本文選題：超聲 + 微泡�。� 參考：《山西醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的:研究超聲微泡破裂法對基因重組人骨形態(tài)發(fā)生蛋白-2(rhBMP-2)質(zhì)粒GV147-rhBMP2-GFP在大鼠骨髓間充質(zhì)干細胞(BMSCs)的轉(zhuǎn)染、表達的促進作用。方法:取8周齡Sprague-Dawley雄性大鼠分離培養(yǎng)骨髓間充質(zhì)干細胞(BMSCs),傳至第3代時細胞懸液分為3組:A組(單純細胞組),B組(質(zhì)粒組,加入含目的基因質(zhì)粒)及C組(微泡+超聲+質(zhì)粒組);轉(zhuǎn)染后48 h用熒光顯微鏡觀察轉(zhuǎn)染情況及細胞生長狀況,RT-PCR檢測細胞轉(zhuǎn)染后的目的基因表達情況,Western-blot檢測目的蛋白翻譯情況。應(yīng)用SPSS13.0軟件對實驗數(shù)據(jù)進行統(tǒng)計學(xué)分析。結(jié)果:(1)轉(zhuǎn)染48 h后可在倒置熒光顯微鏡下觀察到B組及C組轉(zhuǎn)染成功的細胞胞漿內(nèi)發(fā)出綠色熒光,但A組未見明顯綠色熒光表達,C組的綠色熒光表達量明顯高于B組;(2)RT-PCR檢測顯示C組(微泡+超聲+質(zhì)粒組)BMP-2基因mRNA相對表達量較B組(質(zhì)粒組)高;Western-bolt檢測顯示C組(微泡+超聲+質(zhì)粒組)BMP-2蛋白表達量較B組(質(zhì)粒組)高。結(jié)論:超聲微泡破裂法有助于提高外源性rhBMP-2基因在大鼠骨髓間充質(zhì)干細胞轉(zhuǎn)染率與表達水平,有望為骨折提供一種新型基因治療方法。
[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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R68

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