中国组织工程研究

中国组织工程研究 ›› 2021, Vol. 25 ›› Issue (23): 3678-3683.doi: 10.12307/2021.039

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

重组腺病毒介导神经生长因子转染实验性自身免疫性脑脊髓炎模型小鼠少突胶质细胞的凋亡及髓鞘化

谢  阳，吕志宇，张淑江，龙  婷，李作孝   

  1. 西南医科大学附属第一医院神经内科，四川省泸州市   646000
  • 收稿日期:2020-06-10 修回日期:2020-06-16 接受日期:2020-07-29 出版日期:2021-08-18 发布日期:2021-01-26
  • 通讯作者: 李作孝，硕士，教授，西南医科大学附属第一医院神经内科，四川省泸州市 646000
  • 作者简介:谢阳，男，1992年生，四川省成都市人，汉族，2017年西南医科大学毕业，硕士，医师，主要从事神经免疫方面的研究。
  • 基金资助:
    四川省卫生厅科研课题(080192)，项目负责人：李作孝；泸州市人民政府基金课题(2018LZXNYD-ZK17)，项目负责人：李作孝

Effects of recombinant adeno-associated virus mediated nerve growth factor gene transfection on oligodendrocyte apoptosis and myelination in experimental autoimmune encephalomyelitis mice

Xie Yang, Lü Zhiyu, Zhang Shujiang, Long Ting, Li Zuoxiao   

  1. Department of Neurology, the First Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • Received:2020-06-10 Revised:2020-06-16 Accepted:2020-07-29 Online:2021-08-18 Published:2021-01-26
  • Contact: Li Zuoxiao, Master, Professor, Department of Neurology, the First Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • About author:Xie Yang, Master, Physician, Department of Neurology, the First Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • Supported by:
    Research Project of Sichuan Provincial Department of Health, No. 080192 (to LZX); Luzhou Municipal People’s Government Fund Project, No. 2018LZXNYD-ZK17 (to LZX)

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摘要：

文题释义：
神经生长因子：作为神经生长因子家族的成员，它能够调控氨基酸和小分子的摄入、增强功能蛋白和结构蛋白的合成，在神经系统发育及神经细胞再生等方面发挥着重要的作用。中枢神经系统受损后，增加的神经生长因子通过拮抗兴奋性氨基酸的毒性作用维持神经细胞内钙离子浓度的稳定，激活蛋白激酶活性，有效阻断因中枢神经系统损伤后导致的神经细胞凋亡，对神经细胞发挥着保护作用。
少突胶质细胞凋亡：当中枢神经系统中少突胶质细胞发生凋亡后，由它构成的髓鞘会失去维持自身功能结构所需要的髓鞘磷脂糖蛋白等必需营养素，导致髓鞘发生断裂、髓鞘板层出现崩解等病理组织学改变。少突胶质细胞凋亡多见于多发性硬化髓鞘脱失发病机制中的复发缓解型，其发病率约为30%。而Caspase家族作为众多凋亡途径的汇聚点在凋亡调控中发挥着关键作用。

