构建携带人GFAP启动子和p27基因的腺病毒载体及其对胶质增生的影响
发布时间：2019-01-07 20:36
【摘要】：目的：构建携带人GFAP启动子和p27基因的腺病毒表达载体和仅含人p27基因的对照载体。 方法：通过质粒抽提、琼脂糖凝胶电泳、酶切、连接、转化等多种基因工程技术,构建和鉴定真核表达载体pDC315-GFAP-p27-EGFP和对照pDC315-p27-EGFP;利用腺病毒包装系统AdMax和293细胞包装重组腺病毒表达载体Ad-GFAP-p27-EGFP和Ad-p27-EGFP,用酶切、PCR法鉴定重组腺病毒表达载体。 结果：经酶切鉴定和测序,成功鉴定真核表达载体pGFAP-IRES2-EGFP-p27;经酶切鉴定和PCR鉴定,成功构建真核表达载体pDC315-GFAP-p27-EGFP和对照pDC315-p27-EGFP;经包装后,Ad-p27-EGFP可见EGFP表达和细胞致病效应(CPE),而Ad-GFAP-p27-EGFP出现CPE,但EGFP表达微弱。 结论：利用腺病毒包装系统AdMax成功构建了构建携带人GFAP启动子和p27基因的腺病毒表达载体和仅含人p27基因的对照载体,为进一步研究中枢神经系统疾病引起的胶质增生的治疗提供了基因治疗的策略。 目的：明确重组腺病毒载体Ad-GFAP-p27-EGFP和Ad-p27-EGFP对体内外星形胶质细胞增殖的影响。 方法：在体外,用重组腺病毒载体Ad-GFAP-p27-EGFP和Ad-p27-EGFP感染纯化培养的星形胶质细胞,通过免疫荧光化学、Western blot、 MTT和流式细胞术等观察星形胶质细胞感染腺病毒后外源基因EGFP、p27的表达情况以及对星形胶质细胞细胞周期和增殖的影响；重组腺病毒载体Ad-GFAP-p27-EGFP感染神经元和血管内皮细胞,检测GFAP启动子的特异性。 在体内,用重组腺病毒载体Ad-GFAP-p27-EGFP和Ad-p27-EGFP感染大鼠MCAO模型,通过免疫荧光化学、Western blot和行为学NSS评分,观察体内灶周脑组织p27和VEGF的表达情况、星形胶质细胞增生、神经元存活情况和行为学改变。 结果：重组腺病毒载体Ad-GFAP-p27-EGFP和Ad-p27-EGFP体外感染星形胶质细胞后可见星形胶质细胞均有EGFP的表达,并且p27表达水平明显增高,对星形胶质细胞的细胞周期和增殖有抑制作用；重组腺病毒载体Ad-GFAP-p27-EGFP感染神经元和血管内皮细胞,只见到十分微弱的EGFP的表达；在体实验中,重组腺病毒载体Ad-GFAP-p27-EGFP和Ad-p27-EGFP感染大鼠MCAO模型,均可抑制星形胶质细胞的增生,但是Ad-GFAP-p27-EGFP组存活的神经元和新生血管的数目增多,改善了大鼠的NSS评分。 结论：重组腺病毒载体Ad-GFAP-p27-EGFP可以有效抑制反应性胶质细胞增生。 目的：制备改良的三维硫酸肝素-胶原蛋白生物支架并评估硫酸肝素-胶原蛋白生物支架与嗅鞘细胞的生物相容性。 方法：经过改良的干燥冷冻程序,制作三维的硫酸肝素-胶原蛋白生物支架并用扫描电镜进行支架三维结构的检测；将改良的三维硫酸肝素-胶原蛋白生物支架与嗅鞘细胞在体外建立三维共培养体系,在第2、6、10和14天时用MTT法检测细胞增殖活性；将嗅鞘细胞用CFSE标记后以适宜的浓度种植于支架上,通过荧光显微镜和扫描电镜直接追踪和观察细胞在支架上的粘附和生长情况。 结果：改良的三维硫酸肝素-胶原蛋白生物支架具有三维立体的微管结构,可以促进嗅鞘细胞的粘附、增殖以及细胞突起的生长。并且在三维培养体系中,嗅鞘细胞的形态呈现出更具有活性的双极纺锤形,细胞的突起具有一致延伸方向。 结论：改良的三维硫酸肝素-胶原蛋白生物支架与嗅鞘细胞在体外具有很好的生物相容性。因此,改良的三维硫酸肝素-胶原蛋白生物支架可能可以作为细胞载体,与嗅鞘细胞组成三维复合物应用于神经组织工程。
[Abstract]:Objective: To construct an adenovirus expression vector carrying human GFAP promoter and p27 gene and a control vector containing only human p27 gene. Methods: The recombinant adenovirus expression vector pDC315-GFAP-p27-EGFP and control pDC315-p27-EGFP were constructed and identified by a variety of genetic engineering techniques such as plasmid extraction, agarose gel electrophoresis, enzyme digestion, ligation and transformation. Ad-GFAP-p27-EGFP and Ad-p27-EGFP were expressed by recombinant adenovirus expression vector Ad-GFAP-p27-EGFP and Ad-p27-EGFP. identification of recombinant adenovirus expression vector by PCR Results: The true nuclear expression vector pGFAP-IRES2-EGFP-p27 was successfully identified by enzyme-cut identification and sequencing. The true nuclear expression vector pDC315-GFAP-p27-EGFP and the control pDC315-p27-EGFP were successfully constructed by enzyme-cut identification and PCR identification. The expression of EGFP and the cytopathogenic effect (CPE) of Ad-p27-EGFP were observed after the packaging. Conclusion: AdMax successfully constructed an adenovirus expression vector carrying human GFAP promoter and p27 gene and a control vector containing only human p27 gene, and provided a gene therapy for further study of the treatment of gliosis caused by central nervous system diseases. Objective: To clarify the effect of recombinant adenovirus vector Ad-GFAP-p27-EGFP and Ad-p27-EGFP on the proliferation of astrocytes in vitro. Methods: In vitro, the cultured astrocytes were infected by recombinant adenovirus vector Ad-GFAP-p27-EGFP and Ad-p27-EGFP. Expression of EGFP and p27 and the effect on cell cycle and