神经干细胞增殖能力的组织差异性研究

Regional specificity in proliferation capacity of neural stem cells from hippocampus and epithalamus:study with rats

目的从胚胎大鼠海马和上丘脑区中分离、培养神经干细胞,并进行体外增殖能力的比较。方法采用机械分离,无血清传代培养法从胚鼠海马和上丘脑获得神经干细胞。动态观察细胞生长情况,测定其体外生长曲线。使用免疫荧光法对两种来源的细胞克隆进行鉴定,激光共聚焦显微镜三维形态观察。使用扫描电镜观察神经干细胞克隆球的表面形态特征。将神经干细胞与Brdu共孵育,观察体外增殖情况。结果海马源胚胎神经干细胞的体外生长情况与上丘源干细胞基本相同,但前者的增殖速度相对较慢。两种来源的干细胞克隆球具有相似的超微结构。在相差显微镜和激光共聚焦显微镜下,两种来源的神经干细胞形态相似。与Brdu共孵育后,上丘源神经干细胞的阳性率高于海马源干细胞。从细胞生长曲线上可见两种来源的干细胞呈现了相同的增殖趋势,但生长曲线并不相同。从培养第8d起,上丘源胚胎神经细胞的活细胞记数就高于海马源干细胞。结论使用机械分离、无血清培养的方法可以获得胚胎海马源与上丘源神经干细胞,两者具有相似的形态结构,但上丘来源的神经干细胞增殖速度高于海马来源的神经干细胞。

Objective To compare the regional specificity in proliferation capacity of neural stem cells derived from hippocampus and epithalamus ： study with rats. Methods Neural stem cells （NSCs） were separated mechanically from the hippoeampus and the epithalamus of an embryo of SD rat respectively and cultured in serum-free medium to observe the growth status dynamically and their in vitro growth curves were drawn. Single cell clones were established and identified by immunofluorescenee staining. The surface morphology of the clone balls were observed by using scanning electron microscope. The uhrastrueture was observed by scanning electron microscopy. The 3D morphology was observed under a laser confocal microscope. NSCs were co-incubated with 5-bromodeoxyuridine （Brdu） and immunofluoreseenee staining was used to observe their in vitro proliferation potentials. Studies on. Results The in vitro growth of the hippocampus-derived NSCs was basically the same as that of the epithalamus-derived NSCs, however, the culture fluid of the epithalamus-derived NSCs became yellowish 18 hours after the inoculation, significantly earlier than that of the hippoeampus-derived NSCs. Showing a faster multiplication rate of the latter. Scanning electron microscopy showed that these two types of NSCs had a similar ultrastructural feature. Phase contrast microscopy and laser confocal microscopy showed similar form in the NSCs from two different sources. Co-incubation with found that the Brdu positive rate of the epithalamus-derived NSCs was 74. 87%, significantly higher than that of the hippocampus-derived NSCs （ 63.07% , P 〈 0.05 ）. The cell growth curves of the NSCs from both sources displayed the same multiplication trend, however, the NSC count of the epithalamus-derived NSCs at the 8 th, 12 th, 16 th, 20 th, and 24 th days after culture were all higher than those of the corresponding hippocampus-derived NSCs at the same time-points （ all P 〈 0. 05）. Conclusion Both embryonic hippocampus-derived and epithalamus-derived NSCs can be acquired by using mechanical separation and serum-free culture method. The two types of cells have a similar morphological structure and possess the in vitro multiplication capacity. However, the multiplication rate of the epithalamus-derived NSCs is higher than that of the hippocampus-derived NSCs.