【作者】 张华；

【导师】 汪静；

【作者基本信息】 兰州大学 ， 外科学（骨外）， 2018， 硕士

【摘要】 目的:探讨内源性大麻素2-花生四烯酸甘油(2-Arachidonyl glycerol,2-AG)在脂多糖(lipopolysaccharide,LPS)诱导星形胶质细胞凋亡中的保护机制。方法:体外培养星形胶质细胞。细胞随机分为6组:空白组,LPS组(LPS1μg/ml,12 h),2-AG(0.1μM,1 h)组,2-AG(0.1μM)+LPS(1μg/ml)组,ERK阻断组,p38阻断组。用LPS刺激星形胶质细胞后,采用MTT法和Hoechst染色观察2-AG对其活力及凋亡的影响;通过蛋白免疫印迹法(Western Blotting)测定Bax、Bcl-xl、谷氨酰胺合成酶(Glutamine synthetase,GS)、细胞外信号调控激酶(extracellular signal-regulated kinase,ERK)1/2、磷酸化细胞外信号调控激酶(phospho-extracellular signalregulated kinase,p-ERK)1/2、p38丝裂原活化蛋白激酶(p38-Mitogen Activited Protein Kinase,p38-MAPK)和磷酸化p38丝裂原活化蛋白激酶(phospho-p38-Mitogen Activited Protein Kinase,p-p38MAPK)的表达。阻断ERK和p38后,观察其Bax和Bcl-xl蛋白表达量的变化。结果:经鉴定,培养的星形胶质细胞纯度≥95%,符合实验标准。LPS干预12 h,MTT比色法显示细胞活性下降,Hoechst染色显示星形胶质细胞出现典型的凋亡征象,Western Blotting检测发现p-ERK1/2、p-p38MAPK和Bax蛋白表达增加,GS和Bcl-xl蛋白表达下降(P﹤0.05)。给予2-AG后翻转了LPS诱导的星形胶质细胞活性的下降,使星形胶质细胞凋亡数目减少,降低了p-ERK1/2、p-P38MAPK和Bax蛋白表达,升高了GS和Bcl-xl的蛋白表达(P﹤0.05)。在LPS干预情况下,通过阻断ERK和p38,可升高Bcl-xl蛋白的表达,并降低Bax蛋白的表达(P﹤0.05)。结论:LPS作用星形胶质细胞后可诱导其凋亡,2-AG可以抑制其凋亡,具有保护作用。其机制可能是通过阻断ERK1/2/p38MAPK-Bax/Bcl-xl信号通路并升高GS来实现的。更多还原

【Abstract】 Objective: To investigate the neuroprotective mechanisms of endocannabinoid 2-AG on lipopolysaccharide-induced astocytes apoptosis in vitro.Methods: Astrocytes were randomly divided into 6 groups: A na?ve group,an LPS group,an 2-AG group,2-AG+LPS group,blocking ERK group,and blocking p38 group.In order to determined astrocyte cells viability and apoptosis rate,primary cultured astrocytes were processed by Hoechst staining and MTT assay.The expression of B cell lymphoma-2(Bcl-2)-associated X protein(Bax)and B cell lymphoma extra large(Bcl-xl)as well as mitogen-activated protein kinases including extracellular signal-regulated kinase(ERK)1/2,p38,phosphorylated p-ERK1/2 and p-p38 was examined by Western blotting.To furher observe the change of Bax and Bcl-xl proteins by blocking ERK and p38.Results:(1)Morphological results showed that there are more than 95% cultured cells are GFAP staining positive.(2)After treatment with LPS for 12 hours,the MTT ratio decreased and the numbers of astrocytes karyopyknosis significently increased comparing with Na?ve group.In addition,the increased expression of p-ERK1/2,P-p38 and Bax,and decreased expression of GS and Bcl-xl in LPS treated astrocytes(P﹤0.05).However,after treatment with 2-AG for one hour,the decreased MTT ratio is elevated and the numbers of astrocytes karyopyknosis significantly decreased comparing with LPS treated group,as well as the expression of p-ERK1/2,P-p38 and Bax also decreased,and the expression of GS and Bcl-xl increased in 2-AG treated astrocytes(P﹤0.05).The expression of Bax is decreased and Bcl-xl is increased by blocking ERK and p38 in LPS induced astrocytes(P﹤0.05).Conclusion: These findings indicate that 2-AG can promote astrocytes survival and may potentially protect astocytes against LPS-induced apoptotic by blocking the ERK1/2 /p38–Bax/Bcl-xl pathway and raising GS.更多还原