携带神经导向因子netrin-1的腺病毒载体的构建及其在间充质干细胞中的表达

发布时间：2018-02-27 00:15

本文关键词： 神经导向因子腺病毒载体基因重组骨髓间充质干细胞　出处：《南京医科大学》2009年硕士论文　论文类型：学位论文

【摘要】：背景Netrins家族是一类重要的神经导向因子,在神经生长发育过程中通过作用于生长锥上的DCC或UNC5B受体来诱导或排斥轴突的生长,从而决定轴突的生长路线及轴突与特定靶细胞的功能联系。Netrins家族主要包括netrin1,netrin2,netrin3及netrin4,Ntn1是其中研究最为广泛的一员,由底板细胞所分泌。Serafini及其同事在研究脊髓的轴突外生和逆转实验中从鸡胚胎脑组织中首次分离并纯化这一分子。研究表明Ntn1不仅对中枢神经系统中纤维的投射起重要作用,而且在外周神经损伤的修复中也有一定的作用。血管系统和神经系统的发育在胚胎系统中都是十分重要的过程,也具有相似的结构和发育机制,因此二者之间可能存在共同信号进行分子“交谈”。近年的研究表明Ntn1及其受体在血管系统同样有表达。因此提出Ntn1对于糖尿病、脑血管病等同时存在血管和神经病变的疾病的治疗有其优越性。本研究通过将外源的Ntn1基因插入含GEFP标记的腺病毒载体中构建重组腺病毒,经过包装纯化而得到高转录的Ntn1重组腺病毒,为探讨Ntn1对于血管、神经的作用打下基础。方法构建携带Ntn1基因的腺病毒载体分为三个阶段:(1)采用基因克隆技术克隆目的基因Ntn1,并将目的基因克隆入含有报告基因EGFP的穿梭载体pDC316质粒;(2)构建的质粒pDC316-netrin经酶切及测序鉴定正确后,通过脂质体lipofectamine2000的介导与腺病毒包装质粒pBHGlox_E1.3Cre共转染至人胚肾细胞HEK293,经同源重组后得到携带鼠Ntn1的重组腺病毒Ad5-netrin-CMV-EGFP;(3)应用PCR鉴定重组腺病毒,空斑传代纯化病毒并反复冻融扩增病毒,获得大量具有感染能力的重组腺病毒Ad5-netrin-CMV-EGFP颗粒。以50%组织培养感染剂量法(TCID50)测定病毒滴度。原代培养SD大鼠骨髓间充质干细胞,Ad5-netrin-CMV-EGFP感染骨髓间充质干细胞,并用酶联免疫吸附法检测目的基因的表达。结果本试验成功构建了表达神经导向因子Ntn1的重组腺病毒载体Ad5-netrin-CMV-EGFP。MOI50pfu/cell重组腺病毒Ad5-netrin-CMV-EGFP感染原代培养的骨髓间充质干细胞。Ad5-netrin-CMV-EGFP感染间充质干细胞48h后,ELISA检测细胞培养上清中Ntn1蛋白浓度显著高于未感染的细胞培养上清。结论本试验研究显示(1)腺病毒系统可以有效的用于转到目的基因Ntn1进入哺乳动物细胞中; ( 2 )原代培养的间充质干细胞感染重组腺病毒Ad5-netrin-CMV-EGFP后可高水平的分泌Ntn1蛋白;(3)骨髓间充质干细胞以其低免疫性、自我增殖及较易获得等优点,可作为Ntn1基因治疗的有效平台用以促进神经损伤的修复和诱导新生血管的形成。
[Abstract]:Background Netrins family is a kind of important neuronal guidance factor, which induces or repel axonal growth by acting on DCC or UNC5B receptors on the growth cone during nerve growth and development. Thus determining the growth path of axons and the functional relationship between axons and specific target cells. Netrins family mainly includes netrin1netrin2netrin3 and netrin4Ntn1, among which the most widely studied member is netrin 1, netrin 2, netrin2, netrin3 and netrin4. Serafini and his colleagues isolated and purified this molecule from chicken embryonic brain tissue for the first time in the study of axon exophysis and reversal of spinal cord. It has been shown that Ntn1 not only plays an important role in the projection of fibers in the central nervous system. The development of vascular system and nervous system is very important in the embryonic system, and it also has similar structure and development mechanism. So there may be a common signal for molecular "conversation" between the two. Recent studies have shown that Ntn1 and its receptors are also expressed in the vascular system. In this study, we constructed recombinant adenovirus by inserting exogenous Ntn1 gene into adenovirus vector containing GEFP marker. A highly transcriptional Ntn1 recombinant adenovirus was obtained by packaging and purification. In order to investigate the effects of Ntn1 on blood vessels, Methods the adenovirus vector carrying Ntn1 gene was constructed into three stages: 1) the target gene Ntn1 was cloned by gene cloning technique, and the target gene was cloned into the shuttle vector pDC316 containing the reporter gene EGFP. The plasmid pDC316-netrin was identified by restriction endonuclease digestion and sequencing. The recombinant adenovirus Ad5-netrin-CMV-EGFPFP-3 was obtained by co-transfection of liposome lipofectamine2000 and adenovirus packaging plasmid pBHGlox_E1.3Cre into human embryonic kidney cell line HEK293.After homologous recombination, the recombinant adenovirus Ad5-netrin-CMV-EGFPO3 was identified by PCR. A large number of infected recombinant adenovirus Ad5-netrin-CMV-EGFP granules were obtained. The titer of the recombinant adenovirus Ad5-netrin-CMV-EGFP was determined by 50% tissue culture dose method. The primary culture of SD rat bone marrow mesenchymal stem cells (BMSCs) was infected with Ad5-netrin-CMV-EGFP. The expression of the target gene was detected by enzyme-linked immunosorbent assay (Elisa). Results the recombinant adenovirus vector Ad5-netrin-CMV-EGFP.MOI50pfu/cell expressing Ntn1 was successfully constructed to infect primary cultured bone marrow mesenchymal stem cells. Ad5-netrin-CMV-EGFP infected mesenchymal stem cells 48 hours later, the supernatant of cell culture was detected by Elisa. The concentration of Ntn1 protein in the medium was significantly higher than that in the uninfected cell culture supernatant. Conclusion this study shows that the adenovirus system can be used to transfer the target gene Ntn1 into mammalian cells, and (2) the primary cultured mesenchymal stem cells infected with recombinant adenovirus Ad5-netrin-CMV-EGFP can secrete high levels of adenovirus. Bone marrow mesenchymal stem cells (BMSCs) due to their low immunogenicity, The advantages of self-proliferation and easy to obtain can be used as an effective platform for Ntn1 gene therapy to promote the repair of nerve injury and induce the formation of neovascularization.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346

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