重组腺病毒转导的MSC导向的CNTF基因靶向性治疗MS对髓鞘和轴突的保护作用

MSCs transfected by rAd-CNTF-IRES-GFP protect myelin and axon in multiple sclerosis model

目的:探讨重组腺病毒转导的骨髓间充质细胞导向(MSC)的睫状神经营养因子(CNTF)基因靶向性治疗多发性硬化对髓鞘和轴突的保护机制。方法:先构建、扩增、纯化Ad-CNTF-IRES-GFP,在体外培养MSC细胞。将1×108Ad-CNTF-IRES-GFP转染MSCS1×106,测定上清液中CNTF的浓度。再用MOG35-55建立C57BL/6小鼠EAE模型,将Ad-CNTF-IRES-GFP转染的MSC移植治疗EAE小鼠,观察其病情评分;免疫荧光观察其病变部位的外源性MSCs数量;免疫组化观察少突胶质细胞前体细胞(OPC)-NG2(+)细胞在病变部位的数量,Westernblot和免疫组化法观察神经生长因子CNTF表达,免疫组化观察促凋亡蛋白Caspase-3表达;病理观察髓鞘、轴突损伤情况;电镜观察髓鞘和轴突以及ODC损伤情况;所有的数据用x—±s表示。采用SPSS13.0进行统计分析。结果用多因素方差分析,P<0.05差异有显著性。结果:MSC-Ad-CNTF-IRES-GFP治疗的EAE小鼠病情显著减轻。不仅平均发病时间缩短,发病率下降,病情减轻,而且病情严重程度也明显减轻。外源性MSC出现在EAE的脊髓病变部位。治疗后的EAE小鼠脊髓病变部位的NG2(+)表达明显增加,促凋亡蛋白Caspase-3的表达明显降低。治疗后的EAE小鼠病变部位的髓鞘和轴突变性明显减轻。结论:MSC-Ad-CNTF-IRES-GFP可有效治疗EAE动物。其机制可能是:Ad-CNTF-IRES-GFP转染的MSC细胞定向去病变部位,升高其部位CNTF水平和NG2(+)细胞数量,减轻其部位髓鞘、轴突及ODC损伤,减少促凋亡蛋白Caspase-3表达。

Objective： To investigate the efficiency and mechanism for MSCs transfected by rAd-CNTF-IRES-GFP to protect myelin and axon in the model of multiple sclerosis： experimental allergic encephalomyelits（EAE） in C57BL/6 mice.Methods： Ad-CNTF-IRES-GFP vector was constructed with PDC316-CNTF-IRES-GFP and PBHG plasmids, and transfected into 293-LP cells and then MSCs by liposome pathway to amplify the recombinant Ad. The expression of CNTF was confirmed in vitro by immunofluorescence. MSCs transfeeted by Ad-CNTF-IRES-GFP were transplanted to treat the EAE animals,clinical scores of illness were estimated,and the damages of myehn and axons were estimated via pathology. Degeneration of myelin,axons was analyzed under electron microscope. Amount of NG2 positive cells in situ was measured by immunohistochemical staining and the exogenous MSCs in situ were counted under immunoflourescence. Local expression of CNTF was confirmed with immunohistochemistry and Western blot. Expression of the pro-apoptotic caspase was identified with immunohistochemistic staining. All the values were presented as x ± s, and statistically analyzed by SPSS 13.0 software. Results： Ad-CNTF-IRES-GFP vector had been successfully constructed and the PFU of the vector was 2.3 × 10^11. CNTF was expressed in MSCs transfected with Ad-CNTF-IRES-GFP virus. The illness in MSC-Ad-CNTF-IRES-GFP-treated animals was significantly inhered. Not only the incidence and the duration of disease decreased, but also the severity of illness was significantly reduced. Exogenous MSCs was attracted to settle in the spinal cord. CNTF expression and the number of NG2 positive cells in situ dramatically increased due to treatment. The expressing level of pro-apoptotic protein Caspase-3 was significantly reduced in animals of MSC-Ad-CNTF-IRES-GFP group. The and demyelination axonal degeneration were significanty reduced. Conclusion： It is discovered for the first time that the therapy of transplanting MSCs that arc transfected by Ad-CNTF-IRES-GFP is more effective in EAE mice. The mecha- nisms for the therapy include attracting MSC-Ad-CNTF-IRES-GFP to settle in spinal cord and increasing CNTF expression,inducing NG2 positive cells dramatically in the lesions, and reducing Caspase-3 expression significantly, in so much to reducing the demyelination and the axonal degene ration. These fingings suggest that the therapy of transplanting MSCs tmnsfected by Ad-CNTF-IRES-GFP virus benefit in MS.