【摘要】：【目的】構(gòu)建大鼠神經(jīng)營養(yǎng)因子-4(neurotrophin-4，NT-4)基因的真核表達(dá)載體pcDNA-3-NT-4并觀察其穩(wěn)定轉(zhuǎn)染的綠色熒光蛋白(Green flurescent protein，GFP)轉(zhuǎn)基因小鼠骨髓基質(zhì)干細(xì)胞(Bone marrow mesenchymal stem cell，BMSCs)中神經(jīng)營養(yǎng)因子-4(NT-4)表達(dá)的情況。 【方法】用逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-PCR)以大鼠海馬總RNA為模板擴(kuò)增神經(jīng)營養(yǎng)因子-4(NT-4)，應(yīng)用基因重組技術(shù)將NT-4的全長基因克隆到真核表達(dá)載體pcDAN3中，用限制性內(nèi)切酶酶切分析和DNA測序?qū)χ亟M質(zhì)粒載體pcDNA3-NT-4進(jìn)行鑒定。采用陽離子脂質(zhì)體Lipofectamine 2000將重組質(zhì)粒載體轉(zhuǎn)染熒光小鼠骨髓基質(zhì)干細(xì)胞，用G418篩選出抗性陽性克隆并培養(yǎng)至30d。應(yīng)用NT-4免疫細(xì)胞化學(xué)反應(yīng)來分析該細(xì)胞表達(dá)NT-4的情況，并觀察該細(xì)胞培養(yǎng)上清中的NT-4對PC12細(xì)胞的促分化作用。 【結(jié)果】RT-PCR產(chǎn)物瓊脂糖電泳結(jié)果顯示：在預(yù)期位置有陽性條帶。重組載體經(jīng)酶切、測序等分析插入的基因片段為表達(dá)NT-4全長基因。免疫組織化學(xué)染色顯示，轉(zhuǎn)染NT-4基因的BMSCs細(xì)胞呈NT-4陽性反應(yīng)，而轉(zhuǎn)染空載體對照組的細(xì)胞呈NT-4陰性反應(yīng)。加入轉(zhuǎn)染重組載體pcDNA3-NT-4的BMSCs上清液的PC12細(xì)胞，胞體呈梭形，有明顯的突起，，而加入轉(zhuǎn)染空載體pcDNA3的BMSCs上清液的PC12細(xì)胞，胞體基本呈圓形，沒有明顯的突起。
[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.
【學(xué)位授予單位】：昆明醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R346

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