抑制SOCS1联合OK-432刺激的DC疫苗抗肿瘤效应的初步研究

Antitumor Effects and Mechanisms of A Dendritic Cell Vaccine which Silenced SOCS1 by siRNA,Stimulated by OK-432 and Pulsed with Lysate of HepG2 Cells

背景与目的:细胞因子信号抑制因子(suppressor of cytokine signaling1,SOCS1)在肿瘤免疫负反馈调节中起重要作用,下调其表达可以有效增强机体的抗肿瘤免疫反应。本研究应用RNA干扰(RNA interference,RNAi)技术下调SOCS1基因表达联合OK-432刺激成熟的树突细胞(dendritic cell,DC)体外诱导特异性抗肿瘤作用,初步探讨其作用机制。方法:利用特异性小干扰RNA(siRNA)下调DC中SOCS1的表达,同时用肝癌HepG2细胞裂解物负载DC,OK-432刺激DC成熟,观察DC的形态特征,流式细胞仪检测刺激前后DC的表型变化,奥马蓝(AlamarBlue)法检测成熟DC对自身淋巴细胞的激活和增殖作用,LDH法检测其对HepG2、EC109细胞和K562细胞的杀伤作用。结果:DC体外诱导培养成功,设计的siRNA片段能有效下调iDC中SOCS1的表达,OK-432刺激DC成熟,CD80、CD83、CD86、HLA-DR等DC表面抗原明显表达上调,而负载肝癌抗原对DC表型无明显影响;SOCS1的下调可以促进DC成熟,负载肝癌抗原的DC能有效刺激自体淋巴细胞增殖,T细胞增殖率为(110.7±22.2)%,同时产生针对HepG2细胞的特异性杀伤作用,特异性细胞毒T淋巴细胞活性为(54.0±13.2)%。而针对EC109细胞和K562细胞的杀伤率仅为(10.0±30.7)%和(14.5±15.5)%。结论:RNAi下调SOCS1表达,OK432刺激负载肝癌全细胞抗原的成熟DC可以产生高效而特异性的抗肝癌的免疫应答。

BACKGROUND ＆ OBJECTIVE： Suppressor of cytokine signaling 1 （SOCS1） plays a critical role in antitumor immunity. Down- regulating SOCS1 in antigen-presenting dendritic cells （DCs） could enhance antigen-specific antitumor immunity. This study was to investigate the antigenspecific antitumor effect and mechanism of DCs with siRNA-mediated inhibition of SOCS1, stimulated by OK-432 and pulsed with hepatocellular carcinoma cell line HepG2 antigens. METHODS. The expression of SOCS1 in immature DCs was down-regulated by RNA interference （RNAi）. DCs were pulsed with lysate of HepG2 cells and stimulated with OK-432. The morphology of DCs was observed under converted phase microscopy. Phenotypic changes in cells after stimulation were characterized by flow cytometry （FCM）. The Alamar Blue assay was adopted to evaluate the activation and proliferation of autologous lymphocytes induced by mature DCs. The cytotoxicity of cytotoxic T lymphocytes （CTLs） elicited by modified DCs to HepG2, EC109 and K562 cells was tested by the .lactate dehydrogenase （LDH） assay. RESULTS, Cells displaying a typical morphology and phenotypic properties of mature DCs were obtained successfully. The expression of SOCSl in DCs was down-regulated by SOCSl RNAi. Mature DCs showed high expressions of CD80, CD83, CD86, and HLA-DR, Pulsing of DCs with lysate of HepG2 had no influence on the phenotypic properties of DCs. Down-regulating SOCS1 expression enhanced the maturation of DCs, The modified DC tumor vaccine stimulated the proliferation of autologous lymphocytes effectively, and the proliferation rate of T cells was （110.7±22.2）%. After being activated by modified DCs, TCLs exerted a specific and effective killing effect on HepG2 cells, but not on EC109 and K562 cells. CONCLUSION. Mature DCs could induce antigenspecific antitumor immunity against hepatocellular carcinoma after silencing of SOCSl by siRNA, stimulation by OK-432 and pulsing of DCs with HepG2 cell antigens.