本文選題：BV2小膠質(zhì)細(xì)胞　切入點(diǎn)：經(jīng)典激活　出處：《華中科技大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分BV2小膠質(zhì)細(xì)胞經(jīng)典激活及替代激活的鑒定 目的觀察BV2小膠質(zhì)細(xì)胞經(jīng)典激活及替代激活后代謝分子的變化,判斷鑒別M1及M2型極化小膠質(zhì)細(xì)胞的方法。方法小膠質(zhì)細(xì)胞以100ng/ml的LPS及20ng/mL的IL-4分別單獨(dú)經(jīng)典激活及替代激活24小時(shí),以ELISA方法檢測(cè)IL-12、IL-10、TNF-α的分泌量,以實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)檢測(cè)iNOS、Arg-1、IL-10、IL-12的mRNA表達(dá),以流式細(xì)胞術(shù)檢測(cè)細(xì)胞膜MMr蛋白表達(dá)。結(jié)果小膠質(zhì)細(xì)胞被LPS經(jīng)典激活后呈M1型極化,IL-12、TNF-α分泌量、iNOSmRNA、IL-12mRNA表達(dá)量明顯升高,與生理對(duì)照組比較有統(tǒng)計(jì)學(xué)差異(p0.05)。小膠質(zhì)細(xì)胞被IL-4替代激活后呈M2極化,IL-10分泌量、Arg-1mRNA及IL-10mRNA表達(dá)量顯著升高,流式細(xì)胞術(shù)檢測(cè)MMr蛋白熒光強(qiáng)度明顯上升,與生理對(duì)照組比較有統(tǒng)計(jì)學(xué)差異(p0.05)。結(jié)論小膠質(zhì)細(xì)胞被經(jīng)典激活呈M1型極化,替代激活呈M2型極化,不同極化狀態(tài)的小膠質(zhì)細(xì)胞通過檢測(cè)炎性相關(guān)因子、精氨酸代謝分子及細(xì)胞膜特異性蛋白三個(gè)方面得到鑒別。 第二部分Notch信號(hào)通路對(duì)BV2小膠質(zhì)細(xì)胞經(jīng)典激活途徑及替代激活途徑的調(diào)控 目的研究Notch信號(hào)系統(tǒng)對(duì)小膠質(zhì)細(xì)胞經(jīng)典激活及替代激活的影響。 方法1.小膠質(zhì)細(xì)胞以LPS及IL-4分別經(jīng)典激活及替代激活,以實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)檢測(cè)Notch信號(hào)通路分子Notch、Jagged-1、Hes1、Hes5的表達(dá)。2.以DAPT阻斷Notch通道后再以LPS及IL-4分別刺激小膠質(zhì)細(xì)胞,以ELISA方法檢測(cè)IL-12、IL-10、TNF-α的分泌量,以實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)檢測(cè)iNOS、Arg-1、 IL-10、IL-12的mRNA表達(dá),以流式細(xì)胞術(shù)檢測(cè)細(xì)胞膜CD16/32及CD206蛋白表達(dá)。 結(jié)果1.小膠質(zhì)細(xì)胞生理對(duì)照組、LPS誘導(dǎo)組及IL-4刺激組均表達(dá)Notch信號(hào)通路分子,LPS誘導(dǎo)組Notch信號(hào)通路分子Notch1、Jagged-1、Hes1表達(dá)增強(qiáng)。2.以DAPT阻斷Notch信號(hào)通路后,小膠質(zhì)細(xì)胞IL-12、TNF-α分泌量、iNOSmRNA、 IL-12mRNA表達(dá)量較單純LPS誘導(dǎo)組明顯降低(p0.05),IL-10分泌量及Arg-1mRNA表達(dá)量較單純IL-4誘導(dǎo)組提高(p0.05.),流式細(xì)胞術(shù)檢測(cè)膜蛋白CD16/32較單純LPS誘導(dǎo)組減低,CD206較單純LPS誘導(dǎo)組增加。 結(jié)論：BV2小膠質(zhì)細(xì)胞在安靜狀態(tài)、經(jīng)典激活狀態(tài)及替代激活狀態(tài)均表達(dá)Notch信號(hào)分子,并且經(jīng)典激活狀態(tài)下Notch信號(hào)通道也被活化。Notch信號(hào)系統(tǒng)的激活使小膠質(zhì)細(xì)胞M1型極化增強(qiáng),阻斷Notch信號(hào)通路后,BV2小膠質(zhì)細(xì)胞經(jīng)典激活被抑制,M2型極化分子表達(dá)增強(qiáng),表現(xiàn)出一定的M1型級(jí)化狀態(tài)向M2型轉(zhuǎn)變的趨勢(shì)。 第三部分辛伐他汀對(duì)BV2小膠質(zhì)細(xì)胞Notch信號(hào)通路的影響 目的觀察辛伐他汀對(duì)于BV2小膠質(zhì)細(xì)胞Notch信號(hào)通路的影響。方法部分小膠質(zhì)細(xì)胞以20ug/mL的辛伐他汀預(yù)處理后再給予100ng/ml的LPS刺激,部分細(xì)胞給予0.5μg/ml可溶性Jagged1/Fc嵌合蛋白及20ug/mL的辛伐他汀共同預(yù)處理后再給予100ng/ml的LPS刺激,以Western blot檢測(cè)Notch1、Hes1蛋白的表達(dá),以實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)檢測(cè)Notch1、Jagged-1、Hes1、Hes5mRNA的表達(dá)。結(jié)果。LPS誘導(dǎo)組Notch1、Jagged-1、Hes1、Hes5均較生理對(duì)照組顯著增加(p0.05),而辛伐他汀干預(yù)組Notch1、Hesl的蛋白測(cè)定及Notch1、Hes1、Hes5mRNA表達(dá)均較單純LPS誘導(dǎo)組降低(p0.05),而Jagged-1無顯著改變。