促红细胞生成素基因修饰骨髓间充质干细胞移植治疗大鼠脑梗死

摘要

背景：促红细胞生成素具有神经元的保护及促进神经再生的作用。目的：观察促红细胞生成素修饰的骨髓间充质干细胞尾静脉移植对大鼠脑梗死的治疗效果。方法：用Western blot鉴定外源人促红细胞生成素基因在骨髓间充质干细胞中的表达。采用线栓法建立大鼠大脑中动脉阻塞模型，模型组尾静脉注射PBS、骨髓间充质干细胞组注射骨髓间充质干细胞悬液，促红细胞生成素-骨髓间充质干细胞组注射转染了促红细胞生成素的骨髓间充质干细胞悬液。移植后3 d及移植后1，2，3，4周行改良神经功能评分，检测神经功能的损伤情况。移植后4周将大鼠麻醉后断头取脑，RT-PCR 检测脑组织中 bcl-2/bax 基因表达变化，用原位末端标记法测定细胞凋亡情况、苏木精-伊红染色及荧光显微镜观察PKH26标记的骨髓间充质干细胞的存活和分布情况。结果与结论：Western blot 结果显示，转染人促红细胞生成素基因的骨髓间充质干细胞体外能表达促红细胞生成素蛋白。移植后1-4周，骨髓间充质干细胞组和促红细胞生成素-骨髓间充质干细胞组神经缺损评分明显低于模型组(P<0.05，P<0.01)。与骨髓间充质干细胞组及模型组相比，大鼠脑梗死区组织促红细胞生成素-骨髓间充质干细胞组bcl-2基因的表达明显增高(P<0.05)，bax基因的表达明显降低(P<0.05)，凋亡细胞明显减少，PKH26阳性细胞数明显增多(P <0.05)。结果证实，促红细胞生成素修饰的骨髓间充质干细胞尾静脉移植对脑梗死大鼠脑梗死疗效较好。%BACKGROUND:Previous studies have shown that erythropoietin can protect neurons and promote nerve regeneration. OBJECTIVE:To explore the therapeutic effect of erythropoietin gene-modified bone marrow mesenchymal stem celltransplantation via caudal vein on rat cerebral infarction. METHODS:Western blot assay was used to identify the expression of exogenous erythropoietin in bone marrow mesenchymal stem cells. A model of middle cerebral artery occlusion was established in Wistar rats using thread method. And then, model rats were randomly divided into model group (PBS injection via the caudal vein), transplantation group (transplantation of bone marrow mesenchymal stem cellsuspension), erythropoietin group (transplantation of erythropoietin-transfected bone marrow mesenchymal stem cellsuspension). Neurologic function was assessed at 3 days, 1, 2, 3, 4 weeks after celltransplantation. Four weeks after transplantation, the rats were decapitated after anesthesia to take brain tissues for RT-PCR detection of Bcl-2/Bax gene expression. cellapoptosis was measured by TUNEL. Hematoxylin-eosin staining and fluorescence microscopy were employed to observe the survival and distribution of PKH26-labeled bone marrow mesenchymal stem cells. RESULTS AND CONCLUSION:Western blot results showed that erythropoietin-transfected bone marrow mesenchymal stem cells could express the erythropoietin in vitro. At 1, 2, 3, 4 weeks after transplantation, the neurological defect scores in the transplantation group and erythropoietin group were significantly lower than those in the model group (P<0.05, P<0.01). The expression of bcl-2 gene in the infarct region was significantly higher in the erythropoietin group than the transplantation and model groups (P<0.05), but the expression of bax was significantly decreased (P<0.05). In the erythropoietin group, the number of apoptotic cells was reduced, and the number of PKH26 positive cells was increased as compared with the other two groups (P<0.05). These findings indicate that the transplantation of erythropoietin-modified bone marrow mesenchymal stem cells via caudal vein can significantly improve the neurological function in the rats with cerebral infarction.