摘要
背景：胶质瘤细胞与神经干细胞之间可能存在某种内在联系。目前研究表明,胶质瘤细胞可分泌多种细胞因子,可促进NSCs的增殖和迁移。然而胶质瘤细胞是否能维持神经干细胞正常分化还不清楚。本课题组通过体内的实验研究分析神经干细胞在肿瘤细胞的诱导下的迁移现象和分化现象。
体内研究：采用GFP(绿色荧光蛋白)转基因小鼠的神经干细胞在体外扩增后移植脑内进行观察,通过追踪标记细胞分裂、分化后产生细胞谱系构图(lineagemapping),以及对同源标记细胞进行神经元、星形细胞及少突胶质细胞所表达的特异性抗原的流式细胞仪检测,来追踪神经干细胞的存活情况及分化潜能。
实验一GFP转基因鼠NSCs的分离、培养和鉴定
目的：建立GFP转基因胎鼠NSCS的分离、培养和鉴定的方法,为胶质瘤模型鼠脑内移植NSCs研究准备供体细胞。
方法：①分离GFP转基因胎鼠的海马组织,用吸吹打机械方法制成单细胞悬液。采用含B27、碱性成纤维细胞生长因子(bFGF)和表皮生长因子(EGF)的无血清培养基培养。②利用Nestin抗体对原代和传代细胞进行特异性鉴定。③用胎牛血清诱导分化后,荧光显微镜下观察分化细胞的形态和GFP表达情况。利用NeuN、GEAp和04抗体分别对分化后的神经元、星形胶质细胞和少枝胶质细胞进行特异性鉴定。
结果：①从GFP转基因胎鼠海马组织分离的细胞以悬浮方式生长,可形成典型的神经球,能在体外传代培养和连续形成克隆,且原代和传代细胞GFP表达均呈阳性。②免疫细胞化学染色示原代和传代细胞均为Nestin阳性。③血清诱导分化后的细胞可分别表达神经元、星形胶质细胞和少枝胶质细胞特异性抗原NeuN、GEAp和04,且分化后的细胞GFP表达呈阳性。
结论：从GFP转基因胎鼠海马组织成功分离培养出了NSCS。培养出的细胞Nestin阳性且具有增殖、自我更新能力及向神经元及神经胶质细胞分化的潜力,且均可稳定表达GFP,因此可作为NSCs脑内移植实验研究的供体细胞。
实验二神经干细胞体内趋化,分化实验
目的：验证移植GFP转基因神经干细胞在载瘤动物体内的存活能力,向肿瘤组织的趋化能力,并研究神经干细胞在载瘤大鼠移植区域和肿瘤种植区域的相对分布情况,分化情况
方法：首先建立胶质瘤模型,证明胶质瘤模型建立成功后,移植干细胞14天组,21天组,假手术组在相应时间段处死实验动物,无菌新鲜取材鼠脑,定位肿瘤种植区和神经干细胞移植区,采集标记中心约4*4*4mm3大小的脑组织块,匀浆脑组织,过滤,破膜剂破膜后,等分匀浆,兔抗鼠：nestin抗体(1：1),neun抗体(1：1),标记匀浆细胞,采用阿PE荧光二抗标记(羊抗兔1：1),上流式细胞仪检测对比不同区域内神经干细胞的分化情况。
结果：通过流式细胞仪采用抗体荧光标记相关抗体观察到移植的GFP转基因小鼠神经干细胞的生存状态,分布情况,分化情况。
结论：移植的神经干细胞在动物体内具备向胶质瘤细胞的趋化能力,并且增殖；相对于原移植区域,肿瘤周边迁移的神经细胞明显趋向于向神经元分化,但分化为神经元细胞的增值趋势在7-14天逐渐趋缓。
Background
Glioma cells and neural stem cells may have an intrinsic link between. The present study showed that glioma cells secrete various cytokines, can promote the proliferation and migration of NSCs. However, the mechanism of the differentiation and Migration of neural stem cells Induced by glioma cell is unclear. The subject our group study the phenomena of migration and differentiation of neural stem cells induced by glioma cells inn vivo and In vitro.
In vivo:producting a model rat with glioma, and transplant the neural stem cells derived from GFP (green fluorescent protein) transgenic mice into the Parahippocampal area of SD rats with glioma, we observe that cell differentiation of the GFP-positive cells into neurons and astrocytes expressed specific antigens by flow cytometry and immunohistochemistry, and track the survival and the differentiation of neural stem cells
Experiment 1 Isolation,culture and identification of neural stem cells of GFP transgenic mice
Purposes:
separation, culture and identification of NSCs of GFP transgenic mice and provide neural stem cells for the transplantation experment of NSCs and the experment in vitro.
Methods:
①isolated GFP transgenic fetal mouse hippocampus, wind and percussion with a mechanical suction into single cell suspension. The method includes the B27, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) in serum-free culture medium.②The Nestin antibody on primary and passaged cells specific identification.③with fetal bovine serum induced differentiation, the differentiated cells were observed under fluorescence microscope morphology and GFP expression. Using NeuN, GEAp and 04 antibody respectively differentiated neurons, astrocytes and glial cells of small sticks of specific identification.
Results:
①from GFP transgenic fetal rat hippocampus cells isolated form of suspended growth, can be formed typical neurospheres, in culture, and continuous subculture in the formation of cloning, and primary and passage cells were positive for GFP expression.②immunocytochemistry showed primary and passaged cells were Nestin positive.③differentiated cells induced by serum can be separately expressed neurons, astrocytes and Oligodendroglioma specific antigen NeuN, GEAp and 04, and the differentiated cells were positive for GFP expression.
Conclusion: GFP transgenic embryo isolated and cultured rat hippocampus success out NSCS. Nestin-positive cells cultured with proliferation and self-renewal and to the neurons and glial cell differentiation potential, and can be stably expressed GFP, NSCs transplanted into the brain can be used as experimental study of donor cells.
Experiments two chemotaxis and differentiation experiment of neural stem cells in vivo
Purposes:validation transplantation GFP transgenic neural stem cells in the parental animals ability to survive, to tumor tissue chemotaxis and to study the neural stem cells contained tumor rats transplanted tumor growing areas of regional and relative distribution of differentiation.
Methods:First established glioma model, model after successful, stem cell transplantation in 14 days group,21 days group and sham group were killed at the relevant time experimental animals, drawn fresh sterile rat brain tumor growing areas and the positioning of neural stem cell transplantation area, collecting center mark about 4* 4* 4mm3 size of brain tissue, brain tissue homogenate, filtration, breaking Remover rupture of membranes, the attainment homogenate, rabbit anti-mouse nestin antibody (1:1), neun antibody (1:1), labeled cell homogenate, with Alsace red fluorescent secondary antibodies labeled (goat anti-rabbit 1:1), flow cytometry on a comparison of different neural regions stem cell differentiation.
Results:By flow cytometry using fluorescence labeled antibody associated antibodies observed in GFP transgenic mice transplanted neural stem cells in the living state, the distribution and differentiation.
Conclusion:The transplantation of neural stem cells in vivo with glioma cells to chemotaxis and proliferation; relative to the original graft area, migration of nerve cells surrounding tumor clear trend in the neuronal differentiation, but differentiated into neurons value-added trends in 7-14 days, gradually slowing down.
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