【摘要】：目的觀察腦內(nèi)注射脂多糖(LPS)引起的腦內(nèi)M1型小膠質(zhì)細(xì)胞極化對(duì)實(shí)驗(yàn)動(dòng)物焦慮樣行為的影響,并試圖通過Myd88抑制劑MIP逆轉(zhuǎn)LPS帶來(lái)的炎癥反應(yīng)。探究實(shí)驗(yàn)動(dòng)物焦慮樣行為的改變與中樞小膠質(zhì)細(xì)胞極化的關(guān)系。材料與方法本實(shí)驗(yàn)將C57雄性小鼠隨機(jī)分為4組,第一天每?jī)山M分別單側(cè)側(cè)腦室注射Myd88抑制劑(MIP)和Myd88對(duì)照肽(CP),第二天第一組再次注射生理鹽水、第二組注射LPS、第三組注射生理鹽水、第四組注射LPS,即四個(gè)實(shí)驗(yàn)組分別為CP-Con、MIP-Con、CP-LPS、MIP-LPS。最后一次腦內(nèi)注射48 h后觀察實(shí)驗(yàn)動(dòng)物焦慮樣行為的改變,用免疫熒光染色觀察內(nèi)側(cè)前額葉皮質(zhì)和海馬區(qū)i NOS、Arg-1的表達(dá),用Western Blot的方法檢測(cè)內(nèi)側(cè)前額葉皮質(zhì)和海馬區(qū)IL-1β、TNF-α、iNOS、Arg-1、Myd88、P-IRF3等分子的表達(dá)。結(jié)果1.模型建立后行為學(xué)的改變模型建立48 h后,曠場(chǎng)實(shí)驗(yàn)顯示CP-LPS組相較于其他三組小鼠在中央活動(dòng)的距離最短,中央活動(dòng)的時(shí)間最少,這提示CP-LPS組相較于其他三組小鼠表現(xiàn)出更明顯的焦慮樣行為;MIP-LPS組的中央活動(dòng)距離和時(shí)間顯著高于CP-LPS,這提示MIP能夠逆轉(zhuǎn)LPS引起的焦慮樣行為。高架十字迷宮實(shí)驗(yàn)中CP-LPS組相較于其他三組小鼠在開臂活動(dòng)的時(shí)間最少,活動(dòng)的距離最短,這提示CP-LPS相較于其他三組小鼠表現(xiàn)出更明顯的焦慮樣行為;MIP-LPS組在開臂活動(dòng)的時(shí)間和距離顯著高于CP-LPS,這再次提示MIP能夠逆轉(zhuǎn)LPS引起的焦慮樣行為。2.不同處理組實(shí)驗(yàn)動(dòng)物的海馬和內(nèi)側(cè)前額葉皮質(zhì)小膠質(zhì)細(xì)胞的活化情況免疫熒光結(jié)果顯示,在與焦慮樣情緒相關(guān)的腦區(qū),包括海馬的DG、CA1、CA2區(qū)及m PFC內(nèi)CP-LPS組有更多的M1型小膠質(zhì)細(xì)胞的激活,而MIP-Con組和MIP-LPS組有更多的M2型小膠質(zhì)細(xì)胞激活。3.不同處理組實(shí)驗(yàn)動(dòng)物的關(guān)鍵腦區(qū)的重要分子蛋白表達(dá)量的變化Western Blot的結(jié)果顯示,在與焦慮樣情緒相關(guān)的腦區(qū)包括:海馬的DG、CA1、CA2區(qū)及mPFC內(nèi)CP-LPS組有更多的M1小膠質(zhì)細(xì)胞蛋白標(biāo)記物iNOS的表達(dá),而MIP-Con和MIP-LPS組有更多的M2型小膠質(zhì)細(xì)胞蛋白標(biāo)記物Arg-1的表達(dá)。CP-LPS組有更多的促炎因子IL-1β、TNF-α的表達(dá)。MIP-Con組和MIP-LPS組Myd88非依賴通路關(guān)鍵分子P-IRF3的表達(dá)顯著增高。結(jié)論LPS能夠誘導(dǎo)小膠質(zhì)細(xì)胞向M1型極化,并且可能引起實(shí)驗(yàn)動(dòng)物的焦慮樣行為。MIP可以逆轉(zhuǎn)LPS的作用,緩解LPS引發(fā)的實(shí)驗(yàn)動(dòng)物的焦慮樣行為。這種調(diào)控可能通過Myd88非依賴通路實(shí)現(xiàn)且與小膠質(zhì)細(xì)胞向M2型極化密切相關(guān)。
[Abstract]:Objective to observe the effect of intrabrain injection of lipopolysaccharide (LPS) on the anxiety like behavior of M1 microglia in rats and to reverse the inflammatory response induced by LPS by Myd88 inhibitor MIP. To explore the relationship between the changes of anxiety-like behavior and the polarization of central microglia in experimental animals. Materials and methods the C57 male mice were randomly divided into 4 groups. On the first day, each group was given intraventricular injection of Myd88 inhibitor (MIP) and Myd88 control peptide (CP),. The first group was injected with normal saline again on the second day, and the second group was injected with LPS,. The third group was injected with normal saline, the fourth group was injected with LPS, and the four experimental groups were treated with CP-Con,MIP-Con,CP-LPS,MIP-LPS.. The changes of anxiety like behavior were observed 48 hours after the last intracerebral injection, and the expression of I NOS,Arg-1 in medial