目的：比较不同代次的人胚胎骨髓间充质干细胞（ bone marrow mesenchymal stem cells，BM-MSCs）移植治疗大鼠脑梗死模型的疗效，包括梗死体积和行为学改变以及疗效产生机制，包括对梗死区激活的小胶质细胞的抑制、产生营养性细胞因子上的差别。方法体外培养人胚胎骨髓来源间充质干细胞，并通过连续传代制作复制性衰老模型。分别收集传第4代和第10代的MSC用于移植。制作大鼠大脑中动脉远端阻塞（ distal middle cerebral artery occlusion，dMCAO）脑梗死模型。实验分假手术组（无脑梗死）、缺血对照组〔脑梗死模型给予0．9％（质量分数）氯化钠注射液〕和MSC细胞治疗组。移植组在造模后1 h经尾静脉移植1×106不同代次的BM-MSC，移植后2、4 d进行行为学评价；移植后2 d部分动物进行梗死体积测定；部分动物灌注取脑，免疫组织化学法计数并对比皮质梗死区激活的小胶质细胞CD68染色的数量；另一部分动物于移植4 d用ELISA法检测梗死灶区域大鼠来源的脑源性神经营养因子（brain-derived neurotrophic factor，BDNF）、胰岛素样生长因子（insulin like growth factor-1，IGF-1）的浓度。结果只有第4代人BM-MSC能够有效减少梗死体积，并在移植后第2天改善脑梗死模型动物的神经功能。移植后第4天，仅第4代BM-MSC移植组脑内BDNF、IGF-1蛋白浓度明显高于缺血对照组，小胶质细胞激活明显低于对照组；第10代MSC对营养性细胞因子和小胶质细胞的影响和缺血对照组相比，差异无统计学意义。结论脑梗死后1 h移植早代次的BM-MSCs会产生减小梗死体积、改善动物行为的作用，但晚代次细胞的作用不明显，原因可能和晚代次细胞促进脑内神经营养因子表达，抑制脑内小胶质细胞激活的能力下降有关。%Objective To compare the effect of intravascular transplantation of human bone marrow derived mesenchymal stromal cells (hMSCs), which at passage 4 and 10, on the cerebral infarct volume and behavioral improvement of cerebral ischemia rats. The therapeutic mechanisms , including reducing microglia activation and increasing neurotrophic factors will be observed.Methods The SD rats were divided into 4 groups, sham, ischemia vehicle, hMSC at passage 4 and passage 10 transplantation group.The distal middle cerebral artery occlusion(dMCAO) was applied in this study.The ischemia vehicle group received dMCAO surgery and intravascular saline injection.For the transplantation group, 1 ×106 hMSCs were intravascularly transplanted into rats’ tail vein at 1 hour after the ischemia onset.At 2 and 4 days after the ischemia onset, behavioral tests were performed.At 2 d after the ischemia onset, infarct volume was measured.At day 4, CD 68, the symbol of activated microglia, were evaluated in the core of ischemic area, meanwhile, the neurotrophic factors, such as brain-derived neurotrophic factor( BDNF) and insulin-like growth factor( IGF-1) in ipsilateral core ischemia cortex was also observed.Results Only hMSCs at passage 4 reduced infarct volume and improved the behavioral outcome of dMCAO rats significantly.At 4 days after the transplantation, the microglia activation of 4th passage hMSC group was significantly lower(P<0.01) than that of ischemia vehicle group in the core area of ischemia cortex.At day 4, BDNF and IGF-1 in the ischemic core were higher in the group of hMSC at passage 4, than that in ischemia vehicle group.Human MSC at passage 10 did not change the neurotrophic cytokines in the core ischemia cortex.Conclusion Human MSC was transplanted at 1 h after the onset of ischemia to treat the ischemia damaged brain.The hMSC at early passage, but not late passage showed therapeutic effects, including decreasing infarct volume and increasing behavioral deficit.The underlying mechanisms of the difference may closely relate to that the ability of late passage hMSC in reducing microglia activation and increasing neurotrophic factors in ischemia core area was not as strong as the MSC at early passage.
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