异体骨髓间充质干细胞对EAE大鼠神经再生保护作用的研究

Neuroprotection and neuroregeneration of allogenic bone marrow mesenchymal stem cells for experimental autoimmune encephalomyelitis(EAE)in rats

目的观察异体骨髓间充质干细胞(BMSCs)移植后在EAE大鼠体内存活、增殖、分化,探讨BMSCs对EAE的神经再生的修复作用及迁徙分化情况。方法 SD雌性大鼠45只,随机分为3组,其中EAE模型组20只,正常对照组5只,EAE模型BMSCs移植组20只。重组免疫蛋白MBP68-86诱导建立EAE的动物模型,在免疫后早期第5天静脉输注Wistar大鼠的BMSCs(4×106/只,0.4mL)。观察输注后大鼠神经功能变化和组织学变化,利用免疫组化方法检测不同时间脑脊髓神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)、2’,3’-环腺苷酸-3’-磷酸二酯酶(CNPase)表达,观察BMSCs移植后EAE大鼠体内神经元、星形胶质细胞和少突胶质细胞数量。结果 EAE大鼠免疫后早期移植BMSCs,神经功能评分无论是高峰期评分还是平均评分显著低于EAE模型组。BMSCs移植组在第7天NSE阳性细胞数较EAE模型组表达水平增多,但并无显著性差异,而第15天、第21天和第30天NSE阳性细胞数明显高于EAE模型组的表达水平(P<0.05)。BMSCs移植组各时间点CNPase染色阳性细胞数明显高于EAE模型组的表达水平(P<0.05),随着时间迁移,BMSCs移植组CNPase染色阳性细胞数表达逐渐增加。BMSCs移植组各个时间点GFAP阳性细胞数与EAE模型组比较无显著性差异。病理检查显示EAE大鼠免疫后早期移植BMSCs可使脑组织炎症反应减轻,脱髓鞘病灶的数目也明显少于EAE模型组。结论预防性静脉输注BMSCs可能通过免疫调节,抑制免疫反应所致的神经细胞损伤,减少神经元和少突胶质细胞的凋亡,从而发挥神经保护作用,但不能排除仍有部分BMSCs在局部微环境信号的诱导下向脱髓鞘区迁徙并分化为髓鞘形成细胞的可能。

Objective To examine the survival ,proliferation and differentiation of allogenic bone marrow mesenchymal stem cells（BMSCs） in EAE rats and to explore the mechanism of neuroprotection after transplantation with BMSCs. Methods Forty‐five SD female rats were randomly divided into three groups ：EAE model group（n=20） ,control group （n=5） and BM‐SCs transplantation group （n=20）. SD rats were immunized with MBP68‐86. Then BMSCs were administered intravenously on day 5 after immunization ,the dose of BMSCs was 4 × 106 （0.4 mL）for each rat. Throughout the study ,neurological symp‐toms of active disease and histopathologic examination were evaluated in rats. The expressions of NSE ,GFAP and CNPase in the spinal cord and the brain were detected by immunohistochemistry technique at different time ,and the number of neurons , astrocytes and oligodendrocytes in EAE rats after BMSCs transplantation were analyzed.Results Compared with EAE model group ,whether the peak neurofunctional scores or the mean neurofunctional scores of neural function evaluation during the course significantly decreased in BMSCs transplantation group. The number of NSE positive cells at 7th day after transplanta‐tion in BMSCs transplantation group were higher than that in EAE model group ,with no significant difference. The numbers of NSE positive cells at 15th ,21st and 30th day after transplantation in BMSCs transplantation group were higher than that in EAE model group（P〈0.05）. The number of CNPase positive cells at different day after transplantation in BMSCs transplanta‐tion group was higher than that in EAE model group（P〈0.05）. The number of CNPase positive cells increased gradually over time. Compared with EAE model group and control group ,the number of GFAP positive cells at different days after transplan‐tation showed no significant difference in BMSCs transplantation group. Pathologic results showed that the early transplanta‐tion of BMSCs in EAT rats could alleviate the inflammatory reaction participated in cerebral tissue ,and the number of demyeli‐nation decreased in BMSCs transplantation group compared with EAE model group. Conclusion The neuroprotective mecha‐nism of BMSCs by the prophylactic intravenous infusion in EAE rats is that BMSCs may inhibit cells injury ,decrease the apop‐tosis of neurons and oligodendrocytes through immunomodulation. We can’t rule out the possibility that some BMSCs may be migrated to the demyelinated foci and differentiated into oligodendrocytes on the condition of local microenvironment.