摘要
目的从新生鼠大脑皮层分离培养神经干细胞(neuralstemcells,NSC),并将这些NSC植入局灶性缺血损伤鼠的大脑皮层,观察其植入同胞鼠后的细胞替代作用。方法应用含表皮生长因子(EGF)和碱性成纤维细胞生长因子(bFGF或FGF-2)的无血清DMEM/F12(1∶1)培养液获得新生鼠NSC球,酶免疫技术检测NSC标志nestin和诱导分化实验证明所获得的细胞球是NSC。在细胞球的体外培养中掺入BrdU,标记NSC,用于移植实验。鼠出生后第4天,开颅去脑膜阻断脑皮层血液供应建立新生鼠脑皮层局灶性缺血动物模型,术后第4天(出生后第8天)将标记好的NSC直接注射到缺血损伤灶的边缘区。实验鼠分为损伤移植组、损伤对照组和假手术对照组。于移植后第4、7、14、30天杀鼠取脑,进行BrdU标记的酶免疫组化染色,检查植入细胞的存活和迁移情况。结果新生鼠大脑皮层在体外培养一周,可以获得漂浮生长的、表达nestin的、具有分化为神经元和星形胶质细胞潜能的NSC球。移植实验发现:植入细胞有向大脑皮层缺血损伤灶周边迁移的特点,促进损伤灶组织结构的修复,损伤获得了很好的细胞修复作用。镜下可见灶区有满视野BrdU标记的阳性细胞,颗粒层集聚密度最大,未见有远部位的细胞迁移。假手术对照鼠脑内细胞逐渐减少,至移植后第14天只能检测到零星散在阳性细胞,明显较损伤移植鼠少。结论新生鼠大脑皮层内存在NSC。缺血性损伤可刺激移植的NSC增殖与迁移。移植的NSC对于大脑皮层局灶性缺血损伤灶的完整修复发挥了重要作用。
Objective To isolate and culture the neural stem cells （NSCs） from the cerebral cortex of newborn rats, and investigate the cell-eplace responses of the NSCs transplanted into the sibling rats with focal cerebral cortex ischemic lesion. Methods The serum-free medium DMEM/F12 （1 ： 1 ） containing basic flbroblast growth factor （bFGF or FGF2） and epidermal growth factor （EGF） was used to culture the neural stem cell spheres. The NSCs were identified by detecting the neural stem cell marker nestin with enzyme immune assay and inducing neural stem cell spheres to differentiation. The NSCs which would be used in the transplantation experiment were labeled by BrdU incorporation when cultured in vitro. The focal ischemic models were made by opening the skulls and removing the cerebral menings of 4-day-old rats to stop the blood supply for the neopallium. The BrdU-labeled NSCs were transplanted into the cerebral lesion boundary zones of the focal ischemic sibling rat models. The experiment rats were divided into lesion-ransplantation group, lesion-control group and sham-operation-control group. Recipients were killed and the brains were examined by detecting the BrdU-labeled cells with enzyme immunohistochemistry at 4,7,14,30 days postgrafting, indicating the grafts living and migration in the host. Results The neural stem cell spheres, which floated and grew in medium, expressed nestin, as well as gave rise to neurons and astrocytes, could be obtained through culturing the cells derived from the cerebral cortex of newborn rats in vitro for a week. In the transplantation of the NSCs, the grafts were easy to migrate along the boundary zones of the focal ischemic lesions, and promoted the restore of the tissue structures in the damaged areas, the damage recovered well through the cell-replace responses. The BrdU-labeled positive cells in the lesion areas were full of the visual fields under microscope, the greatest density of the positive cells were focused in the granular layer of the injured cerebral cortex and not found in remote sites from the lesion. The number of BrdU-labeled cells gradually decreased in the brains of the sham-operation-control rats, only a few positive cells were found when examined at 14 days postgrafting, significantly less than that in the lesion-transplantation rats. Conclusions NSCs exist in the cerebral cortex of newborn rats. The ischemia can promote proliferation and graft of NSCs. The grafted NSCs play an important role in the recovery of focal cerebral cortex ischemic lesion.
出处
《中华围产医学杂志》
CAS
2005年第4期255-258,i0003,共5页
Chinese Journal of Perinatal Medicine
