大鼠NT-4基因重组载体转染GFP转基因小鼠BMSCs的研究
发布时间：2018-09-17 09:45
【摘要】：【目的】构建大鼠神经营养因子-4(neurotrophin-4，NT-4)基因的真核表达载体pcDNA-3-NT-4并观察其稳定转染的绿色荧光蛋白(Green flurescent protein，GFP)转基因小鼠骨髓基质干细胞(Bone marrow mesenchymal stem cell，BMSCs)中神经营养因子-4(NT-4)表达的情况。 【方法】用逆转录-聚合酶链反应(RT-PCR)以大鼠海马总RNA为模板扩增神经营养因子-4(NT-4)，应用基因重组技术将NT-4的全长基因克隆到真核表达载体pcDAN3中，用限制性内切酶酶切分析和DNA测序对重组质粒载体pcDNA3-NT-4进行鉴定。采用阳离子脂质体Lipofectamine 2000将重组质粒载体转染荧光小鼠骨髓基质干细胞，用G418筛选出抗性阳性克隆并培养至30d。应用NT-4免疫细胞化学反应来分析该细胞表达NT-4的情况，并观察该细胞培养上清中的NT-4对PC12细胞的促分化作用。 【结果】RT-PCR产物琼脂糖电泳结果显示：在预期位置有阳性条带。重组载体经酶切、测序等分析插入的基因片段为表达NT-4全长基因。免疫组织化学染色显示，转染NT-4基因的BMSCs细胞呈NT-4阳性反应，而转染空载体对照组的细胞呈NT-4阴性反应。加入转染重组载体pcDNA3-NT-4的BMSCs上清液的PC12细胞，胞体呈梭形，有明显的突起，，而加入转染空载体pcDNA3的BMSCs上清液的PC12细胞，胞体基本呈圆形，没有明显的突起。
[Abstract]:[objective] to construct the eukaryotic expression vector pcDNA-3-NT-4 of rat neurotrophic factor 4 (neurotrophin-4,NT-4) gene and observe its stable transfection of green fluorescent protein (Green flurescent protein,GFP transgenic mice Expression of neurotrophic factor-4 (NT-4) in bone marrow stromal cells (Bone marrow mesenchymal stem cell,BMSCs). [methods] Neurotrophic factor 4 (NT-4) was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using rat hippocampal total RNA as template. The full-length NT-4 gene was cloned into eukaryotic expression vector pcDAN3 by gene recombination technique. The recombinant plasmid pcDNA3-NT-4 was identified by restriction endonuclease digestion and DNA sequencing. The recombinant plasmid vector was transfected into fluorescent mouse bone marrow stromal stem cells by cationic liposome Lipofectamine 2000. The resistant positive clones were screened by G418 and cultured for 30 days. NT-4 immunocytochemical reaction was used to analyze the expression of NT-4 in the cell. The effect of NT-4 in the supernatant of the cell culture on the differentiation of PC12 cells was observed. [results] the agarose electrophoresis of RT-PCR product showed that there were positive bands in the expected position. The recombinant vector was digested by enzyme and sequenced. The inserted gene fragment was a full-length NT-4 gene. Immunohistochemical staining showed that BMSCs cells transfected with NT-4 gene showed NT-4 positive reaction, while those transfected with empty vector showed NT-4 negative reaction. The PC12 cells in the supernatant of BMSCs transfected with recombinant vector pcDNA3-NT-4 showed spindle shape and obvious protuberance, while the PC12 cells transfected with the BMSCs supernatant of empty vector pcDNA3 had a round cell body and no obvious protrusions.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346
本文编号：2245482
[Abstract]:[objective] to construct the eukaryotic expression vector pcDNA-3-NT-4 of rat neurotrophic factor 4 (neurotrophin-4,NT-4) gene and observe its stable transfection of green fluorescent protein (Green flurescent protein,GFP transgenic mice Expression of neurotrophic factor-4 (NT-4) in bone marrow stromal cells (Bone marrow mesenchymal stem cell,BMSCs). [methods] Neurotrophic factor 4 (NT-4) was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using rat hippocampal total RNA as template. The full-length NT-4 gene was cloned into eukaryotic expression vector pcDAN3 by gene recombination technique. The recombinant plasmid pcDNA3-NT-4 was identified by restriction endonuclease digestion and DNA sequencing. The recombinant plasmid vector was transfected into fluorescent mouse bone marrow stromal stem cells by cationic liposome Lipofectamine 2000. The resistant positive clones were screened by G418 and cultured for 30 days. NT-4 immunocytochemical reaction was used to analyze the expression of NT-4 in the cell. The effect of NT-4 in the supernatant of the cell culture on the differentiation of PC12 cells was observed. [results] the agarose electrophoresis of RT-PCR product showed that there were positive bands in the expected position. The recombinant vector was digested by enzyme and sequenced. The inserted gene fragment was a full-length NT-4 gene. Immunohistochemical staining showed that BMSCs cells transfected with NT-4 gene showed NT-4 positive reaction, while those transfected with empty vector showed NT-4 negative reaction. The PC12 cells in the supernatant of BMSCs transfected with recombinant vector pcDNA3-NT-4 showed spindle shape and obvious protuberance, while the PC12 cells transfected with the BMSCs supernatant of empty vector pcDNA3 had a round cell body and no obvious protrusions.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346
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