α-synuclein对星形胶质细胞内质网—高尔基体系统及凋亡的影响

发布时间：2018-03-05 19:03

本文选题：α-synuclein　切入点：星形胶质细胞　出处：《中南大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：研究过表达α-synuclein野生型和突变型(A53T、A30P)对星形胶质细胞(astrocyte, AST)内质网-高尔基系统及细胞凋亡的影响。方法：分离培养和纯化新生1-3天SD大鼠皮质区的AST,用免疫荧光的方法对其进行鉴定(GFAP), Western blot检测AST内源性α-synuclein的表达。用293FT细胞对前期已经构建好的(WT、A30P、 A53T)α-synuclein-HA-FUIGW-GFP、GFP-fuigw质粒进行慢病毒包装,收获病毒上清后分别感染AST,分为过表达野生型组(WT组)、A30P组、A53T组和对照组(FUIGW组)。5-7天后免疫荧光及Western blot检测AST中(WT、A30P、A53T) α-synuclein-HA融合蛋白的表达。衣霉素处理AST6小时作为阳性对照组,Western blot检测各组内质网应激指标蛋白BIP、CHOP的变化；免疫荧光检测各组GM130观察高尔基体形态的变化；流式细胞仪检测各组细胞凋亡率的变化。结果：1)星形胶质细胞有内源性α-synuclein的表达。 2)慢病毒感染AST5-7天后免疫荧光及Western blot可检测到过表达野生型组(WT组)、A30P组、A53T组α-synuclein-HA融合蛋白的表达,而FUIGW组未检测至α-ynuclein-HA融合蛋白的表达。 3)过表达野生型组(WT组)、A30P组、A53T组BIP、CHOP指标明显高于对照组(FUIGW组)(P0.05)；过表达野生型组(WT组)与A30P组、A53T组相比BIP、CHOP表达无显著性差异(P0.05)。 4)过表达野生型组(WT组)与A30P组、A53T组的高尔基体分散在细胞质中,囊泡碎裂；对照组(FUIGW组)高尔基体分布在细胞核一侧,囊泡完整无碎裂。经χ2检验,过表达野生型组(WT组)、A30P组、A53T组的高尔基体碎裂百分比明显高于对照组(FUIGW组)(P0.01)；过表达野生型组(WT组)与A30P组、A53T组的高尔基体碎百分比差异无显著性(P0.05)。 5)过表达野生型组(WT组)、A30P组、A53T组的AST凋亡率明显高于对照组(FUIGW组)(P0.01)；过表达野生型组(WT组)与A30P组、A53T组的AST凋亡率差异无显著性(P0.05)。结论：过表达a-synuclein野生型和突变型(A53T、A30P)可能通过内质网-高尔基体系统损伤诱导星形胶质细胞的凋亡。
[Abstract]:Aim: to study the effects of overexpression of 伪 -synuclein wild type and mutant A53TnA30P on the endoplasmic reticulum Golgi system and apoptosis of astrocyte astrocyte (AST). Methods: AST3 was isolated and purified from the cortex of neonatal Sprague-Dawley rats (1-3 days), and identified by immunofluorescence. The expression of AST endogenous 伪 -synuclein was detected by Western blot. The previously constructed plasmids were packaged using 293FT cells for the slow virus packaging of the previously constructed plasmids, named as WTA30P, A53T) 伪 -synuclein-HA-FIGW-GFPFPGFP-fuigw, and the expression of 伪 -synuclein-HIGW-GFPGFP-fuigw plasmid was detected by Western blot. The virus supernatants were infected with ASTand were divided into two groups: overexpression wild-type group and control group FUIGW group. Immunofluorescence and Western blot were used to detect the expression of 伪 -synuclein-HA fusion protein in AST after treatment with AST6 for hours. The changes of endoplasmic reticulum stress index protein (BIP-CHOP) were detected by Western blot. The changes of Golgi body morphology and apoptosis rate were detected by immunofluorescence and flow cytometry respectively. Results: 1) Endogenous 伪-synuclein was expressed in astrocytes. 2) the expression of 伪 -synuclein-HA fusion protein was detected by immunofluorescence and Western blot on day after lentivirus infection in wild type AST5-7 group, while the expression of 伪 -ynuclein-HA fusion protein was not detected in FUIGW group. (3) the expression of BIP-CHOP in wild type group was significantly higher than that in control group (P 0.05), and that in wild type group was not significantly different from that in group A30P group (P 0.05), and that in wild type group was higher than that in group A30P group (P < 0.05). 4) the Golgi body of the over-expressed wild type group (WT group) and A30P group (A53T group) were dispersed in the cytoplasm, and the vesicles of the control group (FUIGW group) were scattered in the cytoplasm. The percentage of Golgi body fragmentation in the over-expressed wild type group was significantly higher than that in the control group (FUIGW group) and the wild type group (WT group), and there was no significant difference in the percentage of Golgi body fragmentation between the over-expressed wild type group and the A30P group (P 0.05). 5) the apoptosis rate of AST in the wild type group was significantly higher than that in the control group (P 0.01), and there was no significant difference in the apoptosis rate of AST between the wild type group and the A30P group (P 0.05). Conclusion:. Overexpression of a-synuclein wild-type and mutant A53TnA30Pmay induce apoptosis of astrocytes through endoplasmic reticular-Golgi system injury.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.5

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