發(fā)布時間：2018-02-27 00:15

  本文關(guān)鍵詞： 神經(jīng)導(dǎo)向因子 腺病毒載體 基因重組 骨髓間充質(zhì)干細(xì)胞　出處：《南京醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】：背景Netrins家族是一類重要的神經(jīng)導(dǎo)向因子,在神經(jīng)生長發(fā)育過程中通過作用于生長錐上的DCC或UNC5B受體來誘導(dǎo)或排斥軸突的生長,從而決定軸突的生長路線及軸突與特定靶細(xì)胞的功能聯(lián)系。Netrins家族主要包括netrin1,netrin2,netrin3及netrin4,Ntn1是其中研究最為廣泛的一員,由底板細(xì)胞所分泌。Serafini及其同事在研究脊髓的軸突外生和逆轉(zhuǎn)實驗中從雞胚胎腦組織中首次分離并純化這一分子。研究表明Ntn1不僅對中樞神經(jīng)系統(tǒng)中纖維的投射起重要作用,而且在外周神經(jīng)損傷的修復(fù)中也有一定的作用。血管系統(tǒng)和神經(jīng)系統(tǒng)的發(fā)育在胚胎系統(tǒng)中都是十分重要的過程,也具有相似的結(jié)構(gòu)和發(fā)育機制,因此二者之間可能存在共同信號進(jìn)行分子“交談”。近年的研究表明Ntn1及其受體在血管系統(tǒng)同樣有表達(dá)。因此提出Ntn1對于糖尿病、腦血管病等同時存在血管和神經(jīng)病變的疾病的治療有其優(yōu)越性。本研究通過將外源的Ntn1基因插入含GEFP標(biāo)記的腺病毒載體中構(gòu)建重組腺病毒,經(jīng)過包裝純化而得到高轉(zhuǎn)錄的Ntn1重組腺病毒,為探討Ntn1對于血管、神經(jīng)的作用打下基礎(chǔ)。方法構(gòu)建攜帶Ntn1基因的腺病毒載體分為三個階段:(1)采用基因克隆技術(shù)克隆目的基因Ntn1,并將目的基因克隆入含有報告基因EGFP的穿梭載體pDC316質(zhì)粒;(2)構(gòu)建的質(zhì)粒pDC316-netrin經(jīng)酶切及測序鑒定正確后,通過脂質(zhì)體lipofectamine2000的介導(dǎo)與腺病毒包裝質(zhì)粒pBHGlox_E1.3Cre共轉(zhuǎn)染至人胚腎細(xì)胞HEK293,經(jīng)同源重組后得到攜帶鼠Ntn1的重組腺病毒Ad5-netrin-CMV-EGFP;(3)應(yīng)用PCR鑒定重組腺病毒,空斑傳代純化病毒并反復(fù)凍融擴增病毒,獲得大量具有感染能力的重組腺病毒Ad5-netrin-CMV-EGFP顆粒。以50%組織培養(yǎng)感染劑量法(TCID50)測定病毒滴度。原代培養(yǎng)SD大鼠骨髓間充質(zhì)干細(xì)胞,Ad5-netrin-CMV-EGFP感染骨髓間充質(zhì)干細(xì)胞,并用酶聯(lián)免疫吸附法檢測目的基因的表達(dá)。 結(jié)果本試驗成功構(gòu)建了表達(dá)神經(jīng)導(dǎo)向因子Ntn1的重組腺病毒載體Ad5-netrin-CMV-EGFP。MOI50pfu/cell重組腺病毒Ad5-netrin-CMV-EGFP感染原代培養(yǎng)的骨髓間充質(zhì)干細(xì)胞。Ad5-netrin-CMV-EGFP感染間充質(zhì)干細(xì)胞48h后,ELISA檢測細(xì)胞培養(yǎng)上清中Ntn1蛋白濃度顯著高于未感染的細(xì)胞培養(yǎng)上清。 結(jié)論本試驗研究顯示(1)腺病毒系統(tǒng)可以有效的用于轉(zhuǎn)到目的基因Ntn1進(jìn)入哺乳動物細(xì)胞中; ( 2 )原代培養(yǎng)的間充質(zhì)干細(xì)胞感染重組腺病毒Ad5-netrin-CMV-EGFP后可高水平的分泌Ntn1蛋白;(3)骨髓間充質(zhì)干細(xì)胞以其低免疫性、自我增殖及較易獲得等優(yōu)點,可作為Ntn1基因治療的有效平臺用以促進(jìn)神經(jīng)損傷的修復(fù)和誘導(dǎo)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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 黃其林,陳潔平;Netrins的軸突及神經(jīng)細(xì)胞誘向作用與相關(guān)機制[J];中華神經(jīng)醫(yī)學(xué)雜志;2005年05期

2 孟凡偉,郭慧云,張成崗;軸突生長誘向因子(Netrin)基因家族研究進(jìn)展[J];神經(jīng)解剖學(xué)雜志;2003年01期

3 饒毅,吳瑛;神經(jīng)細(xì)胞遷移導(dǎo)向的分子機制[J];生理科學(xué)進(jìn)展;2000年03期

4 范曉棠,蔡文琴;新型導(dǎo)向分子與海馬內(nèi)聯(lián)系的發(fā)育[J];生理科學(xué)進(jìn)展;2002年04期

5 何旭,劉丹丹,王陽,郭新,李玉林;人骨髓間充質(zhì)干細(xì)胞的分離培養(yǎng)及血管內(nèi)皮生長因子對其體外誘導(dǎo)分化作用[J];中國免疫學(xué)雜志;2004年09期

，

本文編號：1540296