本文選題：神經(jīng)干細(xì)胞 + 嗅鞘細(xì)胞��； 參考：《佳木斯大學(xué)》2010年碩士論文

【摘要】： 目的:本研究旨在探討體外培養(yǎng)的OECs是否具有促進NSCs增殖及向膽堿能神經(jīng)元分化的能力,以及兩者體外共培養(yǎng)最合適的濃度比例,為NSCs和OECs聯(lián)合移植治療AD提供理論依據(jù)。 方法:1.從新生24h內(nèi)大鼠海馬區(qū)機械分離NSCs,在添加無血清培養(yǎng)基(包含bFGF、EGF和B27)中懸浮培養(yǎng),得到原代及傳代NSCs,并進行鑒定。2.從新生1～2天大鼠的嗅球分離培養(yǎng)OECs,分別采用差速貼壁法、化學(xué)抑制法及差速貼壁與化學(xué)抑制相結(jié)合的方法純化培養(yǎng)OECs,并進行鑒定。3. OECs純化培養(yǎng)7天,離心去除原培養(yǎng)液,加入維持培養(yǎng)液(DMEM/F12培養(yǎng)基,2%B27,1%胎牛血清,青霉素100u/ml,鏈霉素100u/ml),機械吹打后調(diào)整細(xì)胞密度分別為1×104 /ml、1×106 /ml、1×108 /ml OECs,取1ml滴入0.1%多聚賴氨酸包被過的培養(yǎng)板中。離心收集第3代NSCs,去除原培養(yǎng)液,加入維持培養(yǎng)液,機械吹打后調(diào)整細(xì)胞密度為5×104 /ml,取1ml滴加入OECs的培養(yǎng)板中共培養(yǎng)。分別用1×104 /ml OECs、1×106/ml OECs、1×108/ml OECs對NSCs進行誘導(dǎo)分化,設(shè)立對照組(NSCs組),觀察NSCs增殖情況,并用免疫細(xì)胞化學(xué)染色方法檢測分化細(xì)胞中膽堿能神經(jīng)元標(biāo)志物ChAT的表達,比較不同濃度OECs誘導(dǎo)NSCs定向分化為膽堿能神經(jīng)元的作用。 結(jié)果:1.從新生24h內(nèi)大鼠海馬區(qū)分離培養(yǎng)的細(xì)胞可持續(xù)分裂增殖,形成許多懸浮生長的神經(jīng)球。獲得的NSCs能在體外長期生存,具有很強的增殖和自我更新能力,呈Nestin抗體陽性,并具有多向分化能力,能分化成神經(jīng)元和膠質(zhì)細(xì)胞。2.體外培養(yǎng)的新生1～2天大鼠嗅球OECs主要為雙極或三級細(xì)胞,其突起細(xì)長。分別采用差速貼壁法、化學(xué)抑制法、差速貼壁+化學(xué)抑制法純化培養(yǎng)OECs,其中差速貼壁+化學(xué)抑制法的純化效率明顯高于其他兩種。培養(yǎng)的OECs行免疫細(xì)胞化學(xué)染色,呈NGFRp75、GFAP染色陽性。3.①分別用1×104 /ml、1×106/ml、1×108/ml三種濃度OECs與NSCs進行共培養(yǎng)。在NSCs與1×106/ml OECs共培養(yǎng)組,神經(jīng)干細(xì)胞滴加到嗅鞘細(xì)胞培養(yǎng)板24 h后,可見重新貼壁生長的OECs;部分NSCs重新形成神經(jīng)細(xì)胞球, 48h后細(xì)胞克隆球逐漸展開,細(xì)胞數(shù)量開始增多,3d后細(xì)胞數(shù)量增多明顯,與對照組比較有統(tǒng)計學(xué)意義(P0.05)。7天后細(xì)胞數(shù)量達到高峰,與對照組、1×104 /ml OECs共培養(yǎng)組、1×108/ml OECs共培養(yǎng)組均有統(tǒng)計學(xué)意義(P0.05)。1×104 /ml OECs共培養(yǎng)組和1×108/ml OECs共培養(yǎng)組加入神經(jīng)干細(xì)胞后,24h后開始貼壁,3d后克隆球中有細(xì)胞呈放射狀遷移出來,5d后細(xì)胞數(shù)量明顯增多,與對照組比較有統(tǒng)計學(xué)意義(P0.05)。三種濃度OECs與NSCs共培養(yǎng)3天后,均促進了NSCs增殖,以1×106/ml OECs共培養(yǎng)組作用最顯著,并且在共培養(yǎng)7天時增殖細(xì)胞數(shù)最多。②分別用1×104 /ml、1×106/ml、1×108/ml三種濃度OECs對NSCs進行誘導(dǎo)。在1×106/ml OECs共培養(yǎng)組,24 h后可見重新貼壁生長的OECs; NSCs重新形成神經(jīng)細(xì)胞球,OECs在懸浮神經(jīng)球下貼壁生長;5d后NSCs開始附著在OECs上生長,神經(jīng)球逐漸展開,有細(xì)小突起長出;7d后可見大量有細(xì)長突起的神經(jīng)元樣細(xì)胞從神經(jīng)球周圍遷出,與嗅鞘細(xì)胞共同生長。1×104 /ml OECs共培養(yǎng)組和1×108/ml OECs共培養(yǎng)組,NSCs附著在OECs上較晚,分化速度較1×106/ml OECs共培養(yǎng)組慢。NSCs經(jīng)不同濃度OECs誘導(dǎo)7天后,與對照組比較,免疫細(xì)胞染色ChAT陽性細(xì)胞數(shù)量較多,胞體較大,突起細(xì)長,顯示成熟度增加。ChAT陽性細(xì)胞率分別為4.60%、5.96%、4.62%,均提高了ChAT陽性細(xì)胞率,其中1×106/ml OECs共培養(yǎng)組作用最明顯,與對照組比較有統(tǒng)計學(xué)意義(P0.05)。 結(jié)論:1.從新生大鼠嗅球中成功地分離并培養(yǎng)出嗅鞘細(xì)胞,差速貼壁法和化學(xué)抑制法相結(jié)合是一種有效的純化嗅鞘細(xì)胞的方法。 2.體外培養(yǎng)的OECs具有促進NSCs增殖及向膽堿能神經(jīng)元分化的作用。
[Abstract]:Objective: the purpose of this study was to investigate whether OECs has the ability to promote NSCs proliferation and differentiation into cholinergic neurons in vitro, as well as the most suitable concentration ratio of both in vitro co culture, and to provide a theoretical basis for the combined transplantation of NSCs and OECs for the treatment of AD.
Methods: 1. the NSCs was mechanically separated from the hippocampus of the newborn 24h rats, and the serum-free medium (including bFGF, EGF and B27) was suspended and cultured to obtain the original and passages NSCs. The identification of.2. from the new 1~2 day rat olfactory bulb was used to isolate and culture OECs, which was combined with differential adherence, chemical inhibition and differential adhesion and chemical inhibition, respectively. Methods the OECs was purified and cultured, and.3. OECs was purified and cultured for 7 days. The original culture solution was removed by centrifugation, and the culture solution was added to maintain culture medium (DMEM/F12 medium, 2%B27,1% fetal bovine serum, penicillin 100u/ml, streptomycin 100u/ml). After mechanical blow, the cell density was 1 x 104 /ml, 1 x 106 /ml, 1 x 108 /ml OECs, and 0.1% polylysine bag was dripped from 1ml. In the culture plate, third generations of NSCs were collected, the culture solution was removed, the culture medium was added, the cell density was 5 x 104 /ml after the mechanical blow, and the culture plate of 1ml drops added to OECs was cultured. The differentiation was induced with 1 x 104 /ml OECs, 1 x 106/ml OECs, 1 x 108/ml OECs, and the control group (NSCs group) was set up to observe the proliferation. The expression of the cholinergic neuron marker ChAT in the differentiated cells was detected by immunocytochemical staining, and the effects of different concentrations of OECs on the differentiation of NSCs into cholinergic neurons were compared.
