【作者】 李立君；

【导师】 王任直；

【作者基本信息】 中国协和医科大学 ， 临床医学， 2008， 博士

【摘要】 研究背景和目的:神经干细胞(Neural Stem Cells,NSCs)移植和基因治疗,是治疗缺血性脑血管疾病的两个重要的研究途径。近年来,神经干细胞已经被用作基因治疗的载体,可将治疗基因携带到病灶,通过治疗基因在缺血性损伤局部表达而发挥作用,有望为中风的治疗带来新的希望。本研究的目的:以慢病毒(Lentivirus,LV)介导,将hNT-3基因转入NSCs,并进行体外培养,探讨NSCs-hNT3在体外分化以及表达NT-3时间上的特点,为体内移植NSCs-hNT3的研究和临床应用提供必要的体外实验依据。方法:实验共分二部分。第一部分:从孕14天Sprague-Dawley(S-D)大鼠胚胎的脑组织分离出神经干细胞,采用无血清培养法,进行体外培养、扩增,并通过免疫荧光化学染色(巢蛋白)进行鉴定。取传至第5～6代神经干细胞,以1×10~5 cells/500μl/孔接种于24孔板,分别按复感染指数(Multiplicities of infection,MOIs值)为0、1、5、10、15、20加入携带报告基因GFP的慢病毒载体(lentiviralvector-GFP,LV/GFP)稀释液,每一滴度加六孔,共六组。2～3天后于倒置荧光显微镜下观察各组GFP的表达效率,并在3天后进行神经干细胞球进行计数,观察LV/GFP对NSCs增殖的影响。并行流式细胞仪检测,得出各组NSCs的GFP阳性转染率。第二部分:构建表达hNT-3基因的慢病毒载体(lentiviral vector-hNT-3,LV/hNT-3),实验组根据最佳MOI用LV/hNT-3转染NSCs,对照组NSCs不转染病毒。两组细胞继续无血清培养,48h～96h后,应用ELISA和免疫荧光染色方法对两组NSCs在体外的分化和NT-3的表达进行检测。结果:第一部分:NSCs以悬浮细胞球方式生长,nestin染色阳性。转染GFP基因后,除MOI值为0的对照组外,2～3天后各孔均有GFP表达。MOI值从0增至10,细胞的阳性表达率逐渐提高(P＜0.05),MOI值为10的组能获得＞85%的转染率。但MOI值从10增至20,形成的神经干细胞球数目却逐渐减少。第二部分:NT-3修饰的神经干细胞早期跟亲代无形态学区别,3-4天后,实验组中神经干细胞分化比例比对照组明显要高,已分化的细胞在形态上与对照组比较:细胞突起长而且数量多,细胞间突触联系多。转NT-3组神经干细胞,向神经元方向分化的比例明显高于对照组。结论:1) LV是将外源基因转入神经干细胞的理想载体。以MOI为10的滴度,LV可将外源基因高效转入神经干细胞内。2)慢病毒介导转染神经营养素-3基因的神经干细胞体外培养,能高效表达NT-3,向神经元方向分化比例增多,且能形成的突起较长,数量多。更多还原

【Abstract】 Background and Objective:Neural stem cells(NSCs) transplantation and gene therapy are the most important approaches to novel strategies for ischemic stroke therapy.In recent years,NSCs,for their pathotropism and their longterm persistence in target tissue have,been used as a promising vehicle for targeted gene deliverys.The innate capacity of NSCs to release protective molecules can further be increased by genetically transfecting NSCs to secrete additional neuroprotective peptides or molecules that can play an important role in hypoxic-ischemic brain injury repair and regeneration.Combined gene therapy and NSCs transplanation brings a novel thinking and pathway for ischemic stroke therapy.NSCs,transduced by hNT-3 gene mediated by LV,were grown,expended and propagated in serum-free medium.We investigated the optimal MOI of LV.The study observed the expression of NSCs modified by NT-3 gene after transfection,as well as tested the neuronal differentiation under the influence of expression of the neuron-inducing factor,NT-3,by genetically modified NSCs.Methods:The study consisted of three parts.PartⅠ:Neural stem cells were extracted from rat embryonic brain(E14),then grown,expended and propagated in serum-free medium.Passage 5～6 neural stem cells were seeded at a density of 1×105/500μl/well in 24-well plates and exposed to lentiviral vector(LV) coding for GFP reporter gene(LV/GFP) at multiplicities of infection(MOIs) of 0,1,5,10,15,20 respectively.There were six groups totally,and each group had six wells seeded.The cells were incubated in a humidified atmosphere with 5%CO2 at 37℃.2～3 days later,the cells were observed under inverted fluorescence microscope.And 3 days later,neurospheres were counted followed by flow cytometer examination to get the percentage of transgene positive NSCs.PartⅡ:The lentiviral vector(LV) coding for Flag-tagged hNT-3(LV/hNT3) gene was constructed by the co-transfection of 293T cells with transfer plasmid coding for hNT-3(pGC-E1/hNT-3) and two help plasmids (pHelper 1.0 and Helper 2.0) using Lipofectamine 2000.NSCs were transduced by hNT-3 gene mediated by LV,grown as partⅠ.Immunofluorencence staining was used to label NSCs-hNT3(MAP2,GFAP,GalC) and newly proliferated cells.The proportion of neuronal differentiation was gotten.We collected the cells and medium at 48h、72h、96h,and observed the expression of NT-3 by ELISA.Results:PartⅠ:7～8 days after isolated from rat embryonic brain,NSCs grew as free-floating nestin-positive neurospheres.2～3 days after GFP gene transduction, GFP-expressing cells cound be seen except in the control group,Over 90%cells were GFP-positive at 3 days after transduction by LV at MOI of 10.A significant dose-response was observed with increasing virus titer for MOI 0 to 10(P＜0.05), whereas the amount of neurospheres decrease with increased virus load for MOI 10 to 20.PartⅡ:When cultured in serum containing media,NSC-NT3 cells,like the parent NSCs,still differentiated into all three fundamental neural cell types((neurons, astrocytes and oligodendrocytes).However,NSCs-NT3’s percentage of neurons was higher than the parent NSCs’.In NSCs-NT3,neuronal differentiation always predominated.The neurons that derived from the NSCs-NT3 cells bore more numerous and longer processes than non-engineered NSCs assayed at the same time in culture.Conclusion:1) LV is an ideal vector for gene transduction and it can transduce a foreign gene into NSCs with high efficency of over 85%at the MOI of 10.2) NSCs-NT3’s percentage of neurons is higher than the controls.The neurons that derived from the NSCs-NT3 cells born more numerous and longer processes than non-engineered NSCs.更多还原