給予可溶性Jagged1/Fc嵌合蛋白后,Notch1及Hes1mRNA表達(dá)較辛伐他汀組上升。結(jié)論LPS激活小膠質(zhì)細(xì)胞Notch信號(hào)通路,而辛伐他汀抑制Notch信號(hào)通路的活性,根據(jù)可溶性Jagged1/Fc嵌合蛋白對(duì)于辛伐他汀抑制作用的逆轉(zhuǎn),判斷辛伐他汀對(duì)Notch信號(hào)通路調(diào)節(jié)可能發(fā)生在細(xì)胞外水平。 第四部分辛伐他汀對(duì)BV2小膠質(zhì)細(xì)胞極化狀態(tài)的調(diào)控 目的研究辛伐他汀對(duì)于BV2小膠質(zhì)細(xì)胞極化狀態(tài)的調(diào)控。方法小膠質(zhì)細(xì)胞給予20ug/ml及5ug/mL的高低濃度的辛伐他汀預(yù)處理,再分別給予LPS經(jīng)典激活及可溶性Jagged1/Fc嵌合蛋白刺激小膠質(zhì)細(xì)胞,以ELISA方法檢測(cè)IL-12、IL-10、TNF-α的分泌量,以實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)檢測(cè)iNOS、Arg-1、IL-10、IL-12的mRNA表達(dá),以流式細(xì)胞術(shù)檢測(cè)細(xì)胞膜蛋白CD206、CD16/32的表達(dá)。結(jié)果。1.辛伐他汀干預(yù)組IL-12、TNF-α分泌量、iNOSmRNA、IL-12mRNA表達(dá)量較LPS經(jīng)典激活組明顯降低,IL-10分泌量顯著升高(p0.05),流式細(xì)胞儀檢測(cè)CD16/32蛋白陽性表達(dá)量減少而CD206表達(dá)量增加。2.辛伐他汀干預(yù)組IL-12、TNF-α分泌量、iNOSmRNA、 IL-12mRNA表達(dá)量較Jagged1/Fc嵌合蛋白刺激組亦明顯降低,Arg-1及IL-10表達(dá)量顯著升高(p0.05)。結(jié)論辛伐他汀抑制LPS誘導(dǎo)的小膠質(zhì)細(xì)胞經(jīng)典激活,同時(shí)也能抑制Notch信號(hào)激活劑Jagged1/Fc嵌合蛋白誘導(dǎo)的小膠質(zhì)細(xì)胞經(jīng)典激活,提示辛伐他汀通過介導(dǎo)Notch信號(hào)途徑調(diào)控小膠質(zhì)細(xì)胞的極化狀態(tài)。
[Abstract]:The identification of the classic activation and alternative activation of BV2 microglia in part 1
Objective To observe the activation of canonical BV2 microglia and metabolic activation of molecular replacement changes, judgment method to identify M1 and M2 polarized microglial cells. Methods the microglial cells with LPS and 20ng/mL 100ng/ml IL-4 respectively classical activation and alternative activation for 24 hours, to detect IL-12, ELISA IL-10, TNF- secretion alpha, to detect iNOS, real-time fluorescence quantitative polymerase chain reaction Arg-1, IL-10, IL-12 expression of mRNA detected by flow cytometry, cell membrane MMr protein. Results the expression of microglia was activated after the classic LPS is M1 type IL-12, TNF- polarization, alpha secretion, iNOSmRNA, expression of IL-12mRNA was significantly higher in comparison there is significant difference between the control group and normal (P0.05). Microglia activation was replaced by IL-4 M2 polarization, the secretion of IL-10, Arg-1mRNA and IL-10mRNA expression increased significantly, flow cytometry was used to detect MMr protein fluorescence intensity increased significantly , there was significant difference between control group and normal (P0.05). Conclusion microglia are classically activated form of M1 type polarization, alternative activation is M2 polarization, different polarization state of microglia by detection of inflammatory related factors, from three aspects of identification of arginine metabolic molecules and cell membrane specific egg.
