分泌型NgR拮抗肽基因修饰嗅鞘细胞促进视神经修复的实验研究

发布时间：2018-05-24 12:32

本文选题：新生大鼠 + 嗅鞘细胞　；参考：《华中科技大学》2011年博士论文

【摘要】：第一部分新生大鼠嗅鞘细胞的分离、培养和纯化研究目的探讨新生大鼠嗅鞘细胞的分离、培养和纯化方法,为后期的基因修饰嗅鞘细胞修复视神经损伤研究奠定基础。方法取2d内的新生大鼠嗅球组织,在解剖显微镜下分离取材,原代培养嗅鞘细胞,运用差速贴壁法和差速贴壁+阿糖胞苷+细胞生长刺激因子法两种方法进行纯化,在倒置显微镜下观察不同培养时间嗅鞘细胞的形态、数量和NGFRp75抗体免疫荧光结果,统计嗅鞘细胞的纯度。结果嗅鞘细胞的纯度分别是差速贴壁组平均为60.41%±4.32%,差速贴壁+阿糖胞苷+细胞生长刺激因子法组平均为84.98%±4.03%。两组数据用t检验进行统计学分析,差异有统计学意义(P0.05)。结论取材于新生大鼠,显微镜下分离脑膜,差速贴壁+阿糖胞苷+细胞生长刺激因子法纯化,都是良好的嗅鞘细胞培养方法。第二部分大鼠NEP1-40基因的扩增及重组慢病毒载体的构建目的构建携带大鼠NEP1-40目的基因的重组慢病毒,并检测其体外表达目的基因的能力。方法PCR扩增出TAT/NEP1-40编码片段,定向克隆入pLV.Des2d.P/puro载体,Lipofectamine2000转染293FT细胞包装携带NEP1-40目的基因的重组慢病毒,测序证实为TAT/NEP1-40后大量扩增,以基因转移单位(GTU)法测定病毒滴度,运用PCR和western blot分别检测NEP1-40在293FT细胞中转录水平和蛋白水平的基因表达。结果成功构建携带大鼠NEP1-40目的基因的重组慢病毒,并证实其在293FT细胞中可表达NEP1-40。结论携带大鼠NEP1-40基因的重组慢病毒可在体外表达其目的基因,为研究NEP1-40在视神经损伤恢复中的作用奠定基础。第三部分慢病毒介导的NEP1-40基因在体外嗅鞘细胞中的表达目的研究慢病毒介导的NEP1-40基因在体外嗅鞘细胞中的表达,并初步观察转染了NEP1-40基因的嗅鞘细胞对视网膜节细胞的作用。方法用NEP1-40慢病毒载体转染体外原代培养嗅鞘细胞,应用PCR及ELISA检测NEP1-40基因在嗅鞘细胞中转录水平和蛋白水平的表达,并将转基因嗅鞘细胞与视网膜神经节细胞共培养,观察节细胞轴突的生长情况。结果NEP1-40慢病毒载体转染体外原代培养嗅鞘细胞后经过PCR和ELISA检测,在转录水平和蛋白表达水平证实NEP1-40基因均有表达,转基因嗅鞘细胞与视网膜神经节细胞共培养时观察到节细胞生长状况良好。结论NEP1-40重组慢病毒载体可以转染体外原代培养嗅鞘细胞,并能在细胞外表达其目的基因,促进视网膜神经节细胞生长,为后期动物实验奠定基础。
[Abstract]:Part one: isolation, culture and purification of olfactory ensheathing cells from neonatal rats Objective to investigate the methods of isolation, culture and purification of olfactory ensheathing cells from newborn rats, so as to lay a foundation for the repair of optic nerve injury by gene modified olfactory ensheathing cells. Methods olfactory ensheathing cells were isolated from the olfactory bulb of newborn rats within 2 days. The olfactory ensheathing cells were cultured under anatomic microscope. The cells were purified by two methods: differential adhesion method and differential adhesion cytosine cell growth stimulating factor method. The morphology and quantity of olfactory ensheathing cells and the results of NGFRp75 antibody immunofluorescence were observed under inverted microscope, and the purity of olfactory ensheathing cells was counted. Results the purity of olfactory ensheathing cells was 60.41% 卤4.32g in differential adhesion group and 84.98% 卤4.03g in differential adhesion cytarabine cell growth factor group. The data of the two groups were analyzed by t test, and the difference was statistically significant (P 0.05). Conclusion it is a good culture method for olfactory ensheathing cells to extract meninges from newborn rats and isolate meninges under microscope and purify cytarabine cell growth stimulating factor by differential adhesion. Part two Amplification of Rat NEP1-40 Gene and Construction of Recombinant Lentivirus Vector Aim to construct a recombinant lentivirus carrying rat NEP1-40 gene and to detect its ability to express the target gene in vitro. Methods TAT/NEP1-40 coding fragment was amplified by PCR and cloned into pLV.Des2d.P/puro vector Lipofectamine2000 to transfect the recombinant lentivirus carrying NEP1-40 target gene in 293FT cell package. The recombinant lentivirus was confirmed to be TAT/NEP1-40 by sequencing. The viral titer was determined by gene transfer unit (GTU) assay. PCR and western blot were used to detect the transcriptional and protein levels of NEP1-40 in 293FT cells. Results Recombinant lentivirus carrying rat NEP1-40 gene was successfully constructed, and NEP1-40 expression was confirmed in 293FT cells. Conclusion Recombinant lentivirus carrying rat NEP1-40 gene can express its target gene in vitro, which lays a foundation for studying the role of NEP1-40 in the recovery of optic nerve injury. The expression of lentivirus-mediated NEP1-40 gene in olfactory ensheathing cells in vitro Objective to investigate the expression of lentivirus mediated NEP1-40 gene in olfactory ensheathing cells in vitro and to investigate the effect of olfactory ensheathing cells transfected with NEP1-40 gene on retinal ganglion cells. Methods the olfactory ensheathing cells were transfected with NEP1-40 lentivirus vector in vitro. The expression of NEP1-40 gene in olfactory ensheathing cells was detected by PCR and ELISA, and the transgene olfactory ensheathing cells and retinal ganglion cells were co-cultured. To observe the growth of ganglion cell axons. Results NEP1-40 lentivirus vector was transfected into olfactory ensheathing cells in vitro and detected by PCR and ELISA. The expression of NEP1-40 gene was confirmed at the level of transcription and protein expression. It was observed that the ganglion cells grew well in co-culture of transgenic olfactory ensheathing cells and retinal ganglion cells. Conclusion Recombinant lentivirus vector of NEP1-40 can transfect olfactory ensheathing cells in vitro, express its target gene in vitro, promote the growth of retinal ganglion cells, and lay a foundation for later animal experiments.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R774.6

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