成纤维细胞生长因子10通过Rho A/ROCK信号通路稳定微管并维持神经元生存  

Fibroblast growth factor 10 stabilizes microtubule and maintains neuronal survival through Rho A/ROCK signal pathway

作　　者：蒋永生 丁哓哓 林建军 Jiang Yongsheng;Ding Xiaoxiao;Lin Jianjun(Science and Education Management Center of Healthcare Group,First People's Hospital of Xiangshan County,Ningbo 315700,China;Department of Pharmacy,Beilun People's Hospital of Ningbo,Ningbo 315826,China)

机构地区：[1]宁波市象山县第一人民医院医疗健康集团科教管理中心,宁波315700 [2]宁波市北仑区人民医院药剂科,宁波315826

出　　处：《中华神经医学杂志》2023年第2期117-126,共10页Chinese Journal of Neuromedicine

基　　金：浙江省卫生健康委员会科技计划项目(2021KY1075);浙江省自然科学基金(LWQ20H170001)。

摘　　要：目的探讨成纤维细胞生长因子10(FGF10)对神经元损伤的保护作用及其潜在的分子机制。方法剥离新生SD乳鼠的脑组织并提取其中的皮层神经元,将其种植于含L-多聚赖氨酸的孔板上进行培养,并分为正常组、髓鞘碎片组和髓鞘碎片+FGF10组,其中髓鞘碎片组神经元在培养4 h后添加一定含量的髓鞘碎片溶液(终浓度为10μg/mL),而髓鞘碎片+FGF10组神经元同时在培养基内添加髓鞘碎片和FGF10溶液(终浓度为4.3 nmol/L)。培养1周后,应用TUNEL染色、流式细胞术检测各组神经元的凋亡情况,应用活/死细胞染色、CCK-8法检测各组神经元的生存情况,应用Western blotting、免疫荧光双标染色检测各组神经元内凋亡相关蛋白、微管相关蛋白和RAS同源基因家族成员A(Rho A)/Rho A相关蛋白激酶(ROCK)信号通路相关蛋白的表达。结果与正常组相比,髓鞘碎片组中TUNEL染色所示神经元凋亡率明显升高,流式细胞术所示早期神经元凋亡率明显升高,活化半胱氨酸蛋白酶-3(caspase-3)蛋白表达明显升高,Bcl-2/Bax值明显降低,活/死细胞染色所示神经元死亡率明显升高,CCK-8法所示神经元生存率明显降低,乙酰化微管蛋白/酪氨酸微管蛋白(Ace/Tyr-tubulin)值明显降低,Tau蛋白表达明显降低,Ace/Tyr-tubulin荧光强度比值明显降低,Rho A、ROCK蛋白表达明显升高,Rho A荧光强度值明显升高,差异均有统计学意义(P<0.05)。与髓鞘碎片组相比,髓鞘碎片+FGF10组中TUNEL染色所示神经元凋亡率明显降低,流式细胞术所示早期神经元凋亡率明显降低,活化caspase-3蛋白表达明显降低,Bcl-2/Bax值明显升高,活/死细胞染色所示神经元死亡率明显降低,CCK-8法所示神经元生存率明显升高,Ace/Tyr-tubulin值明显升高,Tau蛋白表达明显升高,Ace/Tyr-tubulin荧光强度比值明显升高,Rho A、ROCK蛋白表达明显降低,Rho A荧光强度值明显降低,差异均有统计学意义(P<0.05)。结论FGF10可能Objective To investigate the protective effect of fibroblast growth factor 10(FGF10)on neuronal injury and its potential molecular mechanism.Methods Cortical neurons were dissected from brain tissues of newborn SD rats and seeded on Poly-L-Lysine coated plates.These neurons were then divided into control group,myelin group and myelin+FGF10 group;after 4 h of culture,neurons in the myelin group were added with a certain content of myelin solution(final concentration:10μg/mL),while neurons in the myelin+FGF10 group were added with myelin and FGF10 solution(final concentration:4.3 nmol/L).One week after culturing,the neuronal apoptosis was detected by TUNEL and flow cytometry;neuronal survival was evaluated by live/dead assay and CCK-8 assay;expressions of apoptosis-related proteins,microtubule related proteins and RAS homologous gene family member A(Rho A)/Rho a-related protein kinase(ROCK)signaling pathway related proteins were detected by Western blotting and immunofluorescent double-label staining.Results Compared with the control group,the myelin group had significantly increased neuronal apoptosis rate by TUNEL,early neuronal apoptosis rate by flow cytometry,activated cysteine proteinase-3(caspase-3)protein expression and neuronal mortality rate by live/dead assay,and significantly decreased Bcl-2/Bax value,neuronal survival rate by CCK-8 method,value of acetylated tubulin/Tyr-tubulin(Ace/Tyr-tubulin),Tau protein expression and Ace/Tyr-tubulin fluorescent intensity ratio,and statistically increased Rho A and ROCK protein expressions and Rho A fluorescent intensity(P<0.05).Compared with the myelin group,the myelin+FGF10 group had significantly decreased neuronal apoptosis rate by TUNEL,early neuronal apoptosis rate by flow cytometry and activated caspase-3 protein expression,significantly increased Bcl-2/Bax value,neuronal survival rate by CCK-8 method,Ace/Tyr-tubulin value,Tau protein expression and Ace/Tyr-tubulin fluorescent intensity ratio,and statistically decreased Rho A and ROCK protein expressions and

关 键 词：成纤维细胞生长因子10 微管 髓鞘碎片 RAS同源基因家族成员A Rho A相关蛋白激酶 

分 类 号：R741[医药卫生—神经病学与精神病学]