本文選題：神經(jīng)干細(xì)胞 + 大鼠海馬結(jié)構(gòu)�。� 參考：《中國醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 目的 探討表皮生長因子(epidermal growth factor,EGF)對腦源性神經(jīng)生長因子(brain-derived neurotrophic factor,BDNF)促進(jìn)成年大鼠海馬結(jié)構(gòu)神經(jīng)干細(xì)胞(neural stem cells,NSCs)向神經(jīng)元分化的影響及EGF濃度為20ng/mL時,BDNF促進(jìn)成年大鼠海馬結(jié)構(gòu)神經(jīng)干細(xì)胞向神經(jīng)元分化的最佳濃度。 材料和方法 用含堿性成纖維細(xì)胞生長因子(basic fibroblast growth factor,bFGF)、EGF、B27的DMEM/F12體外培養(yǎng)成年大鼠海馬結(jié)構(gòu)神經(jīng)干細(xì)胞,并進(jìn)行傳代培養(yǎng)。當(dāng)?shù)?代細(xì)胞大多數(shù)克隆球直徑約為2μm時,利用有限稀釋法進(jìn)行單細(xì)胞克隆培養(yǎng)。單細(xì)胞克隆培養(yǎng)形成的克隆球直徑約2μm時進(jìn)行傳代培養(yǎng),進(jìn)行以下2組實驗: 1、神經(jīng)干細(xì)胞的鑒定 部分克隆球行巢蛋白(Nestin)免疫細(xì)胞化學(xué)染色;部分克隆球用含10%胎牛血清的DMEM/F12進(jìn)行誘導(dǎo)分化,1w后分別行神經(jīng)元特異性烯醇化酶(neuronespecific enolase,NSE)、膠質(zhì)纖維酸性蛋白(glial fibrillary acidic protein,GFAP)免疫細(xì)胞化學(xué)染色; 2、將單細(xì)胞克隆再傳代而來的神經(jīng)干細(xì)胞分為2部分進(jìn)行誘導(dǎo)分化實驗 (1)根據(jù)培養(yǎng)液中BDNF濃度的不同將細(xì)胞分為5組,0ng/mL組、50ng/mL組、100ng/mL組、150ng/mL組和200ng/mL組,0ng/mL組所用培養(yǎng)液為含10%胎牛血清、20ng/mLEGF的DMEM/F12,其它各組所用的培養(yǎng)液在0ng/mL組的基礎(chǔ)上加入相應(yīng)濃度的BDNF。 (2)按培養(yǎng)液中添加誘導(dǎo)因素的不同分為對照組、EGF組、BDNF組和EGF+BDNF組進(jìn)行誘導(dǎo)分化培養(yǎng),對照組的培養(yǎng)液為含10%胎牛血清的DMEM/F12,另外3組的培養(yǎng)液在對照組所用培養(yǎng)液的基礎(chǔ)上分別加入EGF、BDNF、EGF+BDNF,其中EGF濃度為20ng/mL,BDNF濃度為50ng/mL。 上述2部分細(xì)胞接種于六孔板中,每組6孔,培養(yǎng)1w行NSE免疫細(xì)胞化學(xué)染色,計數(shù)陽性細(xì)胞比例后進(jìn)行統(tǒng)計學(xué)分析。 實驗結(jié)果 1、神經(jīng)干細(xì)胞的鑒定結(jié)果 單細(xì)胞克隆培養(yǎng)形成的克隆球表達(dá)Nestin,誘導(dǎo)分化1w后細(xì)胞表達(dá)NSE、GFAP。 2、誘導(dǎo)因素對神經(jīng)干細(xì)胞分化影響的結(jié)果 (1)0ng/mL組、50ng/mL組、100ng/mL組、150ng/mL組和200ng/mL組大鼠海馬結(jié)構(gòu)神經(jīng)干細(xì)胞向神經(jīng)元分化的比例分別為13.33±3.44%、23.17±2.93%、20.67±2.80%、16.67±2.73%、14.17±3.43%,經(jīng)t檢驗發(fā)現(xiàn),50ng/mL組、100ng/mL組神經(jīng)干細(xì)胞分化為神經(jīng)元的比例明顯增高(P＜0.05)。 (2)對照組、EGF組、BDNF組、EGF+BDNF組細(xì)胞分化為神經(jīng)元的比例分別為17.67±3.27、13.33±3.44、19.67±2.07、23.17±2.93,經(jīng)過t檢驗發(fā)現(xiàn):BDNF組神經(jīng)干細(xì)胞分化為神經(jīng)元的比例明顯高于對照組和EGF組,EGF+BDNF組神經(jīng)干細(xì)胞分化為神經(jīng)元的比例明顯高于BDNF組(P＜0.05)。 結(jié)論 1、EGF可以提高BDNF促進(jìn)成年大鼠海馬結(jié)構(gòu)神經(jīng)干細(xì)胞向神經(jīng)元分化的比例。 2、EGF濃度為20ng/mL時,BDNF促進(jìn)成年大鼠海馬結(jié)構(gòu)神經(jīng)干細(xì)胞向神經(jīng)元分化的最佳濃度為50ng/mL。
[Abstract]:objective
The effect of epidermal growth factor (epidermal growth factor, EGF) on the differentiation of hippocampal neural stem cells (neural stem cells, NSCs) to neuron differentiation in adult rat hippocampal neural stem cells (brain-derived neurotrophic factor, BDNF) and promoting the development of neural stem cells in the hippocampus of adult rats The optimal concentration of neuron differentiation.
