嗅鞘细胞对神经干细胞增殖及向胆碱能神经元分化的实验研究

发布时间：2018-05-12 10:24

  本文选题：神经干细胞 + 嗅鞘细胞　； 参考：《佳木斯大学》2010年硕士论文

【摘要】： 目的:本研究旨在探讨体外培养的OECs是否具有促进NSCs增殖及向胆碱能神经元分化的能力,以及两者体外共培养最合适的浓度比例,为NSCs和OECs联合移植治疗AD提供理论依据。 方法:1.从新生24h内大鼠海马区机械分离NSCs,在添加无血清培养基(包含bFGF、EGF和B27)中悬浮培养,得到原代及传代NSCs,并进行鉴定。2.从新生1～2天大鼠的嗅球分离培养OECs,分别采用差速贴壁法、化学抑制法及差速贴壁与化学抑制相结合的方法纯化培养OECs,并进行鉴定。3. OECs纯化培养7天,离心去除原培养液,加入维持培养液(DMEM/F12培养基,2%B27,1%胎牛血清,青霉素100u/ml,链霉素100u/ml),机械吹打后调整细胞密度分别为1×104 /ml、1×106 /ml、1×108 /ml OECs,取1ml滴入0.1%多聚赖氨酸包被过的培养板中。离心收集第3代NSCs,去除原培养液,加入维持培养液,机械吹打后调整细胞密度为5×104 /ml,取1ml滴加入OECs的培养板中共培养。分别用1×104 /ml OECs、1×106/ml OECs、1×108/ml OECs对NSCs进行诱导分化,设立对照组(NSCs组),观察NSCs增殖情况,并用免疫细胞化学染色方法检测分化细胞中胆碱能神经元标志物ChAT的表达,比较不同浓度OECs诱导NSCs定向分化为胆碱能神经元的作用。 结果:1.从新生24h内大鼠海马区分离培养的细胞可持续分裂增殖,形成许多悬浮生长的神经球。获得的NSCs能在体外长期生存,具有很强的增殖和自我更新能力,呈Nestin抗体阳性,并具有多向分化能力,能分化成神经元和胶质细胞。2.体外培养的新生1～2天大鼠嗅球OECs主要为双极或三级细胞,其突起细长。分别采用差速贴壁法、化学抑制法、差速贴壁+化学抑制法纯化培养OECs,其中差速贴壁+化学抑制法的纯化效率明显高于其他两种。培养的OECs行免疫细胞化学染色,呈NGFRp75、GFAP染色阳性。3.①分别用1×104 /ml、1×106/ml、1×108/ml三种浓度OECs与NSCs进行共培养。在NSCs与1×106/ml OECs共培养组,神经干细胞滴加到嗅鞘细胞培养板24 h后,可见重新贴壁生长的OECs;部分NSCs重新形成神经细胞球, 48h后细胞克隆球逐渐展开,细胞数量开始增多,3d后细胞数量增多明显,与对照组比较有统计学意义(P0.05)。7天后细胞数量达到高峰,与对照组、1×104 /ml OECs共培养组、1×108/ml OECs共培养组均有统计学意义(P0.05)。1×104 /ml OECs共培养组和1×108/ml OECs共培养组加入神经干细胞后,24h后开始贴壁,3d后克隆球中有细胞呈放射状迁移出来,5d后细胞数量明显增多,与对照组比较有统计学意义(P0.05)。三种浓度OECs与NSCs共培养3天后,均促进了NSCs增殖,以1×106/ml OECs共培养组作用最显著,并且在共培养7天时增殖细胞数最多。②分别用1×104 /ml、1×106/ml、1×108/ml三种浓度OECs对NSCs进行诱导。在1×106/ml OECs共培养组,24 h后可见重新贴壁生长的OECs; NSCs重新形成神经细胞球,OECs在悬浮神经球下贴壁生长;5d后NSCs开始附着在OECs上生长,神经球逐渐展开,有细小突起长出;7d后可见大量有细长突起的神经元样细胞从神经球周围迁出,与嗅鞘细胞共同生长。1×104 /ml OECs共培养组和1×108/ml OECs共培养组,NSCs附着在OECs上较晚,分化速度较1×106/ml OECs共培养组慢。NSCs经不同浓度OECs诱导7天后,与对照组比较,免疫细胞染色ChAT阳性细胞数量较多,胞体较大,突起细长,显示成熟度增加。ChAT阳性细胞率分别为4.60%、5.96%、4.62%,均提高了ChAT阳性细胞率,其中1×106/ml OECs共培养组作用最明显,与对照组比较有统计学意义(P0.05)。 结论:1.从新生大鼠嗅球中成功地分离并培养出嗅鞘细胞,差速贴壁法和化学抑制法相结合是一种有效的纯化嗅鞘细胞的方法。 2.体外培养的OECs具有促进NSCs增殖及向胆碱能神经元分化的作用。
[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.

【学位授予单位】：佳木斯大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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