摘要
目的观察β淀粉样蛋白(Aβ)受体PirB对小鼠星形胶质细胞增殖和反应性增生的影响。方法培养小鼠原代星形胶质细胞,将细胞分为对照组、Aβ组、Aβ+0.2μmol/L PEP组、Aβ+0.4μmol/L PEP组、Aβ+Fluspirilene组、Aβ+GFP-LV组和Aβ+mPirB-LV组。分别采用PirB的可溶性胞外段PEP或PirB抑制剂Fluspirilene处理星形胶质细胞,抑制内源性PirB受体;或采用慢病毒转染过表达PirB基因,其后给予Aβ1-42寡聚体处理。采用RTCA和EdU法观察细胞增殖情况,Real-time PCR检测星形胶质细胞反应性增生相关S-100钙结合蛋白B(S-100β)、波形蛋白(Vimentin)、巢蛋白(Nestin)、淀粉样前体蛋白(APP)的mRNA表达水平,Western blotting检测神经胶质纤维酸性蛋白(GFAP)表达水平。结果RTCA法检测结果显示,给药后各组标准化细胞指数(NCI)值均骤降,其后逐步升高并趋于平稳。EdU染色结果显示,与对照组比较,Aβ组星形胶质细胞增殖活性明显增强(P<0.05);与Aβ组比较,Aβ+0.2μmol/L PEP组、Aβ+0.4μmol/L PEP组和Aβ+Fluspirilene组细胞增殖活性均明显降低(P<0.01或P<0.001)。Real-time PCR检测结果显示,与对照组比较,Aβ组GFAP、S-100β、Vimentin、Nestin、APP和PirB的mRNA表达量均明显增高(P<0.05);与Aβ组比较,Aβ+0.4μmol/L PEP组GFAP、S-100β、Vimentin、Nestin、APP和PirB的m RNA表达量均明显下降(P<0.01);与Aβ+GFP-LV组比较,Aβ+mPirB-LV组GFAP、S-100β、Vimentin、Nestin、APP和PirB的mRNA表达量均明显增高(P<0.05)。Western blotting检测结果显示,与对照组比较,Aβ组GFAP表达明显增高(P<0.05);与Aβ组比较,Aβ+0.4μmol/L PEP组GFAP表达明显下降(P<0.05)。结论PirB是调节星形胶质细胞反应性增生和增殖的上游分子,抑制星形胶质细胞PirB受体可能是治疗阿尔茨海默病的潜在方法。
Objective To observe the effects of amyloid-β(Aβ)receptor PirB on mouse astrocyte proliferation and reactive astrogliosis in vitro.Methods Mouse primary astrocytes were cultured,and divided into control group,Aβgroup,Aβ+0.2µmol/L PEP group,Aβ+0.4µmol/L PEP group,Aβ+Fluspirilene group,Aβ+GFP-LV group,and Aβ+mPirB-LV group.The mouse astrocytes were treated with soluble PirB extracellular peptide PEP or PirB inhibitor Fluspirilene,respectively,to inhibit endogenous PirB receptor,or overexpressed PirB gene via lentivirus transfection and then treated with Aβ1-42 oligomers.The proliferation of astrocytes was observed by RTCA and EdU methods,and the mRNA expression levels of S-100 calcium-binding protein B(S-100β),Vimentin,Nestin and amyloid precursor protein(APP)associated with reactive astrogliosis of astrocytes were observed by real-time PCR,and the expression level of glial fibrillary acid protein(GFAP)was detected by Western-blotting.Results The results of RTCA monitoring showed that normalized cell index(NCI)values of each group decreased sharply after treatment,and then increased gradually and tended to be stable.The results of EdU staining showed that the proliferative activity of astrocytes was significantly enhanced in the Aβgroup(P<0.05)compared with control group;Compared with Aβgroup,cell proliferation activity in Aβ+0.2µmol/L PEP group,Aβ+0.4µmol/L PEP group and Aβ+Fluspirilene group were significantly decreased(P<0.01 or P<0.001).The results of real-time PCR showed that compared with control group,mRNA expressions of GFAP,S-100β,Vimentin,Nestin,APP and PirB in Aβgroup were significantly increased(P<0.05);Compared with Aβgroup,mRNA expressions of GFAP,S-100β,Vimentin,Nestin,APP and PirB in Aβ+0.4µmol/L PEP group were significantly decreased(P<0.01);Compared with Aβ+GFP-LV group,mRNA expressions of GFAP,S-100β,Vimentin,Nestin,APP and PirB in Aβ+mPirB-LV group were significantly increased(P<0.05).The results of Western blotting showed that compared with control group,the expressi
作者
赵沅杰
拓振杰
尚培骏
杨锦雯
张晓华
Zhao Yuan-Jie;Tuo Zhen-Jie;Shang Pei-Jun;Yang Jin-Wen;Zhang Xiao-Hua(School of Public Health,Xi'an Medical University,Xi'an,Shaanxi 710021,China;Institute of Neuroscience/Fujian Provincial Key Laboratory of Neurodegenerative Disease and Aging Research,Xiamen University,Xiamen,Fujian 361005,China)
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2024年第1期82-90,共9页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金(81801088)
陕西省高校科协青年人才托举计划(20200313)
陕西省自然科学基础研究计划(2020JQ-881)
国家级大学生创新创业训练计划(202111840011)。
