睫状神经营养因子CNTF基因重组腺病毒的包装

发布时间：2018-04-24 05:12

  本文选题：腺病毒 + 睫状神经营养因子　； 参考：《河北医科大学》2009年硕士论文

【摘要】： 目的:周围神经损伤是临床上常见疾病,周围神经损伤的显微外科修复技术已经十分先进,但长期以来难以获得满意得治疗下效果。随着基因技术的发展和分子生物学不断进步,基因治疗在神经修复的研究中正逐步兴起。 应用复制缺陷型重组腺病毒(Adenovirus,AdV)为载体,将各种神经营养因子基因转入脊髓用来保护和促进受损的周围神经再生的研究,已成为周围神经修复方面的前沿课题。腺病毒的研究和开发进展非常迅猛,已成功应用于多项基因治疗临床试验中。腺病毒载体具有感染效率高、基因装载容量大、能感染分裂期或静息期细胞、宿主范围广、易制备高滴度病毒、遗传毒性较低、不插入宿主基因组等优点。CNTF是由200个氨基酸组成的酸性蛋白,与NTF家族没有同源性,被称为非靶源性营养因子。 CNTF在神经再生过程中起着重要的营养支持作用,能支持感觉、交感及运动神经元存活和分化,防止轴突切断后运动神经元的凋亡以及神经细胞在发育过程中的程序性死亡。它还能促进在体和离体运动神经细胞存活,防止神经组织退变的关键因素。CNTF和其受体在神经系统中分布很广,具有广泛的生物学活性,可以提高体内外各种类型外周神经细胞的存活。在周围神经系统、髓鞘和睫状神经节的雪旺氏细胞(SC),CNTF呈高水平表达,主要位于细胞体和突起,而核中无阳性反应。CNTF主要在于星形胶质细胞中存在;通过逆向传递机制作用机制,睫状神经营养因子(CNTF)的生物功能多样,能促进运动神经元群细胞存活,对中枢神经系统及周围神经系统神经细胞的生长、分化都具有明显的营养作用。利用分子生物技术克隆大鼠睫状神经生长因子(CNTF)基因片段构建腺病毒载体,制备病毒颗粒从而进一步为研究腺病毒载体介导的睫状神经营养因子在周围神经损伤中的应用提供了基础。 方法: 1利用实验室保存的普通感受态制作超级感受态。从感受态大肠杆菌菌种中抽吸菌液培养过夜转接于200mlLB培养基中,在OD600值在0.4左右时收获细菌。加入100ul预冷0.1M CaCL2溶液,冷冻离心用移液枪轻轻上下吸动打匀,使细胞重新悬浮,动作轻柔。细胞悬浮液可立即用于转化试验或加入15%-30%的甘油后-80℃保存待用。(具体见实验步骤) 2利用公司已经包装的含有CNTF的腺病毒载体的质粒在超级感受态中转化从而扩增得到能够转染计量的质粒。在感受态中加入质粒5ul冰上解冻,热击后冰上冷却,加入培养基后震荡培养1小时,在筛选平板上铺板培养16-24小时,长出单克隆菌群后挑菌培养,离心后按质粒提取试剂盒步骤提取质粒。 3扩增后得到的质粒使用脂质体2000转染试剂盒转染人肾胚293细胞,培养细胞,包装带有CNTF基因的重组腺病毒,在-70℃和37℃温度下通过反复冻融制备高滴度腺病毒。 4病毒扩增后在96孔板中感染培养好的人肾胚293细胞,在显微镜下可观察到293细胞有明显的CPE现象,利用50%组织培养感染剂量法测定病毒滴度。 结果: 1将扩增质粒按照实验步骤的体系配好,制胶测定筛选扩增质粒与公司合成质粒酶切结果相同。 2重组质粒转染后的293细胞在培养24小时后即可在倒置荧光显微镜下观察到细胞发出绿色荧光,即检测到报告基因CNTF的表达,细胞表达出携带目的基因的腺病毒颗粒,对照组未发现荧光表达。转染成功。 3在96孔板上培养293细胞,用获得病毒感染293细胞后计数出现CPE孔个数,测定病毒滴度。最后获得病毒滴度为2.5×107TCID50/ml。 结论: 1成功的制备了用于质粒转化扩增的超级感受态,并成功扩增了所需足够计量的质粒。 2成功的包装了CNTF腺病毒,并通过反复冻融提高病毒液滴度,为进一步研究睫状神经营养因子在周围神经修复中的影响奠定了基础。 3病毒扩增后感染人肾胚293细胞,培养细胞14至18天后在显微镜下可观察到293细胞有明显的CPE现象,利用50%组织培养感染剂量法测定病毒滴度,最终得到滴度为2.5×107TCID50/ml的病毒液,为进一步研究睫状神经营养因子在周围神经修复中的影响开辟了道路。
[Abstract]:Objective : Peripheral nerve injury is a common disease in clinic . The microsurgical repair technique of peripheral nerve injury is very advanced , but it is difficult to obtain satisfactory results for a long time . With the development of gene technology and the progress of molecular biology , gene therapy is gradually emerging in the study of nerve repair .



The research and development of adenovirus vector have been successfully applied in many kinds of gene therapy clinical trials . The research and development of adenovirus have been successfully applied in many clinical trials . The adenovirus vector has the advantages of high infection efficiency , large gene loading capacity , low genetic toxicity and no insertion into host genome . The CNTF is an acid protein composed of 200 amino acids , which has no homology with the NTF family , and is called non - target nutrient factor .



CNTF plays an important role in nerve regeneration . It can support sensory , sympathetic and motor neuron survival and differentiation , prevent apoptosis of motor neurons after axonal injury and procedural death of nerve cells during development . The CNTF has a wide distribution in the nervous system . It can promote the survival of motor neuron group cells .



Method :



1 . It was transferred to 200 ml of LB medium overnight by suction of bacterial liquid from competent Escherichia coli strain . 100 ul of pre - cooled 0.1 M CaCL2 solution was added , and the cells were resuspended and the action was gentle . The cell suspension could be immediately used in the conversion test or 15 % to 30 % glycerol was added to the cell suspension for later use . ( See experimental procedure for specific details ) .



2 . The plasmid containing CNTF - containing adenovirus vector which has been packaged by the company is transformed in a super - competent state so as to be amplified to obtain a plasmid capable of transfection and measurement .



3 . The plasmid obtained after amplification was transfected into 293 cells of human kidney embryo with liposome 2000 transfection reagent kit , the cells were cultured , the recombinant adenovirus with CNTF gene was packed , and high titer adenovirus was prepared by repeated freeze - thaw at -70 鈩，

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