Prokineticin2（PK2）對(duì)神經(jīng)干細(xì)胞遷移及向神經(jīng)元定向分化作用的研究
發(fā)布時(shí)間：2019-05-28 17:53
【摘要】： 目的:探討Prokineticin2(PK2)在體外和局灶性腦缺血大鼠腦內(nèi)對(duì)神經(jīng)干細(xì)胞(Neural stem cells,NSCs)遷移及分化的影響。 方法:利用無(wú)血清培養(yǎng)和細(xì)胞克隆培養(yǎng)技術(shù),從新生Wistar大鼠海馬分離NSCs,進(jìn)行體外擴(kuò)增、培養(yǎng)、傳代,采用免疫組織化學(xué)法做Nestin染色鑒定;將傳三代的NSCs分散放置于PK2擴(kuò)散源周圍,PK2擴(kuò)散源由5%的瓊脂和PK2制成,分為1nmol PK2組、30nmol PK2組、60nmolPK組和對(duì)照組。培養(yǎng)于含0.3%瓊脂并撤去bFGF的DMEM/F12培養(yǎng)液中。連續(xù)14天顯微鏡下觀察PK2誘導(dǎo)NSCs遷移情況。并篩選出誘導(dǎo)NSCs遷移作用最佳的PK2劑量。采用線栓法把Wistar大鼠制成大腦中動(dòng)脈梗塞(middle cerebral artery occlusion, MCAO)模型,按照隨機(jī)數(shù)字法將MCAO模型大鼠分成3組:模型組、對(duì)照組(單純移植NSCs)、PK2組(PK2與NSCs聯(lián)合移植),24只/組。運(yùn)用立體定位儀,把經(jīng)過(guò)BrdU標(biāo)記的NSCs移植入MCAO模型大鼠的右側(cè)側(cè)腦室,將體外實(shí)驗(yàn)中篩選的最佳劑量PK2注入MCAO模型大鼠的右側(cè)病灶區(qū)。各組大鼠分別于移植后1d、3d、7d、14d斷頭取腦,以視交叉為中心切片,用免疫組織化學(xué)方法觀察BrdU陽(yáng)性NSCs遷移情況,用免疫熒光方法觀察遷移到病灶區(qū)NSCs的分化情況。 結(jié)果:①體外研究表明,與對(duì)照組比較,不同劑量的PK2均具有誘導(dǎo)NSCs遷移作用,且1nmol PK2誘導(dǎo)遷移的作用最明顯,統(tǒng)計(jì)學(xué)有顯著性差異(p0.01)。②在NSCs移植入MCAO模型大鼠腦內(nèi)1d,在室管膜下區(qū)(sub-ventricular zone,SVZ)PK2組和對(duì)照組BrdU陽(yáng)性細(xì)胞數(shù)與模型組相比明顯增多,差異具有統(tǒng)計(jì)學(xué)意義(p0.05);PK2組與對(duì)照組相比BrdU陽(yáng)性細(xì)胞數(shù)無(wú)明顯差異(p0.05);在病灶區(qū)各組有散在的BrdU陽(yáng)性細(xì)胞,數(shù)量沒有明顯差異(p0.05)。移植后3d、7d和14d,在SVZ,PK2組BrdU陽(yáng)性細(xì)胞明顯多于模型組和對(duì)照組,差異具有統(tǒng)計(jì)學(xué)意義(p0.05);在病灶區(qū),PK2組BrdU陽(yáng)性細(xì)胞數(shù)多于模型組和對(duì)照組,且遷移到病灶區(qū)的BrdU陽(yáng)性NSCs向神經(jīng)元分化,差異具有統(tǒng)計(jì)學(xué)意義(p0.05)。 結(jié)論:PK2對(duì)體外培養(yǎng)的NSCs有誘導(dǎo)遷移作用,以1nmolPK2作用最明顯;PK2對(duì)移植入MCAO模型大鼠側(cè)腦室內(nèi)的NSCs有誘導(dǎo)遷移的作用;PK2促進(jìn)NSCs向神經(jīng)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.
【學(xué)位授予單位】：佳木斯大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R329
[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.
【學(xué)位授予單位】：佳木斯大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R329
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