prefrontal cortex and hippocampus was observed by immunofluorescence staining. The expressions of IL-1 尾, TNF- 偽 and iNOS,Arg-1,Myd88,P-IRF3 in medial prefrontal cortex and hippocampus were detected by Western Blot. Result 1. After the model was established, the open field experiment showed that the distance of central activity was the shortest and the time of central activity was the least in CP-LPS group compared with the other three groups. This suggests that the CP-LPS group showed more obvious anxiety behavior than the other three groups. The distance and time of central activity in MIP-LPS group was significantly higher than that in CP-LPS, which suggested that MIP could reverse the anxiety behavior induced by LPS. In the elevated cross maze test, the CP-LPS group had the least time and the shortest distance in the open arm activity compared with the other three groups, which indicated that the CP-LPS group had more obvious anxiety behavior than the other three groups. The time and distance of open arm activity in MIP-LPS group was significantly higher than that in CP-LPS, suggesting that MIP could reverse the anxious behavior induced by LPS. 2. Activation of microglia in the hippocampus and medial prefrontal cortex of experimental animals in different treatment groups showed that DG,CA1, was found in the brain regions associated with anxiety like emotions, including the hippocampus. M 1 microglia were activated in CA2 region and m PFC in CP-LPS group, while M 2 microglia were activated in MIP-Con group and MIP-LPS group. Changes in the expression of important Molecular proteins in key brain regions of Experimental Animals in different treatment groups the results of Western Blot showed that DG,CA1, in the hippocampus was involved in the brain regions associated with anxiety. In CA2 region and mPFC, the expression of M1 microglial protein marker iNOS was more in CP-LPS group, while in MIP-Con and MIP-LPS group there was more Arg-1 expression of M2 microglial protein marker, while in CP-LPS group, the expression of inflammatory factor IL-1 尾 was higher than that in CP-LPS group. Expression of TNF- 偽. The expression of P-IRF3, a key molecule of Myd88 independent pathway, was significantly increased in MIP-Con group and MIP-LPS group. Conclusion LPS can induce M1 polarization of microglia and may induce anxiety like behavior in experimental animals. MIP can reverse the effect of LPS and alleviate the anxiety like behavior induced by LPS in experimental animals. This regulation may be achieved through Myd88 independent pathway and is closely related to microglial M 2 polarization.
【學(xué)位授予單位】：成都醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R749

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