【作者】 毛成全；

【导师】 邢飞跃；

【作者基本信息】 暨南大学 ， 免疫学， 2010， 硕士

【摘要】 目的：初步探讨靶向Hes-1 siRNA干扰对Th17细胞分化的影响。方法：分离小鼠淋巴结细胞,Trizol法提取总RNA, RT-PCR技术检测不同时间Jagged-1蛋白和γ分泌酶抑制剂DAPT (The y-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylg lycinet butyl ester, DAPT)对外周T淋巴细胞Hes-1和Deltex-1 mRNA表达的影响；WesternBlot分析不同时间和不同浓度Hes-1 siRNA干扰对外周T淋巴细胞Hes-1蛋白表达的影响；Elisa检测靶向Hes-1 siRNA干扰对外周T淋巴细胞IL-17细胞因子分泌的影响；荧光标记单克隆抗体染色技术结合流式细胞仪检测靶向Hes-1 siRNA干扰后外周CD4+T淋巴细胞及其胞内IL-17和IFN-γ的变化。结果：Jagged-1能促进Hes-1 mRNA的表达,以第3d的作用最为明显,第5d作用有所减弱；DAPT组Hes-1 mRNA的表达明显低于空白对照组。Jagged-1组Deltex-1 mRNA表达明显升高,第2d达到高峰；DAPT作用1d后Deltex-1 mRNA的表达低于空白对照组,第2d开始升高,第3d高于空白对照组。Jagged-1和Hes-1siRNA作用12h,Hes-1蛋白表达无明显变化；Jagged-1作用24h,Hes-1蛋白表达开始升高,36h达到高峰,持续至48h，72h后Jagged-1组Hes-1蛋白表达下降。Hes-1 siRNA作用24h后Hes-1蛋白表达下降,在36h降至最低,一直持续至72h；作用36h,100 nmol／LHes-1 siRNA能明显降低Hes-1蛋白表达,与200 nmol／L、250 nmol／LHes-1 siRNA组相比无明显差异。流式分析显示,Jagged-1组CD4+T淋巴细胞胞内IL-17表达明显降低,而Hes-1 siRNA则促进外周CD4+T淋巴细胞胞内IL-17表达。结论：Jagged-1能激活Notch信号通路,抑制外周CD4+T淋巴细胞分泌IL-17。靶向Hes-1 siRNA能下调Jagged-1-Notch信号,促进外周CD4+T淋巴细胞向Th17细胞分化。更多还原

【Abstract】 Objective:To investigate effect of target Hes-1 siRNA on the differentiation of Th17 cells. Methods:Cell suspensions were prepared from mouse lymph nodes, total RNA was extracted by Trizol. The expression of Hes-1 mRNA and Deltex-1 mRNA of peripheral T lymphocytes was checked by RT-PCR method. The expression of protein Hes-1 was checked by Western Blot. The expression of IL-17 of peripheral T lymphocytes was checked by Elisa method. The expression level of intracellular IL-17 and IFN-y in peripheral CD4+T lymphocytes was evaluated by fluorescin-conjugated monoclonal antibody double labeling technique. Results: Jagged-1 can promote the expression of Hes-1 mRNA, and the role of the third day was the most obviously. Its role was reduced on the fifth day. The expression of Hes-1 mRNA in DAPT group was significantly lower than the control group. The expression of Deltex-1 mRNA in Jagged-1 group was significantly increased, peaked at the second day. On the first day, the expression of Deltex-1 mRNA in DAPT group was lower than the control group, The second day began to increase. Hes-1 protein was not affected by Jagged-1 and Hes-1 siRNA effect 12 h. The expression of Hes-1 protein began to increase in Jagged-1 group after 24 h, and it reached peak at 36 h. The expression of Hes-1 protein was reduced after 72 h. The expression of Hes-1 protein decreased to a minimum at 36 h and continued to 48h in siRNA groups.100 nmol/L Hes-1 siRNA could significantly decreased the expression of Hes-1 protein, and had nothing different with 200 nmol/L,250 nmol/L Hes-1 siRNA groups. Flow results showed, Hes-1 siRNA promoted the expression of IL-17 in peripheral CD4+T lymphocytes. Jagged-1 decreased the level of IL-17 in peripheral CD4+T lymphocytes. Conclusions:Jagged-1 activated the Notch pathway, and decreased the expression of IL-17 secreted by peripheral CD4+T lymphocytes. Hes-1 siRNA interfered Notch pathway and promoted peripheral CD4+T lymphocytes to Th17 cells.更多还原