背景：神经生长因子对正常的神经元凋亡具有抑制效应，能够提高其损伤修复的能力，在一些自身免疫性疾病中发挥治疗作用。
目的：观察经外周转染重组腺病毒介导神经生长因子基因对实验性自身免疫性脑脊髓炎小鼠少突胶质细胞凋亡及髓鞘化的影响。
方法：将30只雌性健康的C57BL/6小鼠随机分为3组，每组10只：正常组不进行任何处理；对照组采用髓鞘少突胶质细胞糖蛋白多肽免疫法建立实验性自身免疫性脑脊髓炎模型，建模后第3天尾静脉注射生理盐水，连续注射21 d；转染组同样建立实验性自身免疫性脑脊髓炎模型，建模后第3天尾静脉注射转染重组腺病毒介导神经生长因子基因，连续注射21 d。疾病高峰期处死小鼠，利用LFB染色行髓鞘组织形态病理观察，免疫荧光法观察脊髓组织中凋亡蛋白Caspase3与少突胶质细胞的表达及共定位情况，RT-PCR法检测脊髓组织中神经生长因子、髓鞘碱性蛋白mRNA水平，Western blot法检测脊髓组织中神经生长因子、Caspase3蛋白水平，Elisa法检测脊髓组织髓鞘碱性蛋白水平。实验操作过程通过西南医科大学动物实验伦理审查(201912-8)。
结果与结论：①LFB染色显示，对照组脊髓组织有明显的脱髓鞘改变，转染组髓鞘脱失有显著改善；②免疫荧光显示，对照组脊髓组织少突胶质细胞中Caspase3呈明显的点状聚集，转染组聚集现象不明显；③RT-PCR检测显示，与正常组、转染组比较，对照组的髓鞘碱性蛋白、神经生长因子mRNA表达降低(P < 0.05)；④Western blot检测显示，与正常组、转染组比较，对照组的Caspase3蛋白水平升高(P < 0.05)、神经生长因子蛋白水平降低(P < 0.05)；⑤Elisa检测显示，对照组髓鞘碱性蛋白水平低于正常组、转染组(P < 0.05)；⑥结果表明，经外周转染的重组腺病毒介导神经生长因子基因对实验性自身免疫性脑脊髓炎小鼠模型有防治作用，其机制可能与上调神经生长因子水平、下调少突胶质细胞中凋亡蛋白Caspase3、提高髓鞘碱性蛋白表达而改善髓鞘脱失有关。
https://orcid.org/0000-0001-8475-4966 (李作孝) 

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: 实验, 动物, 鼠, 神经生长因子, 腺病毒, 自身免疫性脑脊髓炎, 少突胶质细胞, 髓鞘, 凋亡, 蛋白

Abstract: BACKGROUND: Nerve growth factor (NGF) has an inhibitory effect on normal neuronal apoptosis, thereby improving the ability to repair cell damage, and playing a therapeutic role in some autoimmune diseases.  
OBJECTIVE: To observe the effect of recombinant adeno-associated virus mediated nerve growth factor (Ad-NGF) on apoptosis and myelination of oligodendrocytes in experimental autoimmune encephalomyelitis (EAE) mice. 
METHODS: Thirty female healthy C57BL/6 mice were randomly divided into normal group, EAE group and transfection group, with 10 mice in each group. In the EAE group and transfection group, EAE models were made in mice using myelin oligodendrocyte glycoprotein peptide immunoassay. Three days after modeling, mice in the EAE group were injected normal saline via the tail vein for 21 continuous days, while those in the transfection group were injected Ad-NGF via the tail vein for 21 continuous days. All the mice were executed at the peak period of the disease. LFB staining was used to observe the morphology and pathology of myelin tissue. Immunofluorescence method was used to observe the expression and co-localization of apoptotic protein Caspase3 and oligodendrocytes in spinal cord tissue. RT-PCR method was used to detect the mRNA levels of NGF and myelin alkali in spinal cord tissue. Western blot assay was used to detect the protein levels of NGF and Caspase3 in spinal cord tissue. ELISA was used to measure the level of myelin basic protein in spinal cord tissue. The study protocol was approved by the Animal Ethics Committee of Southwest Medical University (approval No. 201912-8).
RESULTS AND CONCLUSION: LFB staining showed significant demyelination changes in the EAE group, while the demyelination was significantly improved in the transfection group. Caspase-3 aggregation was obviously observed in oligodendrocytes of EAE group, but not in transfection group. RT-PCR results indicated that the mRNA levels of myelin basic protein and NGF were significantly lower in the EAE group than the normal control and transfection groups (P < 0.05). Western blot results revealed that in the EAE group the level of Caspase3 protein was significantly increased (P < 0.05), while the level of NGF significantly reduced as compared with the normal control and transfection groups (P < 0.05). ELISA results showed that the level of myelin basic protein in the EAE group was significantly lower than those in the normal control and transfection groups (P < 0.05). To conclude, the Ad-NGF transfected by external turnover has preventive and control effects on the EAE mouse model, and its mechanism may be related to upregulation of NGF level, down-regulation of Caspase3 in oligodendrocytes, and promotion of myelin basic protein expression, thereby improving demyelination.

Key words: experiment, animal, mouse, nerve growth factor, adenovirus, autoimmune encephalomyelitis, oligodendrocyte, myelin sheath, apoptosis, protein

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