血红素氧合酶1促进氧化应激条件下神经干细胞向神经元分化  

Heme oxygenase 1 promotes differentiation of neural stem cells into neurons under oxidative stress condition

作　　者：于庆贺 蔡子鸣 田禾 李翩 阮烨 梁金柱 林淑惠 林文平 Yu Qinghe;Cai Ziming;Tian He;Li Pian;Ruan Ye;Liang Jinzhu;Lin Shuhui;Lin Wenping(Department of Spine Surgery,Shenzhen 518118,Guangdong Province,China;Shenzhen Pingle Orthopedic Hospital Affiliated to Guangzhou University of Chinese Medicine,Shenzhen 518118,Guangdong Province,China;Department of Gynaecology,Shenzhen Pingle Orthopedic Hospital Affiliated to Guangzhou University of Chinese Medicine,Shenzhen 518118,Guangdong Province,China;Third School of Clinical Medicine,Guangzhou University of Chinese Medicine,Guangzhou 510006,Guangdong Province,China)

机构地区：[1]广州中医药大学附属深圳平乐骨伤科医院脊柱外科,广东省深圳市518118 [2]广州中医药大学附属深圳平乐骨伤科医院,广东省深圳市518118 [3]广州中医药大学附属深圳平乐骨伤科医院妇科,广东省深圳市518118 [4]广州中医药大学第三临床医学院,广东省广州市510006

出　　处：《中国组织工程研究》2025年第23期4931-4938,共8页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金面上项目(81771323),项目负责人:林文平;广东省自然科学基金面上项目(2021A1515010722),项目负责人:林文平;深圳市自然科学基金面上项目(JCYJ20190813112401660),项目负责人:林文平。

摘　　要：背景:研究表明,血红素氧合酶1能增强细胞的抗氧化、抗凋亡能力,但血红素氧合酶1过表达对氧化应激条件下神经干细胞增殖和分化的影响尚不清楚。目的:探讨血红素氧合酶1过表达对氧化应激条件下神经干细胞存活和分化能力的影响。方法:(1)从新生Balb/c小鼠脊髓组织中分离培养小鼠原代神经干细胞,免疫荧光检测神经干细胞标志物Nestin的表达;(2)采用慢病毒感染神经干细胞以诱导血红素氧合酶1过表达,流式检测绿色荧光蛋白荧光强度,Western blot检测血红素氧合酶1的表达水平;(3)在慢病毒感染神经干细胞培养基中添加H_2O_2以模拟脊髓损伤后的氧化应激微环境,采用细胞增殖检测试剂盒、细胞凋亡检测试剂盒和TUNEL染色试剂盒分析血红素氧合酶1过表达对神经干细胞增殖和凋亡水平的影响;(4)采用试剂盒检测脂质氧化标志物丙二醛、过氧化氢酶、超氧化物歧化酶和谷胱甘肽过氧化物酶水平;(5)采用流式检测细胞内的活性氧水平以及神经干细胞向星形胶质细胞和神经元分化情况;(6)光学和荧光显微镜观察血红素氧合酶1过表达对神经干细胞分化的神经元轴突生长的影响。结果与结论:(1)小鼠神经干细胞球形态稳定,生长状态良好,免疫荧光检测Nestin呈阳性;(2)Western blot检测发现血红素氧合酶1过表达组神经干细胞中血红素氧合酶1的表达显著高于空载体对照组;流式检测血红素氧合酶1过表达组和空载体对照组神经干细胞均表达绿色荧光蛋白;(3)血红素氧合酶1过表达维持了神经干细胞在氧化应激条件下的增殖活力并显著减少了细胞凋亡数量;(4)血红素氧合酶1过表达抑制了氧化应激微环境下神经干细胞的脂质过氧化,增强了维持氧化-还原稳态的相关酶类表达,降低了细胞内的活性氧水平;(5)血红素氧合酶1过表达促进了神经干细胞向神经元分化,并抑制了向星形胶质细BACKGROUND:Studies have shown that upregulation of heme oxygenase-1 expression enhances cellular antioxidant and anti-apoptotic abilities.However,the effects of upregulating heme oxygenase-1 expression on the proliferation and differentiation of neural stem cells under oxidative stress conditions remain unclear.OBJECTIVE:To investigate the influence of heme oxygenase-1 overexpression on the survival and differentiation capacity of neural stem cells under oxidative stress conditions.METHODS:(1)Mouse primary neural stem cells were isolated and cultured from newborn Balb/c mice.Immunofluorescence was used to detect the neural stem cell marker Nestin.(2)Lentivirus was used to infect neural stem cells to induce heme oxygenase-1 overexpression.Flow cytometry was used to assess green fluorescent protein fluorescence.Western blot assay was performed to detect the expression levels of heme oxygenase-1.(3)H2O2 was added to the lentivirus-infected neural stem cell culture medium to simulate the oxidative stress microenvironment after spinal cord injury.Effects of heme oxygenase-1 overexpression on neural stem cell proliferation and apoptosis levels were analyzed using cell proliferation assay kits,cell apoptosis assay kits,and TUNEL staining kits.(4)The levels of lipid oxidation markers malondialdehyde,catalase,superoxide dismutase,and glutathione peroxidase were detected using assay kits.(5)Flow cytometry was used to measure intracellular reactive oxygen species levels,and neural stem cell differentiation into astrocytes and neurons.(6)The effect of heme oxygenase-1 overexpression on neuronal axon growth during neural stem cell differentiation was observed under optical and fluorescence microscopes.RESULTS AND CONCLUSION:(1)Mouse neural stem cells exhibited stable morphology,good growth status,and high expression of Nestin as detected by immunofluorescence.(2)Western blot analysis showed that the overexpression of heme oxygenase-1 in the overexpression group was significantly higher than that in the empty carrier control g

关 键 词：血红素氧合酶1 神经干细胞 氧化应激 脊髓损伤 神经元 凋亡 轴突 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R744