The regulation of the classical activation pathway and alternative activation pathway of BV2 microglia by the second part of Notch signal pathway
Objective to study the effect of Notch signal system on the classical activation and alternative activation of microglia.
Methods 1. microglial cells with LPS and IL-4 respectively, the classical activation and alternative activation, with real-time fluorescence quantitative polymerase chain reaction for detection of Notch signaling pathway molecules Notch, Jagged-1, Hes1,.2. Hes5 expression by DAPT after blocking Notch channels by LPS and IL-4 respectively stimulated microglia, detection of IL-12, ELISA IL-10, secretion the amount of TNF- alpha, detected by iNOS, real-time fluorescence quantitative polymerase chain reaction Arg-1, IL-10, IL-12 expression of mRNA, CD16/32 and CD206 to detect cell membrane protein expression by flow cytometry.
The results of physiological 1. microglial cells in control group, both the expression of Notch signaling molecules induced by LPS group and IL-4 stimulation group, LPS group induced by Notch signaling pathway molecules Notch1, Jagged-1, Hes1.2. expression increased to DAPT after blocking Notch signaling pathway, microglia IL-12, TNF- alpha secretion, iNOSmRNA, expression of IL-12mRNA is higher than LPS the induction group decreased significantly (P0.05), IL-10 secretion and Arg-1mRNA expression compared with IL-4 induced group increased (p0.05.), membrane protein CD16/32 compared with LPS induced group. Flow cytometry, CD206 compared with LPS induced group increased.
Conclusion: BV2 microglia in a quiet state, the classical activation and activation of Notch signaling molecules were expressed in alternative activation state, and classical activation Notch signal channel state is activated by.Notch signaling system that microglia M1 polarization enhancement, blocking the Notch signaling pathway, BV2 classic microglia activation is inhibited, the expression of M2 type molecular polarization enhancement, reflects a change in M1 level of state to M2 trend.
The effect of the third part of simvastatin on the Notch signaling pathway in BV2 microglia
Objective To observe the effect of simvastatin on BV2 Notch signal pathway. Methods microglia with 20ug/mL simvastatin preconditioning after 100ng/ml LPS stimulation, part of cells to 0.5 g/ml soluble Jagged1/Fc chimeric protein and 20ug/mL of simvastatin pretreatment together after the administration of 100ng/ml LPS to Western Notch1 stimulation, blot detection. The expression of Hes1 protein detected by Notch1, real-time fluorescence quantitative polymerase chain reaction Jagged-1, Hes1, Hes5mRNA. Results the expression of.LPS induced group Notch1, Jagged-1, Hes1, Hes5 were compared with normal control group increased significantly (P0.05), and simvastatin group was Notch1, Hesl and Hes1, Notch1 protein and Hes5mRNA expression were compared simple LPS induced group decreased (P0.05), and no significant change in Jagged-1. The administration of soluble chimeric protein Jagged1/Fc, Notch1 and Hes1mRNA expression compared with simvastatin group increased Conclusion LPS activates the Notch signaling pathway of microglia, while simvastatin inhibits the activity of Notch signaling pathway. According to the reversal of inhibitory effect of soluble Jagged1/Fc chimeric protein on simvastatin, it is concluded that simvastatin regulates Notch signaling pathway at extracellular level.
The regulation of the polarization state of BV2 microglia in the fourth part of Simvastatin
Objective to study the regulation of simvastatin on BV2 microglia polarization state. Simvastatin pretreatment method of high concentration of microglial cells treated with 20ug/ml and 5ug/mL, and then were given classic LPS activation and soluble chimeric protein Jagged1/Fc stimulated microglial cells, to detect IL-12, ELISA method IL-10, the secretion of TNF- alpha, detected by iNOS real time fluorescence quantitative polymerase chain reaction of Arg-1, IL-10, mRNA to detect the expression of IL-12, cell membrane protein CD206 by flow cytometry, the expression of CD16/32. Results.1. simvastatin intervention group IL-12, TNF- alpha secretion, iNOSmRNA, IL-12mRNA expression than the classic LPS activation group decreased significantly, IL-10 secretion increased significantly (P0.05). The amount of positive expression of CD16/32 protein was detected by flow cytometry and reduce CD206 expression increased.2. simvastatin intervention group IL-12, TNF- alpha secretion, iNOSmRNA, IL-12mRNA expression than the Jagged1/Fc block Protein stimulation group was obviously decreased, Arg-1 and IL-10 expression increased significantly (P0.05). Activated microglia classic conclusion simvastatin inhibits LPS induced inhibition of the Notch signal, but also can activate the classical microglia inhibitor Jagged1/Fc chimeric protein induced activation, the polarization state of simvastatin mediated through Notch pathway in microglia.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R965

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本文編號(hào)：1638536