Prokineticin2（PK2）对神经干细胞迁移及向神经元定向分化作用的研究

发布时间：2019-05-28 17:53

【摘要】： 目的:探讨Prokineticin2(PK2)在体外和局灶性脑缺血大鼠脑内对神经干细胞(Neural stem cells,NSCs)迁移及分化的影响。 方法:利用无血清培养和细胞克隆培养技术,从新生Wistar大鼠海马分离NSCs,进行体外扩增、培养、传代,采用免疫组织化学法做Nestin染色鉴定;将传三代的NSCs分散放置于PK2扩散源周围,PK2扩散源由5%的琼脂和PK2制成,分为1nmol PK2组、30nmol PK2组、60nmolPK组和对照组。培养于含0.3%琼脂并撤去bFGF的DMEM/F12培养液中。连续14天显微镜下观察PK2诱导NSCs迁移情况。并筛选出诱导NSCs迁移作用最佳的PK2剂量。采用线栓法把Wistar大鼠制成大脑中动脉梗塞(middle cerebral artery occlusion, MCAO)模型,按照随机数字法将MCAO模型大鼠分成3组:模型组、对照组(单纯移植NSCs)、PK2组(PK2与NSCs联合移植),24只/组。运用立体定位仪,把经过BrdU标记的NSCs移植入MCAO模型大鼠的右侧侧脑室,将体外实验中筛选的最佳剂量PK2注入MCAO模型大鼠的右侧病灶区。各组大鼠分别于移植后1d、3d、7d、14d断头取脑,以视交叉为中心切片,用免疫组织化学方法观察BrdU阳性NSCs迁移情况,用免疫荧光方法观察迁移到病灶区NSCs的分化情况。 结果:①体外研究表明,与对照组比较,不同剂量的PK2均具有诱导NSCs迁移作用,且1nmol PK2诱导迁移的作用最明显,统计学有显著性差异(p0.01)。②在NSCs移植入MCAO模型大鼠脑内1d,在室管膜下区(sub-ventricular zone,SVZ)PK2组和对照组BrdU阳性细胞数与模型组相比明显增多,差异具有统计学意义(p0.05);PK2组与对照组相比BrdU阳性细胞数无明显差异(p0.05);在病灶区各组有散在的BrdU阳性细胞,数量没有明显差异(p0.05)。移植后3d、7d和14d,在SVZ,PK2组BrdU阳性细胞明显多于模型组和对照组,差异具有统计学意义(p0.05);在病灶区,PK2组BrdU阳性细胞数多于模型组和对照组,且迁移到病灶区的BrdU阳性NSCs向神经元分化,差异具有统计学意义(p0.05)。 结论:PK2对体外培养的NSCs有诱导迁移作用,以1nmolPK2作用最明显;PK2对移植入MCAO模型大鼠侧脑室内的NSCs有诱导迁移的作用;PK2促进NSCs向神经元分化。
[Abstract]:Aim: to investigate the effects of Prokineticin2 (PK2) on the migration and differentiation of neural stem cells (Neural stem cells,NSCs) in vitro and in rats with focal cerebral ischemia. Methods: NSCs, was isolated from the hippocampus of neonatal Wistar rats by serum-free culture and cell clone culture. NSCs, was amplified, cultured, subcultured and identified by Nestin staining. The third generation of NSCs was scattered around the PK2 diffusion source. The PK2 diffusion source was made of 5% Agar and PK2 and was divided into 1nmol PK2 group, 30nmol PK2 group, 60nmolPK group and control group. Cultured in DMEM/F12 medium containing 0.3% Agar and removing bFGF. The migration of NSCs induced by PK2 was observed under microscope for 14 days. The best dose of PK2 to induce NSCs migration was selected. The (middle cerebral artery occlusion, MCAO) model of middle cerebral artery infarction in Wistar rats was established by suture method. According to the random digital method, the MCAO model rats were divided into three groups: model group, control group (simple transplantation of NSCs), PK2 group (PK2 and NSCs combined transplantation), the model group was divided into three groups: model group, control group (combined transplantation of PK2 and NSCs). 24 rats / group. The BrdU labeled NSCs was transplanted into the right lateral ventricle of MCAO model rats by stereotactic instrument. The best dose of PK2 selected in vitro was injected into the right focal area of MCAO model rats. The brains of rats in each group were decapitated on the 1st day, 3rd day, 7th day and 14th day after transplantation, and the migration of BrdU positive NSCs was observed by immunohistochemistry and the differentiation of NSCs migrated to the focal area was observed by immunofluorescence method with optic chiasma as the central section. Results: 1 compared with the control group, different doses of PK2 could induce NSCs migration, and 1nmol PK2 induced migration was the most obvious. There was significant difference in statistics (p0.01). 2 the number of BrdU positive cells in subependymal area (sub-ventricular zone,SVZ) PK2 group and control group was significantly higher than that in model group 1 day after transplantation of NSCs into the brain of MCAO model rats. The difference was statistically significant (p0.05). There was no significant difference in the number of BrdU positive cells between PK2 group and control group (p0.05), but there was no significant difference in the number of BrdU positive cells in each group (p0.05). On the 3rd day, 7th day and 14th day after transplantation, the number of BrdU positive cells in SVZ,PK2 group was significantly higher than that in model group and control group (p0.05). In the focal area, the number of BrdU positive cells in the PK2 group was higher than that in the model group and the control group, and the BrdU positive NSCs migrated to the focal area differentiated into neurons, the difference was statistically significant (p0.05). Conclusion: PK2 can induce migration of NSCs cultured in vitro, especially 1nmolPK2, PK2 can induce migration of NSCs in lateral ventricle of MCAO model rats, and PK2 promotes NSCs differentiation into neurons.
【学位授予单位】：佳木斯大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

【参考文献】

相关期刊论文 前4条

1 戴宜武;神经干细胞发育过程中分化、迁移调控机制研究现状[J];中华神经医学杂志;2003年02期

2 李巍,李成仁,蔡文琴,周德山,余争平;微波辐射对离体培养人神经干细胞增殖、分化及凋亡的影响[J];第三军医大学学报;2003年01期

3 刘仕勇,张可成,杨辉,蔡文琴,何家全;成年大鼠纹状体区神经干细胞的分离培养[J];解剖学报;2000年01期

4 郑志z，

本文编号：2487240