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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: To study the specific antitumor mechanism of SOCS1-dendritic cells (DC) vaccine and to explore the application prospect of RNAi in gene therapy of laryngeal cancer, and to provide new ideas for the clinical application of DC. 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 , The effective target sequence that inhibited the expression of SOCS1 was screened, the expression of CD83, CD86 and HLA-DR on DC surface were detected by flow cytometry, the content of IFN-γ in supernatant was analyzed by ELISA, and the proliferation of DCs stimulated by DC was evaluated by MTT Ability and cytotoxic T lymphocyte (CTL) killing activity. Results: DC was successfully induced in vitro. The designed RNAi vector was 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)%. This group of DCs can effectively stimulate the proliferation of T cells, increase the secretion of IFN-γ, and finally enhance the specific cytotoxicity of CTL. When the target ratio is 50:1, The difference was statistically significant (P <0.01). CONCLUSIONS: DC vaccines with SOCS1 silencing and loaded with laryngeal cancer Hep-2 antigen can produce efficient and specific anti-laryngeal cancer immune response.