Results: 1. the cells isolated and cultured in the hippocampus of the newborn 24h rat can continue to split and proliferate and form many suspended growth nerve spheres. The obtained NSCs can survive for a long time in vitro, with strong proliferation and self renewal ability, Nestin antibody positive and multidirectional differentiation ability, and can differentiate into neurons and glial cells.2. in vitro culture. The olfactory ball OECs in the 1~2 days of the newborn rats was mainly bipolar or three level cells, and its protuberances were elongated. The differential adherence method, chemical inhibition method, differential adherence + chemical inhibition method were used to purify OECs respectively. The purification efficiency of the differential adherent + chemical inhibition method was obviously higher than that of the other two kinds. The cultured OECs was stained with immunocytochemical staining, NGFRp75, G FAP staining positive.3. (1) was co cultured with 1 x 104 /ml, 1 x 106/ml, 1 x 108/ml three concentrations OECs and NSCs respectively. After NSCs and 1 x 106/ml OECs co culture group, neural stem cells were added to the olfactory ensheathing cell culture plate 24 h. The number of cells began to increase, and the number of cells increased significantly after 3D. The number of cells in the control group was statistically significant (P0.05).7 days after.7, with the control group, 1 x 104 /ml OECs co culture group, and the 1 x 108/ml OECs co culture group had statistical significance (P0.05).1 x 104 /ml OECs co culture group and 1 x 108/ml common culture group added nerve dry fine. After 24h, the cells began to stick to the wall after 3D, and the cells in the cloned spheres moved out in radially, and the number of cells increased significantly after 5D (P0.05). The three concentrations of OECs and NSCs co cultured for 3 days, all promoted the proliferation of NSCs, which was the most significant in the 1 x 106/ml OECs co culture group, and the number of proliferating cells at 7 days co culture. NSCs was induced with 1 x 104 /ml, 1 x 106/ml and 1 x 108/ml, respectively. In the 1 x 106/ml OECs co culture group, the re adherent growth OECs was found after 24 h; NSCs re formed the nerve cell ball, and OECs on the suspended nerve ball. After 7d, a large number of neuron like cells with elongated protuberances were found to move out of the nerve spheres and co grown with the olfactory ensheathing cells in the.1 x 104 /ml OECs co culture group and the 1 x 108/ml OECs co culture group. NSCs attached to OECs later and the differentiation rate was slower than the 1 x 106/ml OECs co culture group for 7 days after the induction of OECs, compared with the control group. Compared with the immune cells, the number of ChAT positive cells was large, the cell body was larger and the protuberance was elongated. The rate of.ChAT positive cells was 4.60%, 5.96% and 4.62%, respectively, which increased the rate of ChAT positive cells, among which 1 x 106/ml OECs co culture group had the most obvious effect, compared with the control group (P0.05).
Conclusion: 1. the olfactory ensheathing cells are successfully isolated and cultured from the olfactory bulb of neonatal rats. The combination of differential adherence and chemical inhibition is an effective method for the purification of olfactory ensheathing cells.
2. OECs in vitro can promote NSCs proliferation and differentiate into cholinergic neurons.

【學(xué)位授予單位】：佳木斯大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

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