proliferation of astrocytes; recombinant adenovirus vector Ad-GFAP-p27-EGFP infected neurons and vascular endothelial cells to detect GFAP promoter In vivo, the expression of p27 and VEGF, the expression of p27 and VEGF, the proliferation of astrocyte and the survival of the neurons were observed by using recombinant adenovirus vector Ad-GFAP-p27-EGFP and Ad-p27-EGFP in the rat MCAO model. Results: Ad-GFAP-p27-EGFP and Ad-p27-EGFP were transfected with Ad-p27-EGFP in vitro. It was found that the recombinant adenovirus vector Ad-GFAP-p27-EGFP and Ad-p27-EGFP were infected by Ad-GFAP-p27-EGFP and Ad-p27-EGFP. The number of surviving neurons and new vessels in the Ad-GFAP-p27-EGFP group increased, and the rats were improved. Conclusion: The recombinant adenovirus vector Ad-GFAP-p27-EGFP can effectively inhibit the reaction. Objective: To prepare the modified three-dimensional heparin-collagen biological scaffold and to evaluate the heparin-collagen biological scaffold and the olfactory system. The method comprises the following steps: a three-dimensional heparin-collagen biological scaffold is prepared through an improved drying and freezing process, and the three-dimensional structure detection is carried out by using a scanning electron microscope; and the modified three-dimensional heparin-collagen biological scaffold and the sniffer cells are in vitro establishing a three-dimensional co-culture system, and detecting the cell proliferation activity by the MTT method on the days 2, 6, 10 and 14; and planting the sniffer cells on the support with a proper concentration after the FSE marker is labeled with the CFSE, and directly tracking and observing the cell on the support through the fluorescence microscope and the scanning electron microscope. Results: The modified three-dimensional heparin-collagen scaffold has three-dimensional and three-dimensional microtubule structure, which can promote the adhesion and increase of the olfactory cells. and in a three-dimensional culture system, the morphology of the olfactory cells exhibits a more active, double-polar, spindle-shaped, cell-like shape, Conclusion: The modified three-dimensional heparin-collagen biological scaffold and the olfactory receptor are in the body. therefore, the improved three-dimensional heparin-collagen biological scaffold can be used as a cell carrier,
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.08;R741
本文编号：2404131
[Abstract]:Objective: To construct an adenovirus expression vector carrying human GFAP promoter and p27 gene and a control vector containing only human p27 gene. Methods: The recombinant adenovirus expression vector pDC315-GFAP-p27-EGFP and control pDC315-p27-EGFP were constructed and identified by a variety of genetic engineering techniques such as plasmid extraction, agarose gel electrophoresis, enzyme digestion, ligation and transformation. Ad-GFAP-p27-EGFP and Ad-p27-EGFP were expressed by recombinant adenovirus expression vector Ad-GFAP-p27-EGFP and Ad-p27-EGFP. identification of recombinant adenovirus expression vector by PCR Results: The true nuclear expression vector pGFAP-IRES2-EGFP-p27 was successfully identified by enzyme-cut identification and sequencing. The true nuclear expression vector pDC315-GFAP-p27-EGFP and the control pDC315-p27-EGFP were successfully constructed by