人胚神经干细胞植入大鼠脑梗死区后的存活和分化

Survival and differentiation of human embryonic neural stem cells in cerebral infarction area of rats following implantation

背景：神经干细胞移植可明显改善受损的神经功能,但在体内外多种因素的影响下,移植的神经干细胞其分化数量和分化方向受到限制。目的：观察人胚神经干细胞植入大鼠脑梗死区后的存活、迁移和分化情况。设计、时间及地点：细胞学体内观察,于2007-04/2008-04在泸州医学院附属医院分子生物实验室完成。材料：SD雌性大鼠30只,由中国医学科学院实验动物研究提供。流产胎儿由泸州医学院附属医院妇产科提供。方法：取流产胎儿大脑皮质,剪碎后消化离心,过滤后取沉淀细胞重新悬浮,加入含成纤维细胞生长因子、表皮细胞生长因子、白血病抑制因子的DMEM/F12培养基,体外培养获得人胚神经干细胞。30只大鼠通过结扎颈外动脉建立脑梗死模型,行走时向右侧转圈者为成功模型,剔除5只无上述明显症状的失败模型鼠。于造模后24h,以前囟尾侧0.8mm,中线右外侧1.2mm为注射点,每只大鼠移植行BrdU标记的1×10^9L^-1人胚神经干细胞悬液10μL,深度为硬脑膜下3.0-3.2mm。主要观察指标：免疫组织化学及免疫荧光染色观察植入的人胚神经干细胞在脑梗死区的存活、迁移和分化状态。结果：人胚神经干细胞体外培养48h后聚集成球悬浮生长,传三四代后加入血清诱导可见巢蛋白免疫荧光染色呈阳性,发出绿色荧光并聚集成球。BrdU阳性细胞为椭圆形棕褐色,移植后第2,4周均可见其在脑梗死区存活并向周围迁移,且第4周时迁移的范围更广。移植后2周,皮质颗粒层和皮质下均见较多的BrdU阳性细胞,且BrdU/微管相关蛋白2双阳性细胞多于BrdU/胶质纤维酸性蛋白双阳性细胞;移植后4周脑梗死区BrdU阳性细胞数明显减少,主要存在于脉络丛和微血管中,且BrdU/胶质纤维酸性蛋白双阳性细胞多于BrdU/微管相关蛋白2双阳性细胞。结论：人胚神经干细胞能存活于脑梗死的环境中,并逐渐分化成神经元或星形胶质细胞。移植后期脑实质内存活的神经干细胞明显减少,大量迁移到侧脑室脉络丛和脑表面微血管内,被内皮吞噬系统消化。

BACKGROUND： Neural stem ceil transplantation can effectively improve nerve function, but differentiated number and direction of transplanted neural stern cells （NSCs） are limited. OBJECTIVE： To observe the survival, migration and differentiation of human embryonic NSCs implanted into rats with cerebral infarction. DESIGN, TIME AND SETTING： This cytology in vivo experiment was performed at the Laboratory of Molecular Organism, Affiliated Hospital of Luzhou Medical College from April 2007 to April 2008. MATERIALS： Thirty female Sprague Dawley rats were supplied by Experimental Animal Institute, Chinese Academy of Medical Sciences. Aborted fetus was obtained from Department of Gynaecology and Obstetrics, Hospital Affiliated to Luzhou Medical College. METHODS： Cerebral cortex from aborted fetus was obtained, cut into pieces, digested and centrifuged. After filtration, precipitated cells were resuspended, and incubated in DMEM/F12, supplemented with fibroblast growth factor, epidermal growth factor and leukaemia inhibitory factor to obtain human embryonic NSCs in vitro. A total of 30 rat models of cerebral infarction were established by deligation of external carotid artery. Rat models that circled to the right suggested successful mode）s. Five failure models were eliminated. 10μL of BrdU-labeled human embryonic NSCs at 1×10^9/L were implanted into infarction area at 0.8 mm from the anterior fontanel caudal, 1.2 mm from right lateral side of the midline after 24 hours and 3.0 3.2 mm below the cerebral dura mater. MAIN OUTCOME MEASURES： Survival, immigration and differentiation of human embryonic NSCs implanted in rats with cerebral infarction were observed by immunohistochemistry and immunofluorescence staining, RESULTS： Human embryonic NSCs gathered and suspended following 48 hours of in vitro culture. At the third to fourth passage, Nestin-positive cells were found by immunofluorescence staining following serum induction. Green fluorescence was detected. Cells gathered in neurospheres. BrdU-positive cells were oval and brown. At 2 and 4 weeks after implantation, BrdU-positive cells survived and migrated, and they migrated more widely at 4 weeks after implantation. At 2 week after implantation, more BrdU-positive cells were found in the subcortical granular layer and subcortex, and BrdU/microtubule-associated protein-2 （MAP-2） -positive cells were more than BrdU/glial fibrillary acidic protein （GFAP）-positive cells. At 4 weeks after implantation, BrdU-positive cells were significantly reduced, and found in choroid plexus and blood capillary, and BrdU/GFAP-positive cells were more than BrdU/MAP-2- positive cells. CONCLUSION： Human embryonic NSCs can survive in the region of infarction; gradually differentiate into neurons or astrocytes during rehabilitation. In later period, NSCs in the brain parenchyma are significantly reduced, and gradually migrates into the choroids plexus of the lateral ventricle and capillary of brain surface, which are digested by endothelial phagocytes.