本文選題：神經(jīng)干細(xì)胞 + 血管內(nèi)皮細(xì)胞生長因子　； 參考：《中南大學(xué)》2009年碩士論文

【摘要】： 研究背景:神經(jīng)干細(xì)胞(neural stem cells,NSCs)的發(fā)現(xiàn)和分離成功,打破了神經(jīng)細(xì)胞不能再生的傳統(tǒng)理論。同時神經(jīng)干細(xì)胞具有自我增殖,多向分化,無免疫原性等特點,已成為轉(zhuǎn)基因治療的理想載體細(xì)胞。近年來研究表明,神經(jīng)干細(xì)胞轉(zhuǎn)基因移植治療中樞神經(jīng)系統(tǒng)疾病作為一種新的治療手段,已經(jīng)在創(chuàng)傷性腦脊髓損傷、腦血管病、退行性疾病、遺傳代謝性疾病、腦腫瘤等動物模型中進(jìn)行了實驗,取得了一定的療效。血管內(nèi)皮細(xì)胞生長因子(Vascular endothelial growthfactor,VEGF)是1989年Ferrara等在牛垂體濾泡星狀細(xì)胞體外培養(yǎng)液中首先純化出來的糖類蛋白質(zhì),其相對分子量為34～45kD,是由兩個相對分子量為17～22kD的亞基通過二硫鍵連接而成的二聚體,能特異性地作用于血管內(nèi)皮細(xì)胞,促進(jìn)血管內(nèi)皮細(xì)胞增殖,加速新生血管的形成,增加血管通透性,并有直接的神經(jīng)保護(hù)和促神經(jīng)發(fā)生作用。 基因治療成功的關(guān)鍵是外源基因的轉(zhuǎn)染效率和表達(dá)強(qiáng)度。目前將外源基因?qū)胝婧思?xì)胞的方法有很多種,可分為兩大類:病毒轉(zhuǎn)染法和非病毒轉(zhuǎn)染法。病毒轉(zhuǎn)染法具有很高的轉(zhuǎn)染效率,但其存在安全和技術(shù)方面的缺點,從而限制了病毒轉(zhuǎn)染法的廣泛應(yīng)用。在非病毒轉(zhuǎn)染法中,最常用的是脂質(zhì)體轉(zhuǎn)染和電穿孔轉(zhuǎn)染。而脂質(zhì)體介導(dǎo)轉(zhuǎn)染神經(jīng)干細(xì)胞,其轉(zhuǎn)染效率僅為5%～15%。 目的:從SD胎鼠腦組織中分離培養(yǎng)出神經(jīng)干細(xì)胞,并尋找一種能高效轉(zhuǎn)染神經(jīng)干細(xì)胞的非病毒轉(zhuǎn)染方法。 方法:本實驗首先從SD胎鼠腦組織中分離NSCs,在體外采用無血清懸浮培養(yǎng)法進(jìn)行培養(yǎng)和擴(kuò)增,利用免疫熒光組織化學(xué)技術(shù)對NSCs及其分化細(xì)胞進(jìn)行鑒定。然后構(gòu)建和鑒定真核表達(dá)質(zhì)粒pEGFP-N1/VEGF165。再使用電穿孔技術(shù)將質(zhì)粒pEGFP-N1轉(zhuǎn)染入NSCs,利用其表達(dá)的綠色熒光蛋白(green fluorescence protein,GFP)作為轉(zhuǎn)染成功的報告物或標(biāo)記物,在熒光顯微鏡下根據(jù)發(fā)出綠色熒光的NSCs數(shù)量,計算出轉(zhuǎn)染效率。 結(jié)果:從SD胎鼠腦組織中分離的細(xì)胞在體外可長時間自我增殖,原代及傳代克隆細(xì)胞均表達(dá)NSCs的特異性抗原Nestin,且誘導(dǎo)分化后可表達(dá)星型膠質(zhì)細(xì)胞的特異性抗原-膠質(zhì)纖維酸性蛋白(Glial fibrillaryacidic protein,GFAP)和神經(jīng)元的特異性抗原-神經(jīng)元特異性烯醇化酶(Neuron-specific enolase,NSE)。通過酶切及測序鑒定,表明pEGFP-N1/VEGF165構(gòu)建成功。同時用電穿孔技術(shù)轉(zhuǎn)染神經(jīng)干細(xì)胞,其效率可達(dá)20%～70%,平均30%。 結(jié)論:本實驗從胎鼠腦組織中分離出了具有自我增殖和多向分化潛能的神經(jīng)干細(xì)胞,并構(gòu)建質(zhì)粒pEGFP-N1/VEGF165。同時證實電穿孔技術(shù)對神經(jīng)干細(xì)胞可進(jìn)行高效轉(zhuǎn)染,其效率可達(dá)20%～70%,平均30%,這為進(jìn)一步研究神經(jīng)干細(xì)胞的轉(zhuǎn)基因治療提供了實驗基礎(chǔ)。
[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).
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R329

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