重组腺病毒介导的β-神经生长因子基因转染大鼠内皮祖细胞的实验研究

发布时间：2018-03-12 20:33

  本文选题：内皮祖细胞　切入点：神经生长因子　出处：《中国矫形外科杂志》2017年02期 　论文类型：期刊论文

【摘要】：[目的]探究携带β-神经生长因子(β-NGF)基因的重组腺病毒转染大鼠骨髓源内皮祖细胞(EPCs)的最佳感染复数(MOI),并从基因转录和蛋白合成两个水平上观察转染后EPCs对β-NGF基因的表达,探讨其对脊髓损伤后神经元细胞微环境的影响。[方法]密度梯度离心法分离、全骨髓贴壁法培养大鼠骨髓源EPCs,免疫荧光法检测EPCs特异性表面分子CD34、CD133和VEGFR-2的表达。用不同MOI值的携带β-NGF基因和绿色荧光蛋白基因(GFP)的重组腺病毒转染EPCs,确定最佳的MOI值。用携带β-NGF基因的重组腺病毒转染的细胞为β-NGF基因组,用空载的重组腺病毒转染的细胞为空载组,未转染的细胞为空白组。RT-PCR法、Western blot法和ELISA法检测β-NGF的表达。[结果]成功分离、培养出大鼠骨髓源EPCs,特异性表面分子CD34、CD133和VEGFR-2经鉴定呈阳性表达;携带β-NGF基因的重组腺病毒转染EPCs最佳的MOI为70;转染成功的细胞β-NGF基因在m RNA和蛋白两个不同的水平上表达都升高,并持续向细胞外分泌。[结论]携带β-NGF基因的重组腺病毒可成功转染EPCs,成功转染β-NGF基因的细胞能持续向细胞外分泌有活性的β-NGF蛋白。
[Abstract]:[objective] to investigate the optimal infection of recombinant adenovirus carrying 尾 -NGF gene into rat bone marrow-derived endothelial progenitor cells (BMSCs), and to observe the expression of 尾 -NGF gene in transfected EPCs at the two levels of gene transcription and protein synthesis. To investigate the effect of the microenvironment of neuron cells after spinal cord injury. [methods] density gradient centrifugation was used to isolate neurons. Rat bone marrow derived EPCs were cultured by whole bone marrow adherent method. The expression of CD34, CD133 and VEGFR-2, a specific surface molecule of EPCs, was detected by immunofluorescence method. The recombinant adenovirus carrying 尾 -NGF gene and green fluorescent protein gene (GFP) with different MOI values was used to transfect EPCs to determine the best. The cells transfected with recombinant adenovirus carrying 尾 -NGF gene were 尾 -NGF genome. The expression of 尾 -NGF was detected by Western blot and ELISA, respectively. Rat bone marrow derived EPCs were cultured, and the specific surface molecules CD34, CD133 and VEGFR-2 were identified to be positive. The optimal MOI of EPCs transfected by recombinant adenovirus carrying 尾 -NGF gene was 70, and the expression of 尾 -NGF gene increased at two different levels of m RNA and protein. [conclusion] Recombinant adenovirus carrying 尾 -NGF gene can transfect EPCssuccessfully, and the cells transfected with 尾 -NGF gene can continuously excrete active 尾 -NGF protein.
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