新型MyD88抑制剂作用机制及其在防治结肠炎相关性结直肠癌中的作用及机理研究

发布时间：2018-05-18 23:25

本文选题：TJ-M2010-5 + MyD88　；参考：《华中科技大学》2016年博士论文

【摘要】：目的探讨新型MyD88抑制剂TJ-M2010-5抑制MyD88同源二聚化和异源二聚化的作用并阐明其机理,为计算机辅助药物设计提供实验依据。方法体外构建Flag-MyD88 pcDNA3.1-, HA-MyD88 pcDNA3.1-和Flag-TIRAP pcDNA3.1-质粒,采用免疫共沉淀方法,将Flag-MyD88 pcDNA3.1-和HA-MyD88 pcDNA3.1-共转染HEK293T细胞,以及Flag-TIRAP pcDNA3.1-和HA-MyD88 pcDNA3.1-共转染HEK293T细胞,并给与不同浓度TJ-M2010-5干预(0μM,10μM,40μM),转染48小时后检测MyD88-MyD88以及TIRAP-MyD88的结合能力。体外诱导培养BALB/c小鼠不成熟骨髓源性树突状细胞,培养第7天给予不同浓度TJ-M2010-5干预(0μM,10μM,40μM)2小时后,给予LPS刺激活化TLR信号通路,48小时后,通过EMSA方法检测细胞核内转录因子NF-κB的含量,ELISA方法检测细胞培养上清中TNF-α和IL-1β表达水平。结果TJ-M2010-5剂量依赖性的抑制MyD88-MyD88和TIRAP-MyD88结合能力,随着剂量增加,抑制作用加强。体外诱导培养BALB/c小鼠,给予不同浓度TJ-M2010-5干预和LPS刺激后,TJ-M2010-5抑制NF-κB入核,以及TNF-α和IL-1β的表达水平,并且随着TJ-M2010-5浓度增加,抑制作用逐渐加强。结论TJ-M2010-5通过干扰MyD88-MyD88同源二聚化和TIRAP-MyD88异源二聚化从而发挥了抑制MyD88二聚化的作用,同时,TJ-M2010-5进一步抑制了TLR/MyD88信号通路的活化。因此,TJ-M2010-5可以作为一种有效的MyD88抑制剂干预依赖MyD88的TLR信号通路,为将来应用于临床奠定理论基础。目的探讨新型MyD88抑制剂TJ-M2010-5在防治AOM/DSS诱发小鼠CAC中的作用及机制,为TJ-M2010-5进一步临床应用提供实验依据。方法通过联合使用AOM和DSS诱导小鼠CAC模型,并给予TJ-M2010-5干预。小鼠腹腔注射大剂量TJ-M2010-5后,通过观察小鼠活动度等指标以评估药物急性毒性：小鼠腹腔注射常规剂量TJ-M2010-5后,通过检测小鼠血常规指标,肝脏、肾脏HE染色以评估药物亚急性毒性。实验分为三组：Normal组,AOM/DSS组,TJ-M2010-5组。观察10周,记录建模后小鼠体重变化,生存率差异。体外培养RAW264.7细胞,TJ-M2010-5干预后给予LPS刺激,检测细胞内IRAK4、p38、Erk磷酸化水平,同时,通过EMSA方法检测结肠组织中NF-κB入核能力,以评估TJ-M2010-5对CAC小鼠TLR/MyD88信号通路的影响。大体观察小鼠结肠肿瘤形成情况并通过HE染色分析肿瘤恶性程度,COX2免疫组织化学染色和Western blot分析评价肿瘤进展情况。结肠HE染色检查评估炎症反应程度,MPO免疫组织化学染色检测中性粒细胞浸润情况。BrdU掺入法和Ki-67染色检查结肠上皮细胞增殖状况,活性caspase-3染色和TUNEL染色检查结肠上皮细胞凋亡状况。Bio-Plex和ELISA方法分析CAC小鼠血清细胞因子蛋白表达水平,qPCR方法分析结肠细胞因子mRNA水平。免疫组织化学染色方法检测结肠CD68+巨噬细胞浸润情况,流式细胞技术分析结肠LPMC中巨噬细胞、DC、CD4+T细胞比例,qPCR方法分析LPMC中巨噬细胞IL-6 mRNA水平。结果联合使用AOM和DSS成功诱发了小鼠CAC模型。药物安全性评估显示TJ-M2010-5不会对小鼠产生急性和亚急性毒性作用。TJ-M2010-5抑制了CAC小鼠TLR/MyD88信号通路活性。与AOM/DSS组相比,长期给予TJ-M2010-5干预后,小鼠死亡率从53%降低至0%,恶性肿瘤发生率从100%降至0%,并进一步抑制了CAC小鼠体重下降的趋势,降低了结肠上皮细胞增殖能力并促进了上皮细胞凋亡,抑制了结肠肿瘤的发生发展并降低了肿瘤恶性程度。同时,TJ-M2010-5干预抑制了细胞因子的分泌以及结肠组织中性粒细胞和免疫细胞的浸润,调节了结肠组织炎性微环境。结论异常的TLR/MyD88信号通路活化诱导结肠组织中促肿瘤发生的炎性微环境形成,给予TJ-M2010-5干预后,通过调节炎性微环境并抑制免疫细胞的浸润,遏制了肿瘤微环境的形成,从而起到防治CAC发生和进展的作用。MyD88抑制剂TJ-M2010-5发挥了强大的抗击CAC发生和进展的效果,具有较好的临床应用价值,将为临床上治疗结肠炎或CAC患者提供更多的选择。
[Abstract]:Objective to investigate the effect of new MyD88 inhibitor TJ-M2010-5 on MyD88 homologous dimerization and heterogenous dimerization and elucidate its mechanism and provide experimental basis for computer aided drug design. Methods Flag-MyD88 pcDNA3.1-, HA-MyD88 pcDNA3.1- and Flag-TIRAP pcDNA3.1- plasmids were constructed in vitro, and Flag-MyD88 pcDNA3 was adopted by immunoprecipitation method. .1- and HA-MyD88 pcDNA3.1- co transfected HEK293T cells, as well as Flag-TIRAP pcDNA3.1- and HA-MyD88 pcDNA3.1- co transfected HEK293T cells, and gave different concentrations of TJ-M2010-5 intervention (0 mu M, 10 micron M, 40 micron). After 48 hours transfection, it was detected and the ability of binding. In vitro induction and culture of immature bone marrow derived dendrites of mice. After seventh days of culture, different concentrations of TJ-M2010-5 intervention (0 M, 10 u M, 40 M) were given for 2 hours, and LPS stimulation activated TLR signaling pathway. After 48 hours, the content of NF- kappa B was detected by EMSA method. TNF- alpha and IL-1 beta expression in cell culture supernatant were detected by ELISA method. Inhibition of the binding ability of MyD88-MyD88 and TIRAP-MyD88, with the increase of dose, the inhibitory effect was strengthened. BALB/c mice were induced and cultured in vitro. After different concentrations of TJ-M2010-5 intervention and LPS stimulation, TJ-M2010-5 inhibited the nucleation of NF- kappa B, as well as the expression level of TNF- alpha and IL-1 beta, and the inhibitory effect gradually strengthened with the increase of TJ-M2010-5 concentration. Conclusion TJ-M 2010-5 by interfering with MyD88-MyD88 homologous dimerization and TIRAP-MyD88 heterologous dimerization, the inhibition of MyD88 dimerization is played, and TJ-M2010-5 further inhibits the activation of the TLR/MyD88 signaling pathway. Therefore, TJ-M2010-5 can be used as an effective MyD88 