慢病毒介导NT-3基因转导神经干细胞体外培养生物学特性研究

发布时间：2018-07-31 11:46

【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。
[Abstract]:Background and objective: neural stem cell (Neural Stem) transplantation and gene therapy are two important approaches in the treatment of ischemic cerebrovascular diseases. In recent years, neural stem cells have been used as gene therapy vectors, which can carry the therapeutic gene to the focus and play a role through the local expression of therapeutic gene in ischemic injury, which may bring new hope for the treatment of apoplexy. The purpose of this study was to transfer hNT-3 gene into NSCs mediated by lentivirus LV and culture in vitro, to explore the characteristics of NSCs-hNT3 differentiation and expression of NT-3 in vitro, and to provide the necessary in vitro experimental basis for the study and clinical application of NSCs-hNT3 transplantation in vivo. Methods: the experiment was divided into two parts. The first part: neural stem cells were isolated from the brain of Sprague-Dawley (S-D) rat embryos on the 14th day of gestation. The neural stem cells were cultured in vitro and amplified by serum-free culture. The neural stem cells were identified by immunofluorescence staining (nestin). Neural stem cells (NSCs) were transferred to the 5th generation and inoculated with 24 well plates with 1 脳 10 ~ (5) cells/500 渭 l / well. According to the multiple infection index (Multiplicities of), they were added to the dilution solution of lentiviralvector-GFP / GFP / GFP carrying the reporter gene GFP, respectively, and were added to the dilution solution of lentiviralvector-GFPV / GFP with six holes in each titer, according to the multiple infection index (Multiplicities of). The expression efficiency of GFP was observed under inverted fluorescence microscope after 3 days in six groups. The neural stem cell spheres were counted 3 days later to observe the effect of LV/GFP on the proliferation of NSCs. The positive transfection rate of NSCs was obtained by flow cytometry. In the second part, lentiviral vector-hNT-3 / LV- / hNT-3 was constructed. The experimental group was transfected with LV/hNT-3 according to the optimal MOI, while the control group (NSCs) was not transfected with the virus. The differentiation of NSCs and the expression of NT-3 in the two groups were detected by ELISA and immunofluorescence staining after 48h and 96h of serum-free culture. Results: in the first part, NSCs grew in the form of suspension cells and positive staining for nestin. After transfection of GFP gene, the expression of GFP was increased from 0 to 10 in every pore 3 days after transfection, and the positive expression rate of the cells increased gradually (P < 0. 05) in the control group with a MOI value of 0. The transfection rate was more than 85% in the control group with a MOI value of 10 (P < 0. 05). However, when MOI increased from 10 to 20, the number of neural stem cell balls gradually decreased. Part two: NT-3 modified neural stem cells had no morphological difference from their parents at the early stage. After 3-4 days, the proportion of neural stem cells differentiated in the experimental group was significantly higher than that in the control group. Compared with the control group, the differentiated cells had longer and more processes and more synaptic connections. The percentage of neural stem cells differentiated into neurons in NT-3 group was significantly higher than that in control group. Conclusion: 1) LV is an ideal vector for the transfer of exogenous gene into neural stem cells. When the titer of MOI was 10, the exogenous gene could be efficiently transferred into neural stem cells. 2) lentivirus mediated neural stem cells transfected with neurotrophin 3 gene could express NT-3 efficiently and differentiate into neurons. And can form a long, large number of protrusions.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329

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