本文選題：人胎腦神經(jīng)干細(xì)胞 + 新生鼠��； 參考：《安徽醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 目的建立人胎腦神經(jīng)干細(xì)胞(hNSC)在新生鼠腦內(nèi)遷移與分化的模型,采用免疫組化的方法比較外源性神經(jīng)干細(xì)胞球和單細(xì)胞懸液在植入宿主體內(nèi)遷移與分化規(guī)律,為選擇合適的hNSC移植方法治療各種新生兒腦病提供實(shí)驗(yàn)基礎(chǔ)。 材料與方法 1.細(xì)胞培養(yǎng)用添加表皮生長(zhǎng)因子(EGF)+堿性成纖維細(xì)胞生長(zhǎng)因子(bFGF)+白血病抑制因子(LIF)的N2培養(yǎng)基從流產(chǎn)的人胚胎前腦組織培養(yǎng)出神經(jīng)干細(xì)胞球;取部分神經(jīng)干細(xì)胞球通過免疫熒光技術(shù)檢測(cè)神經(jīng)干細(xì)胞巢蛋白(Nestin)鑒定其干細(xì)胞屬性,誘導(dǎo)分化神經(jīng)干細(xì)胞,并以抗-人神經(jīng)絲蛋白(NF)和抗-人酸性膠質(zhì)纖維蛋白(GFAP)分別鑒定神經(jīng)干細(xì)胞(NSC)分化成的神經(jīng)元及星形膠質(zhì)細(xì)胞,證實(shí)神經(jīng)干細(xì)胞具有分化能力。 2.將神經(jīng)干細(xì)胞分為細(xì)胞球和單細(xì)胞懸液(采用Dispase酶消化)兩種,調(diào)整濃度為5.0×10~4cells/ul待移植時(shí)用。 3.動(dòng)物分組36只9日齡、體重20-25g的清潔級(jí)雌雄不限的SD大鼠,隨機(jī)分為3組,分別植入細(xì)胞球(細(xì)胞球組)、單細(xì)胞懸液(單細(xì)胞懸液組)和F12培養(yǎng)液(對(duì)照組)。 4.于移植24、48、72 h通過取腦,制作石蠟切片,免疫組織化學(xué)觀察植入細(xì)胞在受體鼠內(nèi)的遷移分化情況,并比較兩組實(shí)驗(yàn)結(jié)果的差異。 結(jié)果 1.在添加EGF等的培養(yǎng)基上培養(yǎng)出來的是典型的神經(jīng)細(xì)胞球,培養(yǎng)6 d,觀察到大量的形態(tài)并不規(guī)整的小細(xì)胞集落,12 d后形成典型的神經(jīng)球。免疫熒光鑒定細(xì)胞網(wǎng)絡(luò)分化的細(xì)胞中有NF(+)、GFAP(+),GalC(+),證實(shí)細(xì)胞球能分化成神經(jīng)元、星形膠質(zhì)細(xì)胞和少突膠質(zhì)細(xì)胞。 2.移植入腦室內(nèi)的兩種類型細(xì)胞,其在腦內(nèi)存活遷移的結(jié)果明顯不同。①細(xì)胞球移植組遷移情況:移植后24 h可在腦室內(nèi)見到大量Anti-hNuc(+)細(xì)胞:48 h后可看到側(cè)腦室、腦室壁上、脈絡(luò)膜及嗅球附近見到大量Anti-hNuc(+)細(xì)胞;72 h大量Anti-hNuc(+)細(xì)胞在鼠腦額葉、枕葉和海馬均有分布。72h后分化情況:大腦皮層的顆粒層和錐體細(xì)胞層及嗅球處有分化成熟的神經(jīng)元,胞體中央有不著色的圓形細(xì)胞核,胞漿和突起均發(fā)出紅色熒光,并可觀察到陽(yáng)性細(xì)胞間有突起-突起間和突起-胞體間的連接;②單細(xì)胞懸液組:移植24 h可在腦室見到大量Anti-hNuc(+)細(xì)胞,72 h在腦室和腦室內(nèi)壁上僅有少量Anti-hNuc(+)細(xì)胞分布;72h后分化情況;未見明顯分化。 結(jié)論 1.體外培養(yǎng)獲得的人神經(jīng)干細(xì)胞具有分化為神經(jīng)元及膠質(zhì)細(xì)胞的潛能。 2.人胎腦神經(jīng)干細(xì)胞(hNSC)移植入鼠側(cè)腦室后不僅能夠存活、遷移和分化為神經(jīng)元和膠質(zhì)細(xì)胞。而且細(xì)胞之間突起-突起間和突起-胞體間的連接。 3.細(xì)胞球移植法遷移分化能力顯著高于單細(xì)胞懸液移植法。
[Abstract]:Objective to establish a model of migration and differentiation of human fetal neural stem cells (hNSC) in the brain of newborn rats, and to compare the migration and differentiation of exogenous neural stem cell balls and single cell suspensions in the implanted host by immunohistochemical method. To provide experimental basis for the selection of appropriate transplantation of hNSC for the treatment of various neonatal encephalopathy. Materials and methods 1. Neural stem cells were cultured in N2 medium supplemented with epidermal growth factor (EGF) basic fibroblast growth factor (bFGF) leukemia inhibitory factor (LIF) from aborted human embryonic forebrain tissue. Some neural stem cell spheres were used to detect the nestin of neural stem cells (NSCs) by immunofluorescence to identify their stem cell properties and to induce the differentiation of neural stem cells (NSCs). Neurons and astrocytes differentiated from neural stem cells (NSC) were identified by anti-human neurofilament protein (NF) and anti-human acidic glial fibrin (GFAP), respectively. Neural stem cells (NSCs) were