GDNF基因修饰的NSCs联合EPO通过Nrf2-COX2/iNOS保护帕金森小鼠神经功能  

Neuroprotection of GDNF gene-modified NSCs combined with EPO for mice with Pakinson’s disease through Nrf2-COX2/iNOS

作　　者：王淑华 周正丽 汤华军 王云 李开荣 孙国刚 胡光强 陈波 WANG Shuhua;ZHOU Zhengli;TANG Huajun;WANG Yun;LI Kairong;SUN Guogang;HU Guangqiang;CHEN Bo(Department of Stomatology,Southwest Medical University,Luzhou 646000,China;Department of Human Anatomy,School of Basic Medical Sciences,Southwest Medical University,Luzhou 646000,China)

机构地区：[1]西南医科大学口腔医学专业,泸州646000 [2]西南医科大学基础医学院人体解剖学教研室,泸州646000

出　　处：《西南医科大学学报》2022年第1期44-50,共7页Journal of Southwest Medical University

基　　金：四川省科技厅项目(2018JY0490);国家级大学生创新创业项目(201710632031);西南医科大学校级课题(00030181)。

摘　　要：目的探讨胶质细胞源性神经营养因子(glial cellline-derivedneurotrophicfactor,GDNF)修饰的神经干细胞(neural stemcells,NSCs)联合促红细胞生成素(erythropoietin,EPO)对1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)诱导帕金森小鼠的神经保护作用。方法C57BL/6雄性小鼠分为Normal组、Sham组、MPTP组、Sham+EPO组、Sham+GDNF-NSCs组、MPTP+EPO组、MPTP+GDNF-NSCs组和MPTP+GDNF-NSCs+EPO组。MPTP腹腔注射诱导建模后分别进行腹腔EPO注射、双侧纹状体移植GDNF-NSCs或者两者同时干预,Sham组在纹状体注射等体积生理盐水。在建模第7、14、28和42 d时检测行为学变化、纹状体多巴胺含量、线粒体膜电位及荧光定量PCR检测细胞内关键抗氧化蛋白NF-E2相关因子2(Nrf2)、环氧合酶2(COX2)和诱导型一氧化氮合酶(iNOS)基因表达变化。结果EPO干预组(14、28、42 d时分别为946.09±48.71、773.88±7.80、703.88±12.45,P<0.001)和GDNF-NSCs干预组(14、28、42 d时分别为1266.32±77.17、1150.80±27.21、1202.48±65.71,P<0.001)较MPTP组(658.14±30.97)多巴胺含量显著增加;线粒体膜电位EPO干预组(14、28、42 d时分别为45.67±4.93、40.33±4.73、42.00±5.29,P<0.001)和GDNF-NSCs组(14、28、42 d时分别为48.33±6.66、53.33±2.89、52.00±6.24,P<0.001)线粒体膜电位较MPTP组(32.33±2.52)显著增加;也显著降低抗氧化相关基因Nrf2、iNOS和COX2的表达,随时间延长统计学差异更显著,但联合干预效果更明显;行为学实验也显示联合干预组较单一干预组行为学改善更佳。结论GDNF基因修饰的NSCs联合EPO可能通过增加氧化应激抵抗发挥帕金森小鼠神经保护作用。Objective To observe the neuroprotective effects of glial cell line-derived neurotrophic factor(GDNF)-modified neural stem cells(NSCs)combined with erythropoietin(EPO)on a mouse model of Parkinson’s disease induced by 1-methyl-4-phe⁃nyl-1,2,3,6-tetrahydropyridine(MPTP).Methods Male C57BL/6 mice were divided into Normal group,Sham group,MPTP group,Sham+EPO group,Sham+GDNF-NSCs group,MPTP+EPO group,MPTP+GDNF-NSCs group,and MPTP+GDNF-NSCs+EPO group.A mouse Parkinson’s disease model was established by intraperitoneal injection of MPTP,followed by intraperitoneal EPO injec⁃tion,GDNF-NSCs implantation into both striata,or both,with injection of an equal volume of normal saline into the striatum in the Sham group.We observed behavioral changes,and measured the content of dopamine in the striatum,mitochondrial transmembrane po⁃tential,and the expression of key antioxidant genes including nuclear factor erythroid 2-related factor 2(Nrf2),cyclooxygenase 2(Cox2),and inducible nitric oxide synthase(iNos)by quantitative fluorescence PCR at 7,14,28,and 42 days after establishing the model.Results The content of dopamine was significantly increased in the MPTP+EPO group(946.09±48.71,773.88±7.80,and 703.88±12.45 at 14,28,and 42 d,respectively,P<0.001)and in the MPTP+GDNF-NSCs group(1266.32±77.17,1150.80±27.21,and 1202.48±65.71 at 14,28,and 42 d,respectively,P<0.001)than in the MPTP group(658.14±30.97).The mitochon drial membrane potential was significantly increased in the MPTP+EPO group(45.67±4.93,40.33±4.73,and 42.00±5.29 at 14,28,and 42 d,respectively,P<0.001)and in the MPTP+GDNF-NSCs group(48.33±6.66,53.33±2.89,and 52.00±6.24,at 14,28,and 42 d,respectively,P<0.001)than in the MPTP group(32.33±2.52).The expression of Nrf2,iNos,and Cox2 genes showed significant decreases,with significant differences over time,and the effects were greater in the combined intervention group.The combined intervention group had better behavioral improvement than the single intervention groups.Conclusion The combination of G

关 键 词：胶质细胞源性神经营养因子 神经干细胞 促红细胞生成素 帕金森病 小鼠 

分 类 号：R742[医药卫生—神经病学与精神病学]