纳洛酮对细菌内毒素脂多糖诱导的视网膜小胶质细胞激活的抑制作用及相关机制的研究

发布时间：2018-07-09 22:09

本文选题：小神经胶质细胞 + 纳洛酮　；参考：《复旦大学》2012年硕士论文

【摘要】：[目的]本研究旨在证明,纳洛酮(naloxone)在体外可以抑制细菌内毒素脂多糖(LPS)诱导的视网膜小胶质细胞的活化,并减少后者分泌的肿瘤坏死因子(TNF-a)和白介素1β (IL-1β)。并且进一步阐述,上述作用是通过抑制p38MAPK这一通路实现的。 [方法]将SD大鼠的视网膜小胶质细胞分离纯化培养。用不同浓度的LPS处理,建立小胶质细胞体外激活模型。不同浓度的naloxone (0.5μM,1.0μM和2.0μM)在LPS激活视网膜小胶质细胞前,预处理30分钟。利用ELISA检测胶质细胞培养上清中TNF-a和IL-1p的分泌量。利用westernblot方法,检测细胞中MAPK通路家族中三个亚家族p38MAPK, ERK, JNK,以及他们活化的形式磷酸化p38MAPK (P-p38),磷酸化ERK (P-ERK),磷酸化JNK (P-JNK)这六种物质的含量。通过免疫荧光的方法,标记OX42(小胶质细胞特异性标记物)和P-p38。利用p38MAPK特异性的抑制剂,SB203580(不作用于ERK, JNK.通常被用来排除p38MAPK的作用)预处理视网膜小胶质细胞12小时,之后再进行上述naloxone预处理,LPS激活这些步骤。利用ELISA再次检测上清液中TNF-α和IL-1p的分泌量,来反向验证我们的推论。 [结果]实验证明naloxone可以减少LPS诱导的TNF-α和IL-1β释放,并呈浓度-效应关系。Naloxone可以有效的减少p38MAPK的磷酸化,而对于ERK, JNK的磷酸化并无作用。对于SB203580处理后的小胶质细胞,naloxone的预处理则不能改变LPS诱导的TNF-α和IL-1β释放增加。 [结论]Naloxone可以抑制小胶质细胞激活和释放炎性因子,这一现象可能是通过对p38MAPK通路实现的。
[Abstract]:[objective] to demonstrate that naloxone (naloxone) can inhibit lipopolysaccharide (LPS) -induced activation of retinal microglia and decrease tumor necrosis factor (TNF-a) and interleukin-1 尾 (IL-1 尾) secreted by naloxone (naloxone) in vitro. Furthermore, the above action is achieved by inhibiting the p38 MAPK pathway. Methods the retinal microglia of SD rats were isolated and cultured. In vitro activation model of microglia was established by different concentration of LPS. Different concentrations of naloxone (0.5 渭 M 1.0 渭 M and 2.0 渭 M) were pretreated with LPS-activated retinal microglia for 30 minutes. The secretion of TNF-a and IL-1p in the supernatant of glial cell culture was detected by Elisa. The contents of three subfamilies p38 MAPK, ERK, JNKand their activated forms of phosphorylated p38 MAPK (P-p38), phosphorylated ERK (P-ERK) and phosphorylated JNK (P-JNK) were detected by westernblot method. OX42 (microglia specific marker) and P-p38 were labeled by immunofluorescence. The specific inhibitor of p38 MAPK was SB203580 (not acting on ERK, JNK). It is usually used to exclude the effects of p38 MAPK) pretreatment of retinal microglia for 12 hours, and then naloxone preconditioning is performed to activate these steps. Elisa was used to detect the secretion of TNF- 伪 and IL-1p in the supernatant to reverse verify our inference. [results] the results showed that naloxone could reduce the release of TNF- 伪 and IL-1 尾 induced by LPS-in a dose-dependent manner. Naloxone could effectively reduce the phosphorylation of p38 MAPK, but had no effect on the phosphorylation of p38 MAPK. Pretreatment of naloxone in microglia treated with SB203580 could not change the increase of LPS-induced TNF- 伪 and IL-1 尾 release. [conclusion] Naloxone can inhibit the activation and release of inflammatory factors by microglia, which may be mediated by p38 MAPK pathway.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R774.1

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