divided into cell spheres and single cell suspensions (digested by Dispase) with an adjusted concentration of 5.0 脳 10~4cells/ul. Thirty-six 9-day-old SD rats weighing 20-25 g were randomly divided into three groups: cell ball group (cell ball group), single cell suspension group (single cell suspension group) and F12 culture medium (control group). After 24 minutes and 48 minutes 72 hours of transplantation, paraffin sections were made and the migration and differentiation of implanted cells in recipient mice were observed by immunohistochemistry, and the differences between the two groups were compared. Result 1. Typical neuronal spheres were cultured on the medium supplemented with EGF. After 6 days of culture, a large number of irregular small cell colonies were observed to form typical neuronal spheres after 12 days. NF-GFAP () GFAP () GalC (), showed that the cells could differentiate into neurons, astrocytes and oligodendrocytes by immunofluorescence. 2. The survival and migration of the two types of cells transplanted into the ventricle were significantly different from those in the 1-cell ball transplantation group: a large number of Anti-hNuc () cells could be seen in the ventricle at 24 h after transplantation, and the lateral ventricle and the wall of the ventricle could be seen at 48 h after transplantation. A large number of Anti-hNuc () cells were observed in the vicinity of choroid and olfactory bulb for 72 h. A large number of Anti-hNuc () cells were distributed in frontal lobe, occipital lobe and hippocampus. 72 h later, there were differentiated neurons in granular layer, pyramidal cell layer and olfactory bulb of cerebral cortex. There are round nuclei in the center of the cell body, and the cytoplasm and process emit red fluorescence, and the connections between the positive cells and the processes between the positive cells can be observed. 2 single cell suspension group: after 24 h transplantation, a large number of Anti-hNuc () cells could be seen in the ventricle. After 72 h, there were only a few Anti-hNuc () cells distributed in the ventricle and the inner wall of the ventricle. Conclusion 1. In vitro cultured human neural stem cells have the potential to differentiate into neurons and glial cells. 2. Human fetal neural stem cells (hNSC) can not only survive, migrate and differentiate into neurons and glial cells, but also be transplanted into the lateral ventricle of rats. And the connections between processes-processes and processes-bodies. 3. The migration and differentiation ability of cell ball transplantation was significantly higher than that of single cell suspension transplantation.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前9條

1 張紅安;;神經(jīng)干細(xì)胞治療脊髓損傷的研究進(jìn)展[J];河北醫(yī)藥;2007年12期

2 梁鵬,衛(wèi)淑靜,徐薇,梁桃,劉恩重,金連弘;人胚神經(jīng)干細(xì)胞體外長(zhǎng)期培養(yǎng)系統(tǒng)的建立[J];中華神經(jīng)外科疾病研究雜志;2005年01期

3 張小東;賈延R，

本文編號(hào)：2062278