脂质体辅助递送CTLA-4 siRNA通过激活系统性抗肿瘤免疫反应抑制肾细胞癌生长

目的:探索携带CTLA-4 siRNA的适配子偶联脂质体颗粒是否可以激活肿瘤部位的抗肿瘤免疫反应，抑制肾细胞癌的生长。方法:采用薄膜水化法制备脂质体；使用透射电子显微镜观察脂质体的形态和结构；用Zetasizer测量Zeta电位；共孵育实验观察靶细胞对Lipo-siRNA的摄取；qPCR检测Lipo-siRNA对CTLA-4基因的沉默；小鼠移植瘤模型检测Lipo-siRNA的体内抑瘤能力；流式细胞术检测肿瘤浸润T细胞的激活状态；免疫荧光法检测肿瘤浸润T细胞的数目。结果:成功制备Lipo-siRNA，电镜结果显示其具有双层球状结构；Zetasizer测得其Zeta电位为(+20.53±2.66)mV；荧光显微镜观察结果表明Lipo-siRNA可以被靶细胞有效摄取，qPCR检测Lipo-siRNA可以显著降低CTLA-4基因的表达（P＜0.001）；小鼠移植瘤模型显示Lipo-siRNA较对照组而言可以显著抑制肿瘤生长（P＜0.001），降低肿瘤细胞中CTLA-4的表达（P＜0.001），提升肿瘤浸润T细胞的数量（P＜0.000 1），并且提高了肿瘤浸润T细胞中IL-2（P＜0.000 1）和IFN-γ（P＜0.000 1）的表达水平。结论:适配子偶联脂质体可以携带CTLA-4 siRNA靶向肿瘤细胞，激活肿瘤部位的抗肿瘤免疫反应，抑制肿瘤的生长，对肾细胞癌的治疗具有潜在的临床应用价值。

Liposome assisted delivery of CTLA-4 siRNA inhibits the growth of renal cell carcinoma by activating systemic anti-tumor immune response

Objective:To explore whether aptamer-coupled liposome particles carrying CTLA-4 siRNA can activate anti-tumor immune response and inhibit the growth of renal cell carcinoma.Methods:Liposomes were prepared by thin film hydration method.The morphology and structure of liposomes were observed by transmission electron microscope.Zeta potential was measured by Zetasizer.The uptake of Lipo-siRNA by target cells was observed in co-incubation experiment.The silencing of Lipo-siRNA to CTLA-4 gene was detected by qPCR.The anti-tumor ability of Lipo-siRNA in vivo was detected by mouse transplanted tumor model.The activation of tumor infiltrating T cells was detected by flow cytometry,and the number of tumor infiltrating T cells was detected by immunofluorescence.Results:The Lipo-siRNA was successfully prepared.It had a double-layer spherical structure,and its Zeta potential was (+20.53±2.66)mV measured by Zetasizer.The results of fluorescence microscope showed that Lipo-siRNA could be effectively absorbed by target cells,and qPCR detection revealed that Lipo-siRNA could significantly reduce the expression of CTLA-4 gene（P＜0.001）.Compared with the control group,mouse transplanted tumor model showed that Lipo-siRNA could significantly inhibit tumor growth（P＜0.001）,reduce the expression of CTLA-4 in tumor cells（P＜0.001）,increase the number of tumor infiltrating T cells（P＜0.000 1）,and increase the expression levels of IL-2（P＜0.000 1） and IFN-γ（P＜0.000 1） in tumor infiltrating T cells.Conclusion:Aptamer-coupled liposomes can carry CTLA-4 siRNA to target tumor cells,activate anti-tumor immune response at tumor site and inhibit tumor growth,which has potential clinical value in the treatment of renal cell carcinoma.