神经干细胞与脑肿瘤干细胞体外培养的生长特性比较

发布时间：2018-04-18 15:22

本文选题：神经干细胞 + 胶质瘤　；参考：《苏州大学》2005年硕士论文

【摘要】：【目的】探索人胚神经干细胞及胶质瘤肿瘤干细胞体外分离培养的条件，并观察比较其在体外分离培养的生长特性。用CD133免疫磁珠分选阳性细胞后为进一步研究提供材料。【方法】神经干细胞：取米非司酮流产8-12周胎龄的人胚胎，从胎脑皮层分离组织，，采用胰蛋白酶消化和机械吹打的方法获得单细胞悬液。采用无血清培养基(DMEM／F12)，加入EGF和bFGF(20ng／ml)细胞因子和N_2添加剂刺激细胞增殖，从而进行体外扩增培养、连续传代。添加10％胎牛血清诱导分化，免疫细胞化学鉴定神经干细胞(Nestin)和分化成熟的神经元(NSE)、星形胶质细胞(GFAP)及少突胶质细胞(CNP)。胶质瘤肿瘤干细胞：将SHG44细胞系和人脑胶质瘤手术标本采用胰蛋白酶消化获得单细胞，用CD133免疫磁珠分离阳性细胞、之后分别应用无血清培养基(DMEM／F12，添加bFGF、EGF)培养。形成肿瘤球后用添加10％胎牛血清诱导分化，免疫细胞化学染色检测肿瘤球细胞及其分化细胞中的Nestin、NSE、GFAP阳性细胞。神经干细胞和肿瘤干细胞在下一步应用之前均行CD133免疫磁珠复选。同一时间点行流式细胞仪检测细胞周期，比较增殖活力。【结果】从人胎脑分离的细胞培养在含有EGF和bFGF的无血清培
[Abstract]:[objective] to explore the conditions of isolation and culture of human embryonic neural stem cells and glioma tumor stem cells in vitro, and to observe and compare the growth characteristics of human embryonic neural stem cells and glioma tumor stem cells in vitro.CD133 immunomagnetic beads were used to separate positive cells for further study.[methods] Neural stem cells (NSCs): single cell suspension was obtained by trypsin digestion and mechanical blow from human embryos of 8-12 weeks of gestational age induced by mifepristone.EGF and bFGF20 ng / ml) cytokines and N2Additives were added to the serum-free medium DMEM / F12 to stimulate the proliferation of the cells, and then the cells were amplified and cultured in vitro and subcultured continuously.Addition of 10% fetal bovine serum to induce differentiation, immunocytochemical identification of neural stem cells (Nestin) and the differentiation of mature neurons, astrocytes (GFAP) and oligodendrocytes (CNP).Glioma tumor stem cells: SHG44 cell lines and human glioma specimens were digested with trypsin to obtain single cells, positive cells were isolated by CD133 immunomagnetic beads, and then cultured in serum-free medium (DMEM / F12) supplemented with bFGFG (EGF).After the tumor ball was formed, 10% fetal bovine serum was added to induce the differentiation. The positive cells of the tumor ball cells and their differentiated cells were detected by immunocytochemical staining.Neural stem cells (NSCs) and tumor stem cells (NSCs) were tested with CD133 immunomagnetic beads before further application.The cell cycle was measured by flow cytometry at the same time point, and the proliferative activity was compared.[results] cells isolated from human fetal brain were cultured in serum-free culture containing EGF and bFGF.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329.2;R739.4

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