enzyme-cut identification and PCR identification. The expression of EGFP and the cytopathogenic effect (CPE) of Ad-p27-EGFP were observed after the packaging. Conclusion: AdMax successfully constructed an adenovirus expression vector carrying human GFAP promoter and p27 gene and a control vector containing only human p27 gene, and provided a gene therapy for further study of the treatment of gliosis caused by central nervous system diseases. Objective: To clarify the effect of recombinant adenovirus vector Ad-GFAP-p27-EGFP and Ad-p27-EGFP on the proliferation of astrocytes in vitro. Methods: In vitro, the cultured astrocytes were infected by recombinant adenovirus vector Ad-GFAP-p27-EGFP and Ad-p27-EGFP. Expression of EGFP and p27 and the effect on cell cycle and proliferation of astrocytes; recombinant adenovirus vector Ad-GFAP-p27-EGFP infected neurons and vascular endothelial cells to detect GFAP promoter In vivo, the expression of p27 and VEGF, the expression of p27 and VEGF, the proliferation of astrocyte and the survival of the neurons were observed by using recombinant adenovirus vector Ad-GFAP-p27-EGFP and Ad-p27-EGFP in the rat MCAO model. Results: Ad-GFAP-p27-EGFP and Ad-p27-EGFP were transfected with Ad-p27-EGFP in vitro. It was found that the recombinant adenovirus vector Ad-GFAP-p27-EGFP and Ad-p27-EGFP were infected by Ad-GFAP-p27-EGFP and Ad-p27-EGFP. The number of surviving neurons and new vessels in the Ad-GFAP-p27-EGFP group increased, and the rats were improved. Conclusion: The recombinant adenovirus vector Ad-GFAP-p27-EGFP can effectively inhibit the reaction. Objective: To prepare the modified three-dimensional heparin-collagen biological scaffold and to evaluate the heparin-collagen biological scaffold and the olfactory system. The method comprises the following steps: a three-dimensional heparin-collagen biological scaffold is prepared through an improved drying and freezing process, and the three-dimensional structure detection is carried out by using a scanning electron microscope; and the modified three-dimensional heparin-collagen biological scaffold and the sniffer cells are in vitro establishing a three-dimensional co-culture system, and detecting the cell proliferation activity by the MTT method on the days 2, 6, 10 and 14; and planting the sniffer cells on the support with a proper concentration after the FSE marker is labeled with the CFSE, and directly tracking and observing the cell on the support through the fluorescence microscope and the scanning electron microscope. Results: The modified three-dimensional heparin-collagen scaffold has three-dimensional and three-dimensional microtubule structure, which can promote the adhesion and increase of the olfactory cells. and in a three-dimensional culture system, the morphology of the olfactory cells exhibits a more active, double-polar, spindle-shaped, cell-like shape, Conclusion: The modified three-dimensional heparin-collagen biological scaffold and the olfactory receptor are in the body. therefore, the improved three-dimensional heparin-collagen biological scaffold can be used as a cell carrier,
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.08;R741
【参考文献】
相关期刊论文 前1条
1 张建祥;胡薇薇;陈忠;;对胶质疤痕所致神经再生障碍的治疗进展[J];浙江大学学报(医学版);2009年06期
，本文编号：2404131
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