本文選題：骨髓間充質(zhì)干細胞　切入點：神經(jīng)干樣細胞　出處：《浙江大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的探討骨髓間充質(zhì)干細胞(bone marrow mesenchymal stem cells,BMSCs)來源的神經(jīng)干樣細胞(bone marrow mesenchymal stemcells-derived neural stem-like cells,MS-NSCs)對急性重癥一氧化碳中毒大鼠腦結(jié)構(gòu)和功能的影響,并對MS-NSCs和BMSCs進行移植后治療效果的比較評價。 方法體外分離90g大鼠長骨干內(nèi)的BMSCs,運用全骨髓法貼壁培養(yǎng),定時換液,祛除懸浮的血細胞和其它類干細胞如造血干細胞;運用生物因子的方法將BMSCs向MS-NSCs定向誘導(dǎo),見克隆增生,并亦定時換液補充營養(yǎng)與能量;運用細胞免疫組化的方法監(jiān)測兩類干細胞在神經(jīng)標(biāo)志蛋白如神經(jīng)特異性核蛋白(neuron-specific nuclearprotein,NeuN)、巢蛋白(nestin)、CD11b等表達與分泌情況;10μmol/l BrdU標(biāo)記預(yù)移植的兩類干細胞(BMSCs和MS-NSCs)。建立成年大鼠中毒模型并復(fù)蘇24h后,將細胞經(jīng)左頸內(nèi)動脈移植入腦內(nèi),以神經(jīng)功能嚴重度評分(neurological severity score,NSS)和Morris水迷宮試驗(Morris Water Maze,MWM)評價腦功能的改善程度,5周后病理學(xué)上評價腦灰白質(zhì)結(jié)構(gòu)的重塑情況,包括HE染色,髓鞘染色,膠質(zhì)纖維酸性蛋白(glial fibrillary acidic protein,GFAP)和2、3-環(huán)核苷酸磷酸二脂酶(2′3′-cyclic nucleotide 3′-phosphodiesterase,CNP)免疫組化染色,BrdU免疫組化染色,BrdU和神經(jīng)標(biāo)志蛋白如GFAP、CNP、CD11b及微管相關(guān)蛋白(microtubule-associatedprotein 2,MAP2)免疫熒光雙染色等。 結(jié)果重癥CO中毒后5周,大鼠腦內(nèi)白質(zhì)存在局灶脫髓鞘現(xiàn)象,灰質(zhì)如皮層、海馬等處散在存在局灶壞死后腔隙;兩類干細胞移植都能夠改善白質(zhì)結(jié)構(gòu)的有序性和致密性及脫髓鞘情況,促進認知記憶功能的恢復(fù),且這種功能恢復(fù)在MS-NSCs移植組中更明顯(p＜0.05);同時在皮層、海馬、基底節(jié)局部受損區(qū)域和疏松白質(zhì)內(nèi)檢測到BrdU陽性細胞,且在MS-NSCs移植組中此陽性細胞數(shù)更多(p＜0.05)。同時MS-NSCs移植組中某些區(qū)域內(nèi)干細胞向下游終末神經(jīng)細胞分化的比例更高。 結(jié)論MS-NSCs能重塑急性重癥一氧化碳中毒大鼠的腦結(jié)構(gòu)和功能,且這種作用較BMSCs更明顯,提示前者可能是更佳的種子細胞。
[Abstract]:Objective to investigate the effects of bone marrow mesenchymal stemcells-derived neural stem-like cells MS-NSCs derived from bone marrow mesenchymal stem cells (BMSCs) on brain structure and function in acute severe carbon monoxide poisoning rats. Methods BMSCs were isolated from 90 g rat long diaphysis in vitro. The whole bone marrow adherent culture was used to remove suspended blood cells and other kinds of stem cells such as hematopoietic stem cells, and BMSCs was induced to MS-NSCs by biological factor method. Clone proliferation was also observed, and nutrition and energy were supplemented by regular fluid exchange. The expression and secretion of neural marker proteins such as neuron-specific nuclear protein (neuron-specific nuclear protein), nestin protein (nestin) and CD11b (CD11b) in neural marker proteins, including neuron-specific nuclear protein (neuron-specific nuclear protein) and CD11b, were detected by immunohistochemical method. Two types of stem cells, BMSCs and MS-NSCs, were labeled with 10 渭 mol/l BrdU to establish adult rats. After 24 hours of resuscitation, The cells were transplanted into the brain through the left internal carotid artery. Neurological severity score (NSS) and Morris water maze test (Morris water maze test) were used to evaluate the degree of improvement of brain function. Five weeks later, the remodeling of the gray matter structure, including HE staining, was evaluated histopathologically. Myelin staining, glial fibrillary acidic protein (GFAP) and 2- (3) -cyclic nucleotide 3- phosphoesterase (CNP) immunocytochemical staining, BrdU immunohistochemical staining and neural marker proteins such as GFAPCNPCD11b and microtubule-associated protein 2MAP2 immunofluorescent double staining, etc. Results at 5 weeks after severe CO poisoning, there was focal demyelination in the white matter of the rat brain, and the gray matter, such as cortex and hippocampus, were scattered in the space after focal necrosis. Both types of stem cell transplantation can improve the order and density of white matter structure and demyelination, promote the recovery of cognitive memory function, and the recovery of this function is more obvious in the MS-NSCs transplantation group than in the control group (p < 0.05), and also in the cortex and hippocampus. BrdU positive cells were detected in the damaged area of basal ganglia and loose white matter, and the number of BrdU positive cells in MS-NSCs transplantation group was more than 0.05, and the proportion of stem cells differentiated into downstream terminal nerve cells in some areas of MS-NSCs transplantation group was higher than that in MS-NSCs transplantation group. Conclusion MS-NSCs can reshape the brain structure and function of acute severe carbon monoxide poisoning rats, and this effect is more obvious than that of BMSCs, suggesting that the former may be a better seed cell.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R329

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