超声微泡破裂法促进骨形态发生蛋白-2在大鼠骨髓间充质干细胞表达的研究

发布时间：2018-05-27 10:32

本文选题：超声 + 微泡　；参考：《山西医科大学》2016年硕士论文

【摘要】：目的:研究超声微泡破裂法对基因重组人骨形态发生蛋白-2(rhBMP-2)质粒GV147-rhBMP2-GFP在大鼠骨髓间充质干细胞(BMSCs)的转染、表达的促进作用。方法:取8周龄Sprague-Dawley雄性大鼠分离培养骨髓间充质干细胞(BMSCs),传至第3代时细胞悬液分为3组:A组(单纯细胞组),B组(质粒组,加入含目的基因质粒)及C组(微泡+超声+质粒组);转染后48 h用荧光显微镜观察转染情况及细胞生长状况,RT-PCR检测细胞转染后的目的基因表达情况,Western-blot检测目的蛋白翻译情况。应用SPSS13.0软件对实验数据进行统计学分析。结果:(1)转染48 h后可在倒置荧光显微镜下观察到B组及C组转染成功的细胞胞浆内发出绿色荧光,但A组未见明显绿色荧光表达,C组的绿色荧光表达量明显高于B组;(2)RT-PCR检测显示C组(微泡+超声+质粒组)BMP-2基因mRNA相对表达量较B组(质粒组)高;Western-bolt检测显示C组(微泡+超声+质粒组)BMP-2蛋白表达量较B组(质粒组)高。结论:超声微泡破裂法有助于提高外源性rhBMP-2基因在大鼠骨髓间充质干细胞转染率与表达水平,有望为骨折提供一种新型基因治疗方法。
[Abstract]:Aim: to investigate the effect of ultrasound microbubble rupture on the transfection and expression of recombinant human bone morphogenetic protein-2rhBMP-2 (rhBMP-2) plasmid GV147-rhBMP2-GFP in rat bone marrow mesenchymal stem cells (BMSCs). Methods: bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured from 8-week-old Sprague-Dawley male rats. At the third passage, the cell suspension was divided into three groups: group A (simple cell group) and group B (plasmid group). The transfection was observed by fluorescence microscope 48 h after transfection and the expression of target gene was detected by RT-PCR. Western-blot was used to detect the translation of target protein. The experimental data were statistically analyzed by SPSS13.0 software. Results after 48 hours of transfection, green fluorescence was observed in the cytoplasm of the cells in group B and group C, respectively. However, the green fluorescence expression in group A was significantly higher than that in group B (P < 0.01). The relative mRNA expression of BMP-2 gene in group C (microbubble ultrasound plasmid group) was higher than that in group B (plasmid group). The expression of BMP-2 protein in microbubble ultrasound plasmid group was higher than that in B group. Conclusion: the method of ultrasonic microbubble rupture can improve the transfection rate and expression level of exogenous rhBMP-2 gene in rat bone marrow mesenchymal stem cells and may provide a new gene therapy for fracture.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R68

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