小鼠非黏附骨髓间充质干细胞移植到缺血性脑损伤部位向神经元样细胞的分化

In vivo differentiation of non-adherent transplanted bone marrow mesenchymal stem cells into neuron-like cells following cerebral ischemia injury in mice

背景:小鼠非黏附骨髓间充质干细胞在体外可以不断形成成纤维细胞集落形成单位,并且可诱导分化为脂肪细胞、成骨细胞和软骨细胞,表现出一定的多分化潜能。目的:探讨小鼠非黏附骨髓间充质干细胞移植到缺血损伤脑内后分化为神经细胞的能力。方法:取β-Gal转基因小鼠,分离双侧股骨、胫骨,全骨髓法分离总骨髓细胞,采用反复转移非黏附的骨髓细胞培养法收集纯化后的第5代非黏附骨髓间充质干细胞,调整细胞浓度为1×1012L-1备用。两组C57BL/6J小鼠均建立大脑中动脉阻塞模型,造模后7d,细胞移植组将第5代非黏附骨髓间充质干细胞悬液3μL定向移植到小鼠脑损伤处,模型组注射等量生理盐水。移植8周后观察供体细胞在缺血脑内微环境中的存活、分布及分化能力。结果与结论:LacZ组织化学染色发现,8周后供体细胞仍可以表达β-Gal蛋白,在缺血侧供体细胞能够存活。免疫组织化学单染和双染后发现,在缺血模型的坏死区及坏死边缘区均可检测到β-Gal阳性的供体细胞,部分细胞还同时表达神经细胞特异性蛋白NeuN及神经胶质细胞特异性标记GFAP。提示小鼠非黏附骨髓间充质干细胞在缺血动物模型的脑内能够存活、迁移,部分细胞还能分化为成熟的神经元样细胞或神经胶质细胞,参与脑损伤修复。

BACKGROUND：Non-adherent mesenchymal stem cells （NA-MSCs） can form colony forming unit of fibroblasts and induce the differentiation into adipocytes,osteoblasts,and chondrocytes.OBJECTIVE：To determine whether non-adherent mesenchymal stem cells （NA-MSCs） in adult mouse bone marrow could differentiate into neuron-like cells in cerebral ischemic region.METHODS：Bilateral femur and tibia of β-Gal transgenic mice was separated,and repeated-transfer was used to collect the fifth-passage purified NA-MSCs which were adjusted at concentration of 1×1012/L.Middle cerebral artery occlusion was established in the two groups.After 7 days,3 μL fifth-passaged NA-MSCs suspension was injected into cerebral ischemic region in the transplantation group,while an equal amount of saline was injected into model group.Survival,distribution,and differentiation of donor cells in cerebral ischemic region were observed at 8 weeks after transplantation.RESULTS AND CONCLUSION：LacZ staining showed that donor cells could express β-Gal protein after 8 weeks and survived in the ischemic region.Simple and double immunohistochemical staining indicated that β-Gal-positive donor cells were detected in necrotic region and at necrotic edge of ischemic model.Additionally,partial cells could express neuro-specific NeuN protein and glial cell-specific GFAP.NA-MSCs are able to survive and migrate in cerebral ischemic region;moreover,partial NA-MSCs can differentiate into mature neuron-like cells or glial cells which participate in repairing brain injury.