本文選題：TJ-M2010-5 + MyD88　； 參考：《華中科技大學(xué)》2016年博士論文

【摘要】：目的探討新型MyD88抑制劑TJ-M2010-5抑制MyD88同源二聚化和異源二聚化的作用并闡明其機(jī)理,為計(jì)算機(jī)輔助藥物設(shè)計(jì)提供實(shí)驗(yàn)依據(jù)。方法體外構(gòu)建Flag-MyD88 pcDNA3.1-, HA-MyD88 pcDNA3.1-和Flag-TIRAP pcDNA3.1-質(zhì)粒,采用免疫共沉淀方法,將Flag-MyD88 pcDNA3.1-和HA-MyD88 pcDNA3.1-共轉(zhuǎn)染HEK293T細(xì)胞,以及Flag-TIRAP pcDNA3.1-和HA-MyD88 pcDNA3.1-共轉(zhuǎn)染HEK293T細(xì)胞,并給與不同濃度TJ-M2010-5干預(yù)(0μM,10μM,40μM),轉(zhuǎn)染48小時(shí)后檢測MyD88-MyD88以及TIRAP-MyD88的結(jié)合能力。體外誘導(dǎo)培養(yǎng)BALB/c小鼠不成熟骨髓源性樹突狀細(xì)胞,培養(yǎng)第7天給予不同濃度TJ-M2010-5干預(yù)(0μM,10μM,40μM)2小時(shí)后,給予LPS刺激活化TLR信號通路,48小時(shí)后,通過EMSA方法檢測細(xì)胞核內(nèi)轉(zhuǎn)錄因子NF-κB的含量,ELISA方法檢測細(xì)胞培養(yǎng)上清中TNF-α和IL-1β表達(dá)水平。結(jié)果TJ-M2010-5劑量依賴性的抑制MyD88-MyD88和TIRAP-MyD88結(jié)合能力,隨著劑量增加,抑制作用加強(qiáng)。體外誘導(dǎo)培養(yǎng)BALB/c小鼠,給予不同濃度TJ-M2010-5干預(yù)和LPS刺激后,TJ-M2010-5抑制NF-κB入核,以及TNF-α和IL-1β的表達(dá)水平,并且隨著TJ-M2010-5濃度增加,抑制作用逐漸加強(qiáng)。結(jié)論TJ-M2010-5通過干擾MyD88-MyD88同源二聚化和TIRAP-MyD88異源二聚化從而發(fā)揮了抑制MyD88二聚化的作用,同時(shí),TJ-M2010-5進(jìn)一步抑制了TLR/MyD88信號通路的活化。因此,TJ-M2010-5可以作為一種有效的MyD88抑制劑干預(yù)依賴MyD88的TLR信號通路,為將來應(yīng)用于臨床奠定理論基礎(chǔ)。目的探討新型MyD88抑制劑TJ-M2010-5在防治AOM/DSS誘發(fā)小鼠CAC中的作用及機(jī)制,為TJ-M2010-5進(jìn)一步臨床應(yīng)用提供實(shí)驗(yàn)依據(jù)。方法通過聯(lián)合使用AOM和DSS誘導(dǎo)小鼠CAC模型,并給予TJ-M2010-5干預(yù)。小鼠腹腔注射大劑量TJ-M2010-5后,通過觀察小鼠活動(dòng)度等指標(biāo)以評估藥物急性毒性：小鼠腹腔注射常規(guī)劑量TJ-M2010-5后,通過檢測小鼠血常規(guī)指標(biāo),肝臟、腎臟HE染色以評估藥物亞急性毒性。實(shí)驗(yàn)分為三組：Normal組,AOM/DSS組,TJ-M2010-5組。觀察10周,記錄建模后小鼠體重變化,生存率差異。體外培養(yǎng)RAW264.7細(xì)胞,TJ-M2010-5干預(yù)后給予LPS刺激,檢測細(xì)胞內(nèi)IRAK4、p38、Erk磷酸化水平,同時(shí),通過EMSA方法檢測結(jié)腸組織中NF-κB入核能力,以評估TJ-M2010-5對CAC小鼠TLR/MyD88信號通路的影響。大體觀察小鼠結(jié)腸腫瘤形成情況并通過HE染色分析腫瘤惡性程度,COX2免疫組織化學(xué)染色和Western blot分析評價(jià)腫瘤進(jìn)展情況。結(jié)腸HE染色檢查評估炎癥反應(yīng)程度,MPO免疫組織化學(xué)染色檢測中性粒細(xì)胞浸潤情況。BrdU摻入法和Ki-67染色檢查結(jié)腸上皮細(xì)胞增殖狀況,活性caspase-3染色和TUNEL染色檢查結(jié)腸上皮細(xì)胞凋亡狀況。Bio-Plex和ELISA方法分析CAC小鼠血清細(xì)胞因子蛋白表達(dá)水平,qPCR方法分析結(jié)腸細(xì)胞因子mRNA水平。免疫組織化學(xué)染色方法檢測結(jié)腸CD68+巨噬細(xì)胞浸潤情況,流式細(xì)胞技術(shù)分析結(jié)腸LPMC中巨噬細(xì)胞、DC、CD4+T細(xì)胞比例,qPCR方法分析LPMC中巨噬細(xì)胞IL-6 mRNA水平。結(jié)果聯(lián)合使用AOM和DSS成功誘發(fā)了小鼠CAC模型。藥物安全性評估顯示TJ-M2010-5不會(huì)對小鼠產(chǎn)生急性和亞急性毒性作用。TJ-M2010-5抑制了CAC小鼠TLR/MyD88信號通路活性。與AOM/DSS組相比,長期給予TJ-M2010-5干預(yù)后,小鼠死亡率從53%降低至0%,惡性腫瘤發(fā)生率從100%降至0%,并進(jìn)一步抑制了CAC小鼠體重下降的趨勢,降低了結(jié)腸上皮細(xì)胞增殖能力并促進(jìn)了上皮細(xì)胞凋亡,抑制了結(jié)腸腫瘤的發(fā)生發(fā)展并降低了腫瘤惡性程度。同時(shí),TJ-M2010-5干預(yù)抑制了細(xì)胞因子的分泌以及結(jié)腸組織中性粒細(xì)胞和免疫細(xì)胞的浸潤,調(diào)節(jié)了結(jié)腸組織炎性微環(huán)境。結(jié)論異常的TLR/MyD88信號通路活化誘導(dǎo)結(jié)腸組織中促腫瘤發(fā)生的炎性微環(huán)境形成,給予TJ-M2010-5干預(yù)后,通過調(diào)節(jié)炎性微環(huán)境并抑制免疫細(xì)胞的浸潤,遏制了腫瘤微環(huán)境的形成,從而起到防治CAC發(fā)生和進(jìn)展的作用。MyD88抑制劑TJ-M2010-5發(fā)揮了強(qiáng)大的抗擊CAC發(fā)生和進(jìn)展的效果,具有較好的臨床應(yīng)用價(jià)值,將為臨床上治療結(jié)腸炎或CAC患者提供更多的選擇。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R574.62;R735.34

