人骨形态蛋白-2基因重组腺病毒载体的构建及修饰骨髓间充质干细胞的实验研究

发布时间：2018-05-26 00:15

本文选题：人骨形态蛋白-2 + 腺病毒载体　；参考：《福建医科大学》2011年硕士论文

【摘要】：目的:(1)构建人骨形态蛋白-2(Human bone morphogenetic protein-2,hBMP-2)基因的重组腺病毒载体并鉴定; (2)检测hBMP-2基因转染对SD大鼠骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)的影响。方法:(1)以人cDNA为模板PCR扩增hBMP-2基因,插入线性化的表达载体pAV-MCMV-EGFP-3FLAG后,转化E.coli感受态细胞,经菌落PCR鉴定阳性转化子、阳性克隆测序无误后,扩增、抽提,将带有目的基因的腺病毒表达载体和携带有腺病毒大部分基因的辅助包装质粒共转染293细胞进行病毒包装扩增,PCR检测目的基因、Western Blot检测目的蛋白及终点稀释法检测病毒滴度; (2)分离纯化SD大鼠BMSCs并体外扩增,通过腺病毒载体介导hBMP-2基因转染BMSCs,分别通过荧光显微镜观察荧光表达情况、蛋白质水平来测定转染后hBMP-2的表达、ALP定量测定鉴定成骨活性及MTT法评估hBMP-2对BMSCs的影响。结果:(1)PCR获得长度为1223bp的hBMP-2目的基因片段,同源重组表达载体经阳性克隆PCR及测序鉴定,结果正确。 (2)293细胞内包装、扩增,经Western blot及PCR鉴定无误后,获得病毒滴度为5x1010pfu/ml的重组腺病毒。 (3)从SD大鼠骨髓提取物中分离培养的细胞形态为梭形,呈铺路石状、漩涡状生长,经流式细胞仪检测及多项分化能力鉴定符合BMSCs的特征。 (4)经转染hBMP-2基因后,BMSCs表达hBMP-2、ALP。MTT法检测转染hBMP-2基因后,BMSCs增殖能力明显增强(P0.05)。结论:成功构建携带有hBMP-2基因的重组腺病毒载体,转染BMSCs后可以表达hBMP-2、ALP,在体外明显促进BMSCs的增殖。
[Abstract]:Objective to construct and identify the recombinant adenovirus vector of human bone morphogenetic protein human bone morphogenetic protein-2hBMP-2 gene. The effect of hBMP-2 gene transfection on bone marrow mesenchymal stem cells (BMSCs) of bone marrow mesenchymal stem cells (BMSCs) in SD rats was detected. Methods the hBMP-2 gene was amplified by PCR using human cDNA as template, inserted into the linearized expression vector pAV-MCMV-EGFP-3FLAG, then transformed into E.coli receptive cells. The positive transformants were identified by colony PCR. The positive clones were sequenced, amplified and extracted. The adenovirus expression vector with the target gene and the auxiliary packaging plasmid carrying most of the adenovirus gene were co-transfected into 293 cells. The virus packaging amplification was carried out. The target protein was detected by Western Blot and the titer of the virus was detected by end-point dilution method. BMSCs was isolated and purified from SD rats and amplified in vitro. HBMP-2 gene was transfected into BMSCs by adenovirus vector. Fluorescence expression was observed by fluorescence microscope. Protein level was used to determine the expression of hBMP-2 after transfection. The osteogenic activity was evaluated by ALP and the effect of hBMP-2 on BMSCs was evaluated by MTT method. Results the target gene fragment of hBMP-2 with length of 1223bp was obtained by PCR. The homologous recombinant expression vector was identified by positive clone PCR and sequencing, and the results were correct. The recombinant adenovirus with 5x1010pfu/ml titer was obtained by Western blot and PCR. The cells isolated from the bone marrow extracts of SD rats were spindle-shaped with paving stone and whirlpool growth. The results of flow cytometry and multiple differentiation were consistent with the characteristics of BMSCs. (4) after transfection of hBMP-2 gene, hBMP-2ALP.MTT assay was used to detect the proliferative ability of hBMP-2 gene. Conclusion: the recombinant adenovirus vector carrying hBMP-2 gene was successfully constructed, which could express hBMP-2ALP after transfection of BMSCs, and promote the proliferation of BMSCs in vitro.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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