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目的研究新生鼠缺氧缺血性脑损伤后经脑室移植人胚胎脑神经干细胞后,植入细胞的存活、迁移及分化情况。方法孕12周人胚胎神经干细胞在含有碱性成纤维细胞生长因子、表皮细胞生长因子和白血病细胞抑制因子的N2培养基(D/F12+N2+B27)中培养11天。生后7天的新生SD大鼠32只建立缺氧缺血性脑病模型,3天后将培养的人神经干细胞制成悬液(1.0×105/μl),采用立体定向移植至实验动物损伤侧脑室。移植后1、2、4周及3个月(每时间点各8只)取脑组织行HE染色和免疫组织化学分析。结果移植后1周大量抗人细胞核蛋白抗体阳性的植入细胞从脑室沿胼胝体向外迁移并到达损伤区,以损伤侧皮层、海马多见,在不同区域可见与宿主细胞类似的分化的植入细胞:移行至皮层的植入细胞85%分化为抗人细胞核蛋白神经丝蛋白或抗人核蛋白神经元管蛋白抗体双阳性的皮层细胞,而位于移行区域的植入细胞60%表达为抗人核蛋白胶质纤维酸性蛋白抗体双阳性的星形胶质细胞,其余细胞仍在迁移中未分化。移植后2周更多的移植细胞到达损伤区,移植后4周及3个月植入细胞数较前明显减少。结论人胚胎神经干细胞经脑室移植至缺氧缺血性脑损伤的鼠脑后能存活、迁移,并依迁移区域的不同分化为神经元和星形胶质细胞。
Objective To investigate the survival, migration and differentiation of transplanted human neural stem cells in neonatal rats after hypoxic-ischemic brain damage. Methods Human embryonic neural stem cells were cultured for 11 days in N2 medium (D / F12 + N2 + B27) containing basic fibroblast growth factor, epidermal growth factor and leukemia cell inhibitory factor. Thirty-two newborn SD rats aged 7 days were established hypoxic-ischemic encephalopathy model. Three days later, the cultured human neural stem cells were suspended in 1.0 × 10 5 / μl suspension and fixed in lateral ventricle of experimental animals . At 1, 2, 4 weeks and 3 months after transplantation (8 for each time point), brain tissue was harvested for HE staining and immunohistochemical analysis. Results A week after transplantation, a large number of anti-human nucleoprotein antibody-positive implanted cells migrated outward from the ventricle along the corpus callosum and reached the lesion to injure the lateral cortex. The hippocampus was more common. Differentiated implants similar to the host cells were observed in different regions Cells: 85% of implanted cells migrating into the cortex differentiate into double-positive cortical cells that are anti-human nuclear protein neurofilament or anti-human nuclear protein neuronal tubulin antibody while 60% of implanted cells located in the transitional region are expressed as anti-human Nuclear protein glial fibrillary acidic protein antibody double positive astrocytes, the remaining cells are still undifferentiated during migration. Two weeks after transplantation, more transplanted cells reached the lesion area, and the number of implanted cells at 4 weeks and 3 months after transplantation was significantly reduced compared with that before transplantation. Conclusion Human embryonic neural stem cells can survive and migrate transplanted into the brain of rats with hypoxic-ischemic brain injury via brain ventricle, and differentiate into neurons and astrocytes according to their migration regions.