本文選題：內(nèi)皮祖細(xì)胞　切入點(diǎn)：神經(jīng)生長因子　出處：《中國矯形外科雜志》2017年02期 　論文類型：期刊論文

【摘要】：[目的]探究攜帶β-神經(jīng)生長因子(β-NGF)基因的重組腺病毒轉(zhuǎn)染大鼠骨髓源內(nèi)皮祖細(xì)胞(EPCs)的最佳感染復(fù)數(shù)(MOI),并從基因轉(zhuǎn)錄和蛋白合成兩個(gè)水平上觀察轉(zhuǎn)染后EPCs對β-NGF基因的表達(dá),探討其對脊髓損傷后神經(jīng)元細(xì)胞微環(huán)境的影響。[方法]密度梯度離心法分離、全骨髓貼壁法培養(yǎng)大鼠骨髓源EPCs,免疫熒光法檢測EPCs特異性表面分子CD34、CD133和VEGFR-2的表達(dá)。用不同MOI值的攜帶β-NGF基因和綠色熒光蛋白基因(GFP)的重組腺病毒轉(zhuǎn)染EPCs,確定最佳的MOI值。用攜帶β-NGF基因的重組腺病毒轉(zhuǎn)染的細(xì)胞為β-NGF基因組,用空載的重組腺病毒轉(zhuǎn)染的細(xì)胞為空載組,未轉(zhuǎn)染的細(xì)胞為空白組。RT-PCR法、Western blot法和ELISA法檢測β-NGF的表達(dá)。[結(jié)果]成功分離、培養(yǎng)出大鼠骨髓源EPCs,特異性表面分子CD34、CD133和VEGFR-2經(jīng)鑒定呈陽性表達(dá);攜帶β-NGF基因的重組腺病毒轉(zhuǎn)染EPCs最佳的MOI為70;轉(zhuǎn)染成功的細(xì)胞β-NGF基因在m RNA和蛋白兩個(gè)不同的水平上表達(dá)都升高,并持續(xù)向細(xì)胞外分泌。[結(jié)論]攜帶β-NGF基因的重組腺病毒可成功轉(zhuǎn)染EPCs,成功轉(zhuǎn)染β-NGF基因的細(xì)胞能持續(xù)向細(xì)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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本文編號：1603188