辛伐他汀调控小胶质细胞极化状态的实验研究

发布时间：2018-03-20 09:44

本文选题：BV2小胶质细胞　切入点：经典激活　出处：《华中科技大学》2014年博士论文　论文类型：学位论文

【摘要】：第一部分BV2小胶质细胞经典激活及替代激活的鉴定目的观察BV2小胶质细胞经典激活及替代激活后代谢分子的变化,判断鉴别M1及M2型极化小胶质细胞的方法。方法小胶质细胞以100ng/ml的LPS及20ng/mL的IL-4分别单独经典激活及替代激活24小时,以ELISA方法检测IL-12、IL-10、TNF-α的分泌量,以实时荧光定量聚合酶链反应检测iNOS、Arg-1、IL-10、IL-12的mRNA表达,以流式细胞术检测细胞膜MMr蛋白表达。结果小胶质细胞被LPS经典激活后呈M1型极化,IL-12、TNF-α分泌量、iNOSmRNA、IL-12mRNA表达量明显升高,与生理对照组比较有统计学差异(p0.05)。小胶质细胞被IL-4替代激活后呈M2极化,IL-10分泌量、Arg-1mRNA及IL-10mRNA表达量显著升高,流式细胞术检测MMr蛋白荧光强度明显上升,与生理对照组比较有统计学差异(p0.05)。结论小胶质细胞被经典激活呈M1型极化,替代激活呈M2型极化,不同极化状态的小胶质细胞通过检测炎性相关因子、精氨酸代谢分子及细胞膜特异性蛋白三个方面得到鉴别。第二部分Notch信号通路对BV2小胶质细胞经典激活途径及替代激活途径的调控目的研究Notch信号系统对小胶质细胞经典激活及替代激活的影响。方法1.小胶质细胞以LPS及IL-4分别经典激活及替代激活,以实时荧光定量聚合酶链反应检测Notch信号通路分子Notch、Jagged-1、Hes1、Hes5的表达。2.以DAPT阻断Notch通道后再以LPS及IL-4分别刺激小胶质细胞,以ELISA方法检测IL-12、IL-10、TNF-α的分泌量,以实时荧光定量聚合酶链反应检测iNOS、Arg-1、 IL-10、IL-12的mRNA表达,以流式细胞术检测细胞膜CD16/32及CD206蛋白表达。结果1.小胶质细胞生理对照组、LPS诱导组及IL-4刺激组均表达Notch信号通路分子,LPS诱导组Notch信号通路分子Notch1、Jagged-1、Hes1表达增强。2.以DAPT阻断Notch信号通路后,小胶质细胞IL-12、TNF-α分泌量、iNOSmRNA、 IL-12mRNA表达量较单纯LPS诱导组明显降低(p0.05),IL-10分泌量及Arg-1mRNA表达量较单纯IL-4诱导组提高(p0.05.),流式细胞术检测膜蛋白CD16/32较单纯LPS诱导组减低,CD206较单纯LPS诱导组增加。结论：BV2小胶质细胞在安静状态、经典激活状态及替代激活状态均表达Notch信号分子,并且经典激活状态下Notch信号通道也被活化。Notch信号系统的激活使小胶质细胞M1型极化增强,阻断Notch信号通路后,BV2小胶质细胞经典激活被抑制,M2型极化分子表达增强,表现出一定的M1型级化状态向M2型转变的趋势。第三部分辛伐他汀对BV2小胶质细胞Notch信号通路的影响目的观察辛伐他汀对于BV2小胶质细胞Notch信号通路的影响。方法部分小胶质细胞以20ug/mL的辛伐他汀预处理后再给予100ng/ml的LPS刺激,部分细胞给予0.5μg/ml可溶性Jagged1/Fc嵌合蛋白及20ug/mL的辛伐他汀共同预处理后再给予100ng/ml的LPS刺激,以Western blot检测Notch1、Hes1蛋白的表达,以实时荧光定量聚合酶链反应检测Notch1、Jagged-1、Hes1、Hes5mRNA的表达。结果。LPS诱导组Notch1、Jagged-1、Hes1、Hes5均较生理对照组显著增加(p0.05),而辛伐他汀干预组Notch1、Hesl的蛋白测定及Notch1、Hes1、Hes5mRNA表达均较单纯LPS诱导组降低(p0.05),而Jagged-1无显著改变。给予可溶性Jagged1/Fc嵌合蛋白后,Notch1及Hes1mRNA表达较辛伐他汀组上升。结论LPS激活小胶质细胞Notch信号通路,而辛伐他汀抑制Notch信号通路的活性,根据可溶性Jagged1/Fc嵌合蛋白对于辛伐他汀抑制作用的逆转,判断辛伐他汀对Notch信号通路调节可能发生在细胞外水平。第四部分辛伐他汀对BV2小胶质细胞极化状态的调控目的研究辛伐他汀对于BV2小胶质细胞极化状态的调控。方法小胶质细胞给予20ug/ml及5ug/mL的高低浓度的辛伐他汀预处理,再分别给予LPS经典激活及可溶性Jagged1/Fc嵌合蛋白刺激小胶质细胞,以ELISA方法检测IL-12、IL-10、TNF-α的分泌量,以实时荧光定量聚合酶链反应检测iNOS、Arg-1、IL-10、IL-12的mRNA表达,以流式细胞术检测细胞膜蛋白CD206、CD16/32的表达。结果。1.辛伐他汀干预组IL-12、TNF-α分泌量、iNOSmRNA、IL-12mRNA表达量较LPS经典激活组明显降低,IL-10分泌量显著升高(p0.05),流式细胞仪检测CD16/32蛋白阳性表达量减少而CD206表达量增加。2.辛伐他汀干预组IL-12、TNF-α分泌量、iNOSmRNA、 IL-12mRNA表达量较Jagged1/Fc嵌合蛋白刺激组亦明显降低,Arg-1及IL-10表达量显著升高(p0.05)。结论辛伐他汀抑制LPS诱导的小胶质细胞经典激活,同时也能抑制Notch信号激活剂Jagged1/Fc嵌合蛋白诱导的小胶质细胞经典激活,提示辛伐他汀通过介导Notch信号途径调控小胶质细胞的极化状态。
[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.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R965

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