inhibitor to interfere with the TLR signaling pathway of MyD88 and to be applied in the future for future applications. The bed lays a theoretical foundation. Objective to explore the role and mechanism of a new MyD88 inhibitor TJ-M2010-5 in the prevention and control of AOM/DSS induced mouse CAC, and to provide experimental basis for further clinical application of TJ-M2010-5. Methods the mouse CAC model was induced by combined use of AOM and DSS, and TJ-M2010-5 intervention was given. After intraperitoneal injection of a large dose of TJ-M2010-5, the mice passed through the peritoneal injection of a large dose of TJ-M2010-5. Observe the activity of mice and other indexes to assess the acute toxicity of drugs: after intraperitoneal injection of conventional dose of TJ-M2010-5 in mice, the subacute toxicity of the drug was evaluated by detecting the blood routine index, liver and kidney HE staining in mice. The experiment was divided into three groups: group Normal, group AOM/DSS, TJ-M2010-5 group. The body weight changes and survival of the mice after modeling were recorded. Rate difference. In vitro culture of RAW264.7 cells, TJ-M2010-5 stem prognosis was given to LPS stimulation to detect the level of IRAK4, p38, Erk phosphorylation in cells. Meanwhile, the EMSA method was used to detect the NF- kappa B nucleation ability in colon tissue, in order to evaluate the effect of TJ-M2010-5 on TLR/MyD88 signal pathway in CAC mice. Color analysis of tumor malignancy, COX2 immunohistochemical staining and Western blot analysis to evaluate tumor progression. Colonic HE staining was used to assess the degree of inflammatory response. MPO immunohistochemical staining was used to detect neutrophils infiltration,.BrdU incorporation and Ki-67 staining were used to examine the proliferation of upper colonic cells, reactive caspase-3 staining and TUN EL staining was used to detect the apoptosis of colonic epithelial cells,.Bio-Plex and ELISA methods were used to analyze the level of cytokine protein expression in serum of CAC mice. QPCR method was used to analyze the level of colonic cytokine mRNA. Immunohistochemical staining was used to detect the infiltration of colon CD68+ macrophages. Flow cytometry was used to analyze macrophages in colon LPMC, DC, CD4+T fine. Cell ratio, qPCR method was used to analyze the IL-6 mRNA level of macrophages in LPMC. Results combined with AOM and DSS, the mouse CAC model was successfully induced. The drug safety assessment showed that TJ-M2010-5 did not produce acute and subacute toxicity to mice..TJ-M2010-5 inhibited the TLR/MyD88 signaling activity of CAC mice. Compared with the AOM/DSS group, it was given a long time. After M2010-5, the death rate of mice decreased from 53% to 0%, and the incidence of malignant tumor decreased from 100% to 0%. It further inhibited the trend of weight loss in CAC mice, reduced the proliferation of colonic epithelial cells and promoted the apoptosis of epithelial cells, inhibited the development of colonic tumor and reduced the malignancy of the tumor. At the same time, TJ-M2010-5 intervention was used. Inhibiting the secretion of cytokines and the infiltration of neutrophils and immune cells in colon tissue and regulating the inflammatory microenvironment of colon tissue. Conclusion abnormal TLR/MyD88 signaling pathway is activated to induce the formation of inflammatory microenvironment in colonic tissue, and after the intervention of TJ-M2010-5, the inflammatory microenvironment is regulated and the immunization is inhibited. Cell infiltration, which contain the formation of tumor microenvironment, and thus play a role in preventing and controlling the occurrence and progress of CAC,.MyD88 inhibitor TJ-M2010-5 exerts a strong effect against the occurrence and progress of CAC. It has good clinical application value and will provide more choice for clinical treatment of colitis or CAC patients.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R574.62;R735.34

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