大鼠胎脑神经干细胞的分离培养及电穿孔转染

发布时间：2018-06-12 18:29

本文选题：神经干细胞 + 血管内皮细胞生长因子　；参考：《中南大学》2009年硕士论文

【摘要】： 研究背景:神经干细胞(neural stem cells,NSCs)的发现和分离成功,打破了神经细胞不能再生的传统理论。同时神经干细胞具有自我增殖,多向分化,无免疫原性等特点,已成为转基因治疗的理想载体细胞。近年来研究表明,神经干细胞转基因移植治疗中枢神经系统疾病作为一种新的治疗手段,已经在创伤性脑脊髓损伤、脑血管病、退行性疾病、遗传代谢性疾病、脑肿瘤等动物模型中进行了实验,取得了一定的疗效。血管内皮细胞生长因子(Vascular endothelial growthfactor,VEGF)是1989年Ferrara等在牛垂体滤泡星状细胞体外培养液中首先纯化出来的糖类蛋白质,其相对分子量为34～45kD,是由两个相对分子量为17～22kD的亚基通过二硫键连接而成的二聚体,能特异性地作用于血管内皮细胞,促进血管内皮细胞增殖,加速新生血管的形成,增加血管通透性,并有直接的神经保护和促神经发生作用。基因治疗成功的关键是外源基因的转染效率和表达强度。目前将外源基因导入真核细胞的方法有很多种,可分为两大类:病毒转染法和非病毒转染法。病毒转染法具有很高的转染效率,但其存在安全和技术方面的缺点,从而限制了病毒转染法的广泛应用。在非病毒转染法中,最常用的是脂质体转染和电穿孔转染。而脂质体介导转染神经干细胞,其转染效率仅为5%～15%。目的:从SD胎鼠脑组织中分离培养出神经干细胞,并寻找一种能高效转染神经干细胞的非病毒转染方法。方法:本实验首先从SD胎鼠脑组织中分离NSCs,在体外采用无血清悬浮培养法进行培养和扩增,利用免疫荧光组织化学技术对NSCs及其分化细胞进行鉴定。然后构建和鉴定真核表达质粒pEGFP-N1/VEGF165。再使用电穿孔技术将质粒pEGFP-N1转染入NSCs,利用其表达的绿色荧光蛋白(green fluorescence protein,GFP)作为转染成功的报告物或标记物,在荧光显微镜下根据发出绿色荧光的NSCs数量,计算出转染效率。结果:从SD胎鼠脑组织中分离的细胞在体外可长时间自我增殖,原代及传代克隆细胞均表达NSCs的特异性抗原Nestin,且诱导分化后可表达星型胶质细胞的特异性抗原-胶质纤维酸性蛋白(Glial fibrillaryacidic protein,GFAP)和神经元的特异性抗原-神经元特异性烯醇化酶(Neuron-specific enolase,NSE)。通过酶切及测序鉴定,表明pEGFP-N1/VEGF165构建成功。同时用电穿孔技术转染神经干细胞,其效率可达20%～70%,平均30%。结论:本实验从胎鼠脑组织中分离出了具有自我增殖和多向分化潜能的神经干细胞,并构建质粒pEGFP-N1/VEGF165。同时证实电穿孔技术对神经干细胞可进行高效转染,其效率可达20%～70%,平均30%,这为进一步研究神经干细胞的转基因治疗提供了实验基础。
[Abstract]:Background: the discovery and isolation of neural stem cells have broken the traditional theory that nerve cells can not regenerate. At the same time, neural stem cells with the characteristics of self-proliferation, multi-differentiation, no immunogenicity and so on, have become the ideal vector cells for transgenic therapy. Recent studies have shown that neural stem cell transplants for the treatment of central nervous system diseases, as a new treatment, have been traumatic brain and spinal cord injury, cerebrovascular disease, degenerative diseases, genetic metabolic diseases, Experiments were carried out in animal models such as brain tumors, and certain curative effects were obtained. Vascular endothelial growth factor (VEGF) is the first glycoprotein purified from bovine pituitary follicular stellate cells in 1989 by Ferrara et al. Its relative molecular weight is 34 ~ 45kD. it is a dimer formed by two relative molecular weight (17 ~ 22 KD) subunits connected by disulfide bond. It can act specifically on vascular endothelial cells, promote the proliferation of vascular endothelial cells, and accelerate the formation of new vessels. Increased vascular permeability and direct neuroprotective and neurogenic effects. The key to the success of gene therapy is the transfection efficiency and expression intensity of exogenous genes. At present, there are many methods to transfer foreign genes into eukaryotic cells, which can be divided into two categories: viral transfection and non-viral transfection. Virus transfection has high transfection efficiency, but it has some shortcomings in safety and technology, which limits the wide application of virus transfection. Among non-viral transfection methods, liposome transfection and electroporation transfection are the most commonly used methods. However, the transfection efficiency of neural stem cells mediated by liposome was only 5%. Aim: to isolate and culture neural stem cells (NSCs) from the brain tissue of SD fetus and to find a nonviral transfection method for neural stem cells (NSCs). Methods: NSCs were isolated from the brain tissue of SD fetus and cultured and amplified by serum-free suspension culture in vitro. Immunofluorescence histochemical technique was used to identify NSCs and their differentiated cells. Then the eukaryotic expression plasmid pEGFP-N1 / VEGF165was constructed and identified. Then the plasmid pEGFP-N1 was transfected into NSCsby electroporation, and the green fluorescence protein (GFP) was used as the successful reporter or marker. The transfection efficiency was calculated under fluorescence microscope according to the number of green fluorescent NSCs. Results: the cells isolated from the brain tissue of SD fetus could proliferate for a long time in vitro. Both primary and subculture clones expressed NSC-specific antigen Nestin, and after induction of differentiation, they expressed glial fibrillaryacidic protein (Glial fibrillaryacidic protein) and neuron-specific enolase (Neuron-specific enolase). Restriction endonuclease digestion and sequencing showed that pEGFP-N 1 / VEGF165 was successfully constructed. At the same time, electroporation technology transfected neural stem cells, its efficiency can reach 20, 70, an average of 30. Conclusion: neural stem cells with self-proliferation and multi-differentiation potential were isolated from fetal brain tissue and constructed plasmid pEGFP-N1 / VEGF165. At the same time, it was proved that electroporation technology could transfect neural stem cells efficiently, and its efficiency could reach 200.70g, with an average of 30%, which provided the experimental basis for further study on the transgenic therapy of neural stem cells (NSCs).
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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