【相似文獻(xiàn)】

相關(guān)期刊論文 前9條

1 魏妙妙;楊卓;;Toll樣受體2、4及其轉(zhuǎn)導(dǎo)分子MyD88和炎性復(fù)合體在腎小管間質(zhì)性腎病中的重要作用[J];天津醫(yī)藥;2012年06期

2 高輝;張海玉;單玉興;;電離輻射對小鼠腹腔巨噬細(xì)胞TLR4和轉(zhuǎn)接蛋白MyD88表達(dá)的影響[J];吉林大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2009年01期

3 牟慧;蔡輝;姚茹冰;趙智明;;甲氨喋呤對佐劑性關(guān)節(jié)炎大鼠滑膜髓樣分化因子MyD88表達(dá)的影響[J];安徽醫(yī)藥;2013年05期

4 姜華;姜玉姬;;阿托伐他汀對人臍靜脈內(nèi)皮細(xì)胞TLR4及下游MyD88依賴性信號轉(zhuǎn)導(dǎo)通路的影響[J];重慶醫(yī)學(xué);2012年11期

5 楊綺紅;舒建昌;鄧延梅;呂霞;傅妹伢;宋慧東;張曉燕;;姜黃素抑制肝星狀細(xì)胞MyD88及信號通路細(xì)胞因子表達(dá)的研究[J];胃腸病學(xué)和肝病學(xué)雜志;2014年08期

6 陳建國;蘇兆亮;柳迎昭;王勝軍;黃新祥;邵啟祥;許化溪;;MyD88-銅綠假單胞菌表位核酸疫苗的構(gòu)建及其真核表達(dá)[J];細(xì)胞與分子免疫學(xué)雜志;2009年03期

7 張夢澤;葛春林;張雨京;;MyD88在重癥急性胰腺炎胰腺組織中表達(dá)及意義的實(shí)驗(yàn)研究[J];中國普外基礎(chǔ)與臨床雜志;2014年03期

8 呂游;仇麗鴻;;口腔致病菌脂多糖誘導(dǎo)的MyD88依賴性信號轉(zhuǎn)導(dǎo)途徑相關(guān)機(jī)制研究進(jìn)展[J];中國實(shí)用口腔科雜志;2011年12期

9 ;[J];;年期

相關(guān)會(huì)議論文 前1條

1 張利民;周平;;一種新型MyD88抑制劑作用功能的探討[A];2013中國器官移植大會(huì)論文匯編[C];2013年

相關(guān)博士學(xué)位論文 前2條

1 張利民;新型MyD88抑制劑作用機(jī)制及其在防治結(jié)腸炎相關(guān)性結(jié)直腸癌中的作用及機(jī)理研究[D];華中科技大學(xué);2016年

2 辛光大;非酒精性脂肪性肝炎性別間差異與MyD88依賴性IL-6信號通路的關(guān)系[D];吉林大學(xué);2015年

相關(guān)碩士學(xué)位論文 前3條

1 楊海燕;TLR4及MyD88在急性閉角型青光眼患者虹膜中的表達(dá)[D];吉林大學(xué);2015年

2 田堯夫;家兔MyD88基因多態(tài)性和表達(dá)量與腸炎易感性的關(guān)聯(lián)分析[D];四川農(nóng)業(yè)大學(xué);2012年

3 宋鵬;納米載體介導(dǎo)抑制大鼠肝臟MyD88基因表達(dá)的實(shí)驗(yàn)研究[D];天津醫(yī)科大學(xué);2013年

，

本文編號：1907682