發(fā)布時(shí)間：2018-05-13 10:46

  本文選題：神經(jīng)干細(xì)胞 + 星形膠質(zhì)細(xì)胞　； 參考：《中國(guó)醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 前言 神經(jīng)干細(xì)胞neural stem cells, NSCs)的特點(diǎn)：能夠自我更新和增殖；具有向神經(jīng)元和神經(jīng)膠質(zhì)細(xì)胞多向分化的潛能；免疫原性低；可長(zhǎng)期培養(yǎng)。 神經(jīng)干細(xì)胞的作用：促進(jìn)神經(jīng)組織的修復(fù)；作為神經(jīng)系統(tǒng)疾病基因治療的載體。 神經(jīng)干細(xì)胞存在于哺乳動(dòng)物中樞神經(jīng)系統(tǒng)的多個(gè)部位。大鼠海馬是神經(jīng)干細(xì)胞的主要聚集區(qū),利于獲取神經(jīng)干細(xì)胞。新生大鼠海馬所占腦體積的比例較成鼠大,便于取材。 星形膠質(zhì)細(xì)胞能合成和分泌多種神經(jīng)因子,促進(jìn)神經(jīng)干細(xì)胞的定向分化,對(duì)神經(jīng)元的正�；顒�(dòng)與代謝都有重要作用。大腦皮質(zhì)是星形膠質(zhì)細(xì)胞與神經(jīng)元密切接觸的主要場(chǎng)所,從大腦皮質(zhì)獲取星形膠質(zhì)細(xì)胞,有利于認(rèn)識(shí)星形膠質(zhì)細(xì)胞的生物學(xué)作用。 神經(jīng)干細(xì)胞作為治療神經(jīng)系統(tǒng)疾病理想的供體細(xì)胞,其作用主要是通過(guò)神經(jīng)干細(xì)胞分化的神經(jīng)元實(shí)現(xiàn)。神經(jīng)干細(xì)胞的分化是神經(jīng)干細(xì)胞的重要屬性之一,受諸多因素影響,但在很大程度上取決于微環(huán)境中神經(jīng)因子的作用。 目的 探討星形膠質(zhì)細(xì)胞(Astrocytes)對(duì)誘導(dǎo)新生大鼠海馬神經(jīng)干細(xì)胞分化為神經(jīng)元的影響。 材料和方法 一、材料 新生2-3d的正常Wistar大鼠12只。 二、方法 (一)神經(jīng)干細(xì)胞的培養(yǎng)和鑒定 新生2-3d的Wistar大鼠,無(wú)菌分離海馬組織,制成細(xì)胞懸液,在含堿性成纖維生長(zhǎng)因子(basic fibroblast growth factor, bFGF)、表皮生長(zhǎng)因子(epidermal growth factor, EGF)和B27的DMEM／F12(1：1)無(wú)血清培養(yǎng)基中進(jìn)行神經(jīng)干細(xì)胞的原代培養(yǎng)。原代培養(yǎng)細(xì)胞大多數(shù)細(xì)胞球直徑達(dá)200gm時(shí)行傳代培養(yǎng),傳3代的細(xì)胞球行單克隆培養(yǎng)�？寺∏虻牟糠旨�(xì)胞行巢蛋白(Nestin)免疫細(xì)胞熒光染色；部分細(xì)胞用含10%胎牛血清的DMEM/F12進(jìn)行誘導(dǎo)分化,5d后分別行神經(jīng)元特異性烯醇化酶(neurone specific enolase, NSE)、膠質(zhì)纖維酸性蛋白(glial fibrillary acidic protein, GFAP)、半乳糖腦苷脂(galactocerebroside, Galc)免疫細(xì)胞熒光染色。 (二)星形膠質(zhì)細(xì)胞條件培養(yǎng)液的收集 新生2-3d的Wista大鼠,無(wú)菌分離出大腦皮質(zhì),制成細(xì)胞懸液,在含DMEM/F12(1：1)、15%胎牛血清的培養(yǎng)液中行星形膠質(zhì)細(xì)胞的原代培養(yǎng)。用差速貼壁和室溫震蕩法純化星形膠質(zhì)細(xì)胞。傳3代后部分細(xì)胞行GFAP免疫細(xì)胞熒光染色,標(biāo)記星形膠質(zhì)細(xì)胞的純度；另一部分細(xì)胞換成NSCs培養(yǎng)液繼續(xù)培養(yǎng)48h,收集細(xì)胞培養(yǎng)液即為星形膠質(zhì)細(xì)胞的條件培養(yǎng)液(ACM)。 (三)神經(jīng)干細(xì)胞的誘導(dǎo)分化培養(yǎng) 將單克隆培養(yǎng)的NSCs分為三組：對(duì)照組(A組)、ACM:NSCs (1:2)培養(yǎng)組(B組)和Astrocytes和NSCs共培養(yǎng)組(C組)。各組細(xì)胞分別接種于含不同培養(yǎng)液的六孔培養(yǎng)板的板孔中,進(jìn)行誘導(dǎo)分化培養(yǎng)。采用免疫細(xì)胞熒光染色及免疫蛋白印跡兩種檢測(cè)方法,檢測(cè)各組NSCs分化為神經(jīng)元的比例及其NSE蛋白的表達(dá)情況。 (四)統(tǒng)計(jì)學(xué)分析 應(yīng)用SPSS13.0統(tǒng)計(jì)分析軟件,對(duì)神經(jīng)元的比例進(jìn)行統(tǒng)計(jì)學(xué)分析,所有數(shù)據(jù)采用均數(shù)±標(biāo)準(zhǔn)差(x±s)表示,組間比較采用t檢驗(yàn),P0.05有統(tǒng)計(jì)學(xué)意義。 實(shí)驗(yàn)結(jié)果 一、神經(jīng)干細(xì)胞的鑒定結(jié)果 單細(xì)胞克隆培養(yǎng)的細(xì)胞Nestin陽(yáng)性表達(dá),誘導(dǎo)分化的細(xì)胞NSE、GFAP和GalC陽(yáng)性表達(dá)。 二、星形膠質(zhì)細(xì)胞鑒定結(jié)果 GFAP免疫細(xì)胞熒光染色顯示：96.5%細(xì)胞GFAP陽(yáng)性,細(xì)胞純度適合做后續(xù)實(shí)驗(yàn)。 三、各組神經(jīng)干細(xì)胞向神經(jīng)元分化的結(jié)果 各組神經(jīng)干細(xì)胞誘導(dǎo)分化3d時(shí),細(xì)胞貼壁分化的程度不同：B組、C組明顯比A組快；誘導(dǎo)分化7d時(shí),免疫細(xì)胞熒光染色統(tǒng)計(jì)學(xué)結(jié)果表明：A組、B組、C組NSE陽(yáng)性細(xì)胞的比例分別為：14.7%±3.5%,35.2%±4.1,40.3%±3.7。B組、C組誘導(dǎo)分化為神經(jīng)元的比例明顯高于A組(P0.05),C組最高。B組、C組間無(wú)顯著性差異(P0.05)。免疫印跡結(jié)果顯示：B組、C組NSE的表達(dá)量較A組明顯增加,B組、C組間無(wú)明顯差異。 結(jié)論 1、星形膠質(zhì)細(xì)胞具有促進(jìn)體外培養(yǎng)新生大鼠海馬神經(jīng)干細(xì)胞向神經(jīng)元分化的作用。 2、星形膠質(zhì)細(xì)胞對(duì)神經(jīng)干細(xì)胞分化的影響與其分泌的活性物質(zhì)有關(guān)。
[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.

【學(xué)位授予單位】：中國(guó)醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329.28

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