星形胶质细胞与神经干细胞共培养对神经干细胞分化影响的实验研究

发布时间：2018-05-13 10:46

本文选题：神经干细胞 + 星形胶质细胞　；参考：《中国医科大学》2010年硕士论文

【摘要】： 前言神经干细胞neural stem cells, NSCs)的特点：能够自我更新和增殖；具有向神经元和神经胶质细胞多向分化的潜能；免疫原性低；可长期培养。神经干细胞的作用：促进神经组织的修复；作为神经系统疾病基因治疗的载体。神经干细胞存在于哺乳动物中枢神经系统的多个部位。大鼠海马是神经干细胞的主要聚集区,利于获取神经干细胞。新生大鼠海马所占脑体积的比例较成鼠大,便于取材。星形胶质细胞能合成和分泌多种神经因子,促进神经干细胞的定向分化,对神经元的正常活动与代谢都有重要作用。大脑皮质是星形胶质细胞与神经元密切接触的主要场所,从大脑皮质获取星形胶质细胞,有利于认识星形胶质细胞的生物学作用。神经干细胞作为治疗神经系统疾病理想的供体细胞,其作用主要是通过神经干细胞分化的神经元实现。神经干细胞的分化是神经干细胞的重要属性之一,受诸多因素影响,但在很大程度上取决于微环境中神经因子的作用。目的探讨星形胶质细胞(Astrocytes)对诱导新生大鼠海马神经干细胞分化为神经元的影响。材料和方法一、材料新生2-3d的正常Wistar大鼠12只。二、方法 (一)神经干细胞的培养和鉴定新生2-3d的Wistar大鼠,无菌分离海马组织,制成细胞悬液,在含碱性成纤维生长因子(basic fibroblast growth factor, bFGF)、表皮生长因子(epidermal growth factor, EGF)和B27的DMEM／F12(1：1)无血清培养基中进行神经干细胞的原代培养。原代培养细胞大多数细胞球直径达200gm时行传代培养,传3代的细胞球行单克隆培养。克隆球的部分细胞行巢蛋白(Nestin)免疫细胞荧光染色；部分细胞用含10%胎牛血清的DMEM/F12进行诱导分化,5d后分别行神经元特异性烯醇化酶(neurone specific enolase, NSE)、胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)、半乳糖脑苷脂(galactocerebroside, Galc)免疫细胞荧光染色。 (二)星形胶质细胞条件培养液的收集新生2-3d的Wista大鼠,无菌分离出大脑皮质,制成细胞悬液,在含DMEM/F12(1：1)、15%胎牛血清的培养液中行星形胶质细胞的原代培养。用差速贴壁和室温震荡法纯化星形胶质细胞。传3代后部分细胞行GFAP免疫细胞荧光染色,标记星形胶质细胞的纯度；另一部分细胞换成NSCs培养液继续培养48h,收集细胞培养液即为星形胶质细胞的条件培养液(ACM)。 (三)神经干细胞的诱导分化培养将单克隆培养的NSCs分为三组：对照组(A组)、ACM:NSCs (1:2)培养组(B组)和Astrocytes和NSCs共培养组(C组)。各组细胞分别接种于含不同培养液的六孔培养板的板孔中,进行诱导分化培养。采用免疫细胞荧光染色及免疫蛋白印迹两种检测方法,检测各组NSCs分化为神经元的比例及其NSE蛋白的表达情况。 (四)统计学分析应用SPSS13.0统计分析软件,对神经元的比例进行统计学分析,所有数据采用均数±标准差(x±s)表示,组间比较采用t检验,P0.05有统计学意义。实验结果一、神经干细胞的鉴定结果单细胞克隆培养的细胞Nestin阳性表达,诱导分化的细胞NSE、GFAP和GalC阳性表达。二、星形胶质细胞鉴定结果 GFAP免疫细胞荧光染色显示：96.5%细胞GFAP阳性,细胞纯度适合做后续实验。三、各组神经干细胞向神经元分化的结果各组神经干细胞诱导分化3d时,细胞贴壁分化的程度不同：B组、C组明显比A组快；诱导分化7d时,免疫细胞荧光染色统计学结果表明：A组、B组、C组NSE阳性细胞的比例分别为：14.7%±3.5%,35.2%±4.1,40.3%±3.7。B组、C组诱导分化为神经元的比例明显高于A组(P0.05),C组最高。B组、C组间无显著性差异(P0.05)。免疫印迹结果显示：B组、C组NSE的表达量较A组明显增加,B组、C组间无明显差异。结论 1、星形胶质细胞具有促进体外培养新生大鼠海马神经干细胞向神经元分化的作用。 2、星形胶质细胞对神经干细胞分化的影响与其分泌的活性物质有关。
[Abstract]:Preface
The characteristics of neural stem cells neural stem cells, NSCs) can be self renewing and proliferating; they have the potential for multiple differentiation into neurons and glial cells; the immunogenicity is low; it can be cultured for a long time.
The role of neural stem cells is to promote the repair of nerve tissue, as a carrier of gene therapy for nervous system diseases.
Neural stem cells exist in many parts of the central nervous system of mammals. The hippocampus is the main aggregation area of neural stem cells, which is beneficial to obtain neural stem cells. The proportion of the brain volume in the hippocampus of newborn rats is larger than that of the adult rat.
Astrocytes can synthesize and secrete a variety of nerve factors, promote the directional differentiation of neural stem cells, and play an important role in the normal activity and metabolism of neurons. The cerebral cortex is the main place for astrocytes to be closely contacted with neurons. Astrocytes are obtained from the cerebral cortex, which is beneficial to the understanding of astrocytes. The role of physical science.
Neural stem cells are the ideal donor cells for the treatment of nervous system diseases. The function of neural stem cells is realized mainly through neurons differentiated by neural stem cells. The differentiation of neural stem cells is one of the important attributes of neural stem cells. It is influenced by many factors, but it is largely dependent on the role of neural factors in the microenvironment.
objective
Objective to investigate the effect of astrocytes (Astrocytes) on the differentiation of neural stem cells into neurons in neonatal rat hippocampus.
Materials and methods
First, material
There were 12 normal Wistar rats of newborn 2-3D.
Two, method
(1) the culture and identification of neural stem cells
