarkably.After Panc-1 cells silencing DcR3 in mixed lymphocyte tumor cell cultured,the number of CD3 ＋,CD4 ＋ T lymphocyte was increased （P 〈 0.05） and the number of CD8 ＋ T lymphocyte was unchanged statistically （P 〉 0.05）.Additionally,the number of Th1 cells was increased [（7.87 ± 1.07） % vs.（5.57 ± 0.70） %,P 〈 0.05],while the number of Th2 cells was decreased [（0.91 ± 0.15） % vs.（2.83 ± 0.21） %,P 〈 0.05] as compared with negative control group （NC-siRNA）.Conclusion The silencing DcR3 may reverse immune regulating of T lymphocyte and Correct the imbalance of Th1/Th2.