SOCS1沉默的树突状细胞疫苗在喉癌治疗中的实验研究

Research of SOCS1 silent DC vaccine on laryngocarcinoma therapy

目的:研究SOCS1沉默的树突状细胞(DC)疫苗特异性抗肿瘤作用机制,并探讨RNAi技术在喉癌基因治疗中的应用前景,为DC的临床应用提供新思路。方法:以细胞因子GM-CSF、IL-4和TNF-α体外诱导扩增外周血单核细胞来源的DC,倒置显微镜下观察DC形态特征;构建RNAi载体转染DC,Western blot检测SOCS1的表达情况,筛选抑制SOCS1表达的有效靶序列;流式细胞术检测DC表面分子CD83、CD86和HLA-DR的表达;ELISA法分析上清中IFN-γ的含量;MTT法评估DC刺激T细胞增殖的能力及诱导细胞毒性T细胞(CTL)的杀伤活性。结果:DC体外诱导培养成功;设计的RNAi载体经测序验证无误。干扰序列5可显著下调SOCS1表达水平;SOCS1沉默联合喉癌Hep-2抗原致敏的DC可显著上调表面分子标志CD83(85.61±0.96)%、CD86(96.86±1.20)%和HLA-DR(98.02±0.94)%的表达;该组DC能有效刺激T细胞增殖,增加IFN-γ的分泌量,最终增强CTL的特异性杀伤作用,效靶比为50∶1时其杀伤活性与对照组之间的差异有统计学意义(P<0.01)。结论:SOCS1沉默并负载喉癌Hep-2抗原的DC疫苗可以产生高效而特异性的抗喉癌免疫应答。

Objective：Investigate the specific antitumor mechanism of SOCS1 silent DC vaccine and discuss the prospect of RNAi in the gene therapy for laryngocarcinoma in order to provide novel ideas of DC＇s clinical applications.Method：Dendritic cells derived from peripheral blood monocytes were cultured in vitro in the presence of GM-CSF,IL-4 and TNF-α.The morphological feature of DC was observed with inverted microscope.RNAi vector were transfected into DC.The expression of SOCS1 protein was detected with Western blot.The effective target sequences of siRNA against SOCS1 were screened out.The surface markers of mature DC,including CD83,CD86 and HLA-DR,were detected with flow cytometry.The concentration of IFN-γ in the supernatant was assayed by ELISA.The proliferative ability of T cell stimulated by DC and the specific killing activity of cytotoxic T lymphocyte（CTL） induced by DC were evaluated by MTT assay.Result：Dendritic cells were obtained successfully.The RNAi vector was proved to be right by sequencing.The expression of SOCS1 decreased significantly under the influence of the 5th interference sequence.SOCS1 silent dendritic cells which were loaded with Hep-2 antigen had high expressions of CD83（85.61±0.96）%,CD86（96.86±1.20）% and HLA-DR（98.02±0.94）%.It could also stimulate the proliferation of T cells effectively as well as could increase the production of IFN-γ,eventually enhanced the specific killing effect of CTL.The killing activity was more higher than that in control group when the effect cells and target cells were mixed up at the ratio of 50∶1（P0.01）.Conclusion：SOCS1 silent DC vaccines which were loaded with Hep-2 antigen could induce effective and specific anti-laryngocarcinoma immune responses.