The Wistar rats of newborn 2-3D were aseptic separation of hippocampal tissue to make cell suspension. The primary culture of neural stem cells was carried out in the serum containing basic fibroblast growth factor (basic fibroblast growth factor, bFGF), epidermal growth factor (epidermal growth factor, EGF) and B27. Most of the primary culture cells were cultured. The number of cell spheres was cultured at 200gm, and the 3 generation cell spheres were McAb. Some cells of the cloned ball were stained with Nestin, and some cells were induced to differentiate with the DMEM/F12 containing 10% fetal bovine serum. After 5D, the neuron specific enolase (neurone specific enolase, NSE) and colloid were performed respectively. Glial fibrillary acidic protein (GFAP), galactoside (galactocerebroside, Galc) immunofluorescence staining.
(two) collection of conditioned medium for astrocytes
Neonatal 2-3D Wista rats were aseptic separated from the cerebral cortex to form cell suspension, the primary culture of planetary glial cells in the medium containing DMEM/F12 (1:1) and 15% fetal bovine serum. The astrocytes were purified by differential adhesion and room temperature concussion. After the 3 generation, some cells were stained with GFAP immunofluorescence to mark astrocytes. The other cells were replaced with NSCs medium and continued to grow 48h, and the conditioned medium (ACM) of astrocytes was collected.
(three) induction and differentiation of neural stem cells
The monoclonal cultured NSCs was divided into three groups: control group (group A), ACM:NSCs (1:2) culture group (B group) and Astrocytes and NSCs co culture group (C group). The cells were inoculated in the plate hole of six hole culture plate containing different culture liquid, and the differentiation culture was induced. The immunofluorescence staining and immunoblotting were used to detect the cells, and the detection methods were detected by immunofluorescence staining and immunoblotting. The proportion of NSCs differentiated into neurons and the expression of NSE protein in each group were measured.
(four) statistical analysis
The statistical analysis software of SPSS13.0 was used to analyze the proportion of neurons. All the data were expressed by mean number + standard deviation (x + s), and t test was used for comparison among groups. P0.05 had statistical significance.
experimental result
First, the identification results of neural stem cells
Nestin was positive in single cell clone culture, and NSE, GFAP and GalC were positive in differentiated cells.
Two, astrocyte identification results
GFAP immunofluorescence staining showed that 96.5% cells were GFAP positive, and cell purity was suitable for follow-up experiments.
Three, the differentiation of neural stem cells into neurons in each group.
When the neural stem cells were induced to differentiate 3D, the degree of cell adhesion differentiation was different: B group, C group was significantly faster than group A, and when induced 7d, the immunofluorescence staining statistics showed that the proportion of NSE positive cells in A group, B group and C group were 14.7% + 3.5%, 35.2% + 4.1,40.3% + 3.7.B group, and C group induced the proportion of differentiation into neurons. Significantly higher than group A (P0.05), the highest.B group in group C, and no significant difference between group C (P0.05). The results of immunoblotting showed that the expression of NSE in group B was significantly increased in group C than in A group, and there was no significant difference between B group and C group.
conclusion
1, astrocytes can promote the differentiation of hippocampal neural stem cells into neurons in vitro.
2, the effect of astrocytes on the differentiation of neural stem cells is related to the active substances secreted by them.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329.28

【参考文献】