Materials and methods
The adult rat hippocampal neural stem cells were cultured with basic fibroblast growth factor (bFGF), EGF, B27 DMEM/F12 in vitro and carried out in vitro. When most of the cloned spheres in the fourth generation cells were about 2 micron in diameter, the single cell clone culture was carried out by the finite dilution method. Single cell clone culture was formed. The diameter of the cloned sphere was about 2 m, and the following 2 groups of experiments were carried out.
1, identification of neural stem cells
Some cloned spherical nestin (Nestin) immunocytochemical staining was cloned, and some cloned spheres were induced by DMEM/F12 containing 10% fetal bovine serum. After 1W, neuron specific enolase (neuronespecific enolase, NSE), glial fibrillary acidic protein (glial fibrillary acidic protein, GFAP) were immunocytochemical staining, respectively.
2, the neural stem cells from single cell clone were divided into 2 parts to induce differentiation experiments.
(1) according to the different concentration of BDNF in the medium, the cells were divided into 5 groups, group 0ng/mL, group 50ng/mL, group 100ng/mL, 150ng/mL group and 200ng/mL group. The medium used for 0ng/mL group was 10% fetal bovine serum, 20ng/mLEGF DMEM/F12, and the culture solution of other groups was added to the 0ng/mL group based on the BDNF. concentration.
(2) the control group was divided into control group according to the induction factors added in the culture solution, the EGF group, BDNF group and EGF+BDNF group were induced and cultured. The culture solution of the control group was DMEM/F12 containing 10% fetal bovine serum, and the other 3 groups were added to EGF, BDNF, EGF+BDNF respectively on the basis of the culture solution in the control group. The concentration of EGF was 20ng/mL, and the BDNF concentration was 5. 0ng/mL.
The above 2 parts of cells were inoculated in six hole plates, 6 holes in each group, cultured 1W, NSE immunocytochemical staining, counting the proportion of positive cells, statistical analysis.
experimental result
1, identification of neural stem cells
The cloned spheres of single cell clone culture expressed Nestin, and after induction of 1W, the cells expressed NSE, GFAP.
2, the effect of inducing factors on the differentiation of neural stem cells.
(1) the proportion of neural stem cells differentiated into neurons in the hippocampus of 0ng/mL group, group 50ng/mL, group 100ng/mL, group 150ng/mL and 200ng/mL group was 13.33 + 3.44%, 23.17 + 2.93%, 20.67 + 2.80%, 16.67 + 2.73%, 14.17 + 3.43%. The proportion of neural stem cells differentiated into neurons in group 50ng/mL was significantly higher (P < 0.05).
(2) the proportion of cells differentiated into neurons in group EGF, group BDNF and EGF+BDNF group was 17.67 + 3.27,13.33 + 3.44,19.67 + 2.07,23.17 + 2.93 respectively. After t test, it was found that the proportion of neural stem cells differentiated into neurons in BDNF group was significantly higher than that of control group and EGF group, and the proportion of neural stem cells differentiated into neurons in EGF+BDNF group was significantly higher than that of BDNF. Group (P < 0.05).
conclusion
1, EGF can increase the ratio of BDNF to the differentiation of neural stem cells into neurons in adult rats.
2, when EGF concentration is 20ng/mL, BDNF promotes the differentiation of adult rat hippocampal formation neural stem cells into neurons, the best concentration is 50ng/mL.
【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R329.28

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 陶軼;李立新;傅震;胡衛(wèi)星;魯艾林;謝青松;黃保勝;史德志;;腦源性神經(jīng)營養(yǎng)因子與胰島素樣生長因子Ⅰ促進(jìn)神經(jīng)干細(xì)胞向神經(jīng)元定向分化的作用差異[J];中國臨床康復(fù);2006年09期

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本文編號：1954314