脑缺血后移植骨髓神经组织定向干细胞的分布与分化

Distribution and differentiation of transplanted neural tissue committed stem cells to the brain after ischemic stroke

目的观察骨髓神经组织定向干细胞(NTCSCs)经静脉移植后在脑缺血大鼠脑内的分布和分化情况。方法采用DMEM/F12(1:1)无血清神经干细胞条件培养基,从SD大鼠骨髓中分离、培养NTCSCs,应用免疫组化法对所获细胞及其分化情况进行鉴定,RT-PCR检测Nestin、CXCR4、CD31、βⅢ-tubulin及GFAP mRNA的表达水平。采用改良Zea-Longa线栓法制作SD大鼠大脑中动脉栓塞(MCAO)模型,造模成功24h后经尾静脉注射1ml DAPI标记的NTCSCs。移植后第1、3、5、7d处死大鼠,制备脑组织冰冻切片,荧光显微镜观察NTCSCs在脑组织的分布,激光共聚焦显微镜观察NTCSCs的分化情况。结果分离培养获得的NTCSCs在神经干细胞培养条件下形成神经球,并表达神经干细胞标志巢蛋白Nestin及SDF-1受体CXCR4,可分化成βⅢ-tubulin、GFAP阳性细胞;RT-PCR检测结果显示NTCSCs表达Nestin、CXCR4、CD31、βⅢ-tubulin、GFAP mRNA。静脉移植后NTCSCs能够进入大鼠脑内,移植后第3天观察到DAPI标记的NTCSCs主要分布在缺血损伤部位,第7天时有少量NTCSCs分化为GFAP阳性细胞。结论分离培养的NTCSCs具有神经干细胞特性,移植后可能进入缺血脑组织,部分细胞表达GFAP。

Objective To investigate the distribution and differentiation of neural tissue committed stem cells （NTCSCs） by venous transplantation to the brain after middle cerebral artery occlusion in rats. Methods Monocytes were collected from bone marrow and cultured in DMEM/F12 serum free medium plus EGF, bFGF and 2% B27 to obtain NTCSCs. Expressions of nestin, CXCR4, CD31, β - III-tubulin and GFAP mRNA in NTCSCs were detected by KT-PCR. Modified Zea-Longa suture method was used to prepare middle cerebral artery occlusion （MCAO） model. The NTCSCs were labeled with DAPI and were injected through the caudal vein of the rats. At 1,3,5,7 days after injection, the rats were sacrificed and the brain tissues were prepared with frozen section in transplantation group. The distribution and differentiation of transplanted NTCSCs were observed under fluorescent microscope and laser confocal microscope respectively after immunofluorescent staining. Results The cultured NTCSC formed neural stem cell spheres in neural stem cell culture medium, and they expressed Nestin and SDF-1 receptor CXCK4 at protein level. RT-PCK showed that NTCSCs expressed Nestin, CXCR4, CD31, β-III-tubulin and GFAP mRNA. DAPI-labelled NTCSCs could be observed in the brain 3 days after transplantation and a few GFAP expressing cells could be found on the 7th day. Conclusion The NTCSCs by intravenous transplantation can enter rat brain, and they distribute mainly in ischemic tissues. Some NTCSCs labelled with DAPI in the brain tissue could express GFAP.