本文關鍵詞： 骨髓間充質干細胞 股骨頭壞死 桿狀病毒 供體質粒 反應質粒 轉染 肝生長因子 強力霉素 四環(huán)素基因表達調控系統(tǒng) 增殖　出處：《南昌大學》2014年碩士論文　論文類型：學位論文

【摘要】：目的：建立轉染高效可調控的攜帶肝生長因子(hepatocytegrowthfactor，HGF）為目的基因的重組桿狀病毒vAcrtTA2s-Ptight-HGF,并探尋其在轉染骨髓間充質干細胞（bonemarrowmesenchymalstemcells,BM-MSCs）后的DOX誘導濃度、轉染后細胞形態(tài)和增殖速率的變化。 方法：采用全骨髓培養(yǎng)法培養(yǎng)兔BM-MSCs,基因工程方法構建重組桿狀病毒vAcrtTA2s-Ptight-HGF，隨后轉染BM-MSCs，用ElISA和WesternBlot檢測不同濃度強力霉素（doxycycline，DOX）誘導體外培養(yǎng)的兔BM-MSCs分泌的HGF的濃度，MTT方法檢測轉染后BM-MSCs的增殖變化。 結果：成功獲得兔BM-MSCs第5代細胞，成功構建了高效可調控重組桿狀病毒載體vAcrtTA2s-Ptight-HGF，并且成功轉染兔BM-MSCs，用系列濃度DOX誘導時ELISA和Westernblot均檢測到BM-MSCs中HGF的表達有DOX誘導劑量依賴關系并且連續(xù)7天持續(xù)表達，，且實驗組的HGF的表達量明顯高于對照組（P0.001），轉染后的BM-MSCs仍然呈渦旋形生長，誘導產生序列濃度的HGF的BM-MSCs的增殖速度明顯高于空白對照組（P0.001）。 結論：成功構建一次性轉染、高效可調控的重組桿狀病毒vAcrtTA2s-Ptight-HGF,并且實現(xiàn)對HGF表達的調控,當強力霉素誘導濃度為1ug/ml時為其體外最佳誘導濃度，并證實HGF可促進BM-MSCs的增殖,轉染后BM-MSCs的細胞形態(tài)沒有明顯改變。
[Abstract]:Objective: to establish an efficient and controllable hepatocyte growth factor carrying hepatocyte growth factor. The recombinant baculovirus vAcrtTA2s-Ptight-HGF with HGF as the target gene. To explore the DOX induction concentration after transfection of bone marrow mesenchymal stem cells (BM-MSCs) into bone marrow mesenchymal stem cells (BM-MSCs). Changes of cell morphology and proliferation rate after transfection. Methods: BM-MSCswere cultured by whole bone marrow culture. Recombinant baculovirus vAcrtTA2s-Ptight-HGFwas constructed by genetic engineering method and then transfected into BM-MSCs. ElISA and WesternBlot were used to detect the concentration of HGF secreted by rabbit BM-MSCs in vitro induced by doxycycline doxycycline doxycycline (doxycycline doxycycline dox). MTT method was used to detect the proliferation of BM-MSCs after transfection. Results: the fifth passage of rabbit BM-MSCs cells were successfully obtained and the recombinant baculovirus vector vAcrtTA2s-Ptight-HGF was successfully constructed. Rabbit BM-MSCs was transfected successfully. The expression of HGF in BM-MSCs was found to be in a dose-dependent manner by DOX induction in both ELISA and Westernblot induced by serial concentrations of DOX and continued for 7 days. The expression of HGF in the experimental group was significantly higher than that in the control group (P 0.001), and the BM-MSCs still grew in a vortex shape after transfection. The proliferation rate of BM-MSCs induced by sequence concentration of HGF was significantly higher than that of control group (P 0.001). Conclusion: the recombinant baculovirus vAcrtTA2s-Ptight-HGFwas successfully constructed, and the expression of HGF was regulated by the recombinant baculovirus vAcrtTA2s-Ptight-HGF. The optimal concentration of doxycycline was 1ugrml in vitro, and HGF could promote the proliferation of BM-MSCs, but the cell morphology of BM-MSCs was not changed after transfection.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R329.2

【參考文獻】

相關期刊論文 前7條

1 潘志敏;馬勇;程細高;;常用病毒載體的基礎研究及臨床應用[J];中國生化藥物雜志;2011年05期

2 劉正山;張成;盧錫林;李勇;許勇峰;熊符;馮善偉;李玲;;桿狀病毒轉導不同哺乳動物骨髓來源間充質干細胞[J];生理學報;2008年03期

3 ;A rapid and efficient method to express target genes in mammalian cells by baculovirus[J];World Journal of Gastroenterology;2004年11期

4 許辰煜,程通,盧五迅,陳敏,吳婷,王穎彬,張軍,夏寧邵;桿狀病毒對不同哺乳動物細胞基因轉移及表達效率的研究[J];生物工程學報;2004年01期

5 盛璞義,廖威明,李曉艷,李佛保,何智勇,黃綱,譚美珍;Tet-on基因表達系統(tǒng)轉染骨髓基質干細胞和誘導調控效應檢測[J];中華骨科雜志;2003年03期

6 ;Construction of transposon-mediated baculovirus vector and expression of green fluorescent protein in insect cells and larvae[J];Chinese Science Bulletin;1999年02期

7 岳莉莉,齊義鵬,杜全勝,李燕;用Bac-to-Bac桿狀病毒表達系統(tǒng)高效表達綠色熒光蛋白標記的HBVe抗原[J];病毒學報;1998年03期

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