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目的研究SOCS1沉默的树突状细胞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,为树突状细胞的临床应用提供新思路和理论依据。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时其杀伤活性显著高于对照组(P<0.01)。结论 SOCS1沉默并负载喉癌Hep-2抗原的DC可以产生高效而特异性的抗喉癌免疫应答。
Objective To study the specific antitumor mechanism of SOCS1-silenced dendritic cells and to explore the application prospect of RNAi in the gene therapy of laryngeal cancer, and to provide new ideas and theoretical basis for the clinical application of dendritic cells. Methods DCs derived from peripheral blood mononuclear cells were induced by cytokines GM-CSF, IL-4 and TNF-α in vitro. The morphology of DCs was observed under inverted microscope. The expression of SOCS1 was detected by Western blot , And the effective target sequences that inhibited the expression of SOCS1 were screened. The expression of CD83, CD86 and HLA-DR were detected by flow cytometry. The content of IFN-γ in the supernatant was analyzed by ELISA. The ability of DCs to stimulate the proliferation of T cells was assessed by MTT assay And induce cytotoxic T cell killing activity. Results The DCs were successfully induced in vitro. The designed RNAi vectors were verified by sequencing. Interference sequence 5 significantly down-regulated SOCS1 expression; SOCS1 silencing combined with laryngeal cancer Hep-2 antigen-sensitized DC significantly upregulated the expression of surface markers CD83 (85.61 ± 0.96)%, CD86 (96.86 ± 1.20)% and HLA-DR ± 0.94)%. The DCs can effectively stimulate the proliferation of T cells and increase the secretion of IFN-γ, and finally enhance the specific cytotoxicity of CTLs. The cytotoxicity of CTLs was significantly higher than that of the control group (P <0.01). Conclusion DCs with SOCS1 silencing and loaded with Hep-2 antigen of laryngeal squamous cell carcinoma can produce efficient and specific anti-laryngeal cancer immune response.