本文關(guān)鍵詞： 骨髓間充質(zhì)干細(xì)胞 股骨頭壞死 桿狀病毒 供體質(zhì)粒 反應(yīng)質(zhì)粒 轉(zhuǎn)染 肝生長(zhǎng)因子 強(qiáng)力霉素 四環(huán)素基因表達(dá)調(diào)控系統(tǒng) 增殖　出處：《南昌大學(xué)》2014年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的：建立轉(zhuǎn)染高效可調(diào)控的攜帶肝生長(zhǎng)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(zhuǎn)染骨髓間充質(zhì)干細(xì)胞（bonemarrowmesenchymalstemcells,BM-MSCs）后的DOX誘導(dǎo)濃度、轉(zhuǎn)染后細(xì)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(gòu)建重組桿狀病毒vAcrtTA2s-Ptight-HGF，隨后轉(zhuǎn)染BM-MSCs，用ElISA和WesternBlot檢測(cè)不同濃度強(qiáng)力霉素（doxycycline，DOX）誘導(dǎo)體外培養(yǎng)的兔BM-MSCs分泌的HGF的濃度，MTT方法檢測(cè)轉(zhuǎn)染后BM-MSCs的增殖變化。 結(jié)果：成功獲得兔BM-MSCs第5代細(xì)胞，成功構(gòu)建了高效可調(diào)控重組桿狀病毒載體vAcrtTA2s-Ptight-HGF，并且成功轉(zhuǎn)染兔BM-MSCs，用系列濃度DOX誘導(dǎo)時(shí)ELISA和Westernblot均檢測(cè)到BM-MSCs中HGF的表達(dá)有DOX誘導(dǎo)劑量依賴(lài)關(guān)系并且連續(xù)7天持續(xù)表達(dá)，，且實(shí)驗(yàn)組的HGF的表達(dá)量明顯高于對(duì)照組（P0.001），轉(zhuǎn)染后的BM-MSCs仍然呈渦旋形生長(zhǎng)，誘導(dǎo)產(chǎn)生序列濃度的HGF的BM-MSCs的增殖速度明顯高于空白對(duì)照組（P0.001）。 結(jié)論：成功構(gòu)建一次性轉(zhuǎn)染、高效可調(diào)控的重組桿狀病毒vAcrtTA2s-Ptight-HGF,并且實(shí)現(xiàn)對(duì)HGF表達(dá)的調(diào)控,當(dāng)強(qiáng)力霉素誘導(dǎo)濃度為1ug/ml時(shí)為其體外最佳誘導(dǎo)濃度，并證實(shí)HGF可促進(jìn)BM-MSCs的增殖,轉(zhuǎn)染后BM-MSCs的細(xì)胞形態(tài)沒(méi)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(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R329.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 潘志敏;馬勇;程細(xì)高;;常用病毒載體的基礎(chǔ)研究及臨床應(yīng)用[J];中國(guó)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(zhuǎn)導(dǎo)不同哺乳動(dòng)物骨髓來(lái)源間充質(zhì)干細(xì)胞[J];生理學(xué)報(bào);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(duì)不同哺乳動(dòng)物細(xì)胞基因轉(zhuǎn)移及表達(dá)效率的研究[J];生物工程學(xué)報(bào);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(dá)系統(tǒng)轉(zhuǎn)染骨髓基質(zhì)干細(xì)胞和誘導(dǎo)調(diào)控效應(yīng)檢測(cè)[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(dá)系統(tǒng)高效表達(dá)綠色熒光蛋白標(biāo)記的HBVe抗原[J];病毒學(xué)報(bào);1998年03期

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