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. (Chinese)

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

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. [ABSTRACT FROM AUTHOR]

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。 [ABSTRACT FROM AUTHOR]

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