本文選題：可誘導(dǎo)慢病毒載體 + hBMP-2基因　； 參考：《青島大學(xué)》2010年碩士論文

【摘要】： 目的構(gòu)建可誘導(dǎo)表達(dá)人骨形態(tài)發(fā)生蛋白-2(hBMP-2)基因的慢病毒載體,轉(zhuǎn)染人胚胎腎上皮細(xì)胞系293FT細(xì)胞獲得相應(yīng)的病毒顆粒,感染并獲得在強(qiáng)力霉素誘導(dǎo)條件下高效表達(dá)hBMP-2的人臍帶間充質(zhì)干細(xì)胞,為下一步可誘導(dǎo)表達(dá)hBMP-2的人臍帶間充質(zhì)干細(xì)胞移植治療骨壞死奠定堅(jiān)實(shí)的實(shí)驗(yàn)基礎(chǔ)。 方法以pcDNA-hBMP-2為模板,采用聚合酶鏈反應(yīng)法擴(kuò)增前后端帶有attB重組位點(diǎn)的人骨形態(tài)發(fā)生蛋白-2(hBMP-2)全長(zhǎng)序列。利用Gateway載體構(gòu)建技術(shù),將擴(kuò)增產(chǎn)物通過(guò)BP反應(yīng)克隆至pDown載體中,測(cè)序正確后,采用LR重組酶將pDown-hBMP-2, pUp-TRE和pLV/Des2-Neo進(jìn)行重組反應(yīng),構(gòu)建慢病毒載體表達(dá)質(zhì)粒pLV/EXPN2-Neo-TRE-hBMP-2.將pLV/EXPN2-Neo-TRE-hBMP-2與包裝質(zhì)粒(pLV/helper-SL3、pLV/helper-SL4、pLV/helper-SL5)混合,利用脂質(zhì)體共同轉(zhuǎn)染293FT細(xì)胞,包裝攜帶hBMP-2基因的慢病毒顆粒,感染人臍帶間充質(zhì)干細(xì)胞,在強(qiáng)力霉素(doxcycline)誘導(dǎo)下,用ELISA檢測(cè)目的細(xì)胞hBMP-2的表達(dá)情況。 結(jié)果通過(guò)酶切、聚合酶鏈?zhǔn)椒磻?yīng)及測(cè)序驗(yàn)證可誘導(dǎo)hBMP-2真核表達(dá)慢病毒載體構(gòu)建成功；pLV/EXPN2-Neo-TRE-hBMP-2與包裝質(zhì)粒共轉(zhuǎn)染包裝細(xì)胞293FT細(xì)胞,成功獲得攜帶hBMP-2基因的慢病毒顆粒,繼之感染人臍帶間充質(zhì)干細(xì)胞,在強(qiáng)力霉素誘導(dǎo)條件下,使其高效表達(dá)hBMP-2。 結(jié)論成功構(gòu)建出可誘導(dǎo)表達(dá)hBMP-2基因的慢病毒載體,獲得濃縮lentiviral-hBMP-2病毒液,在此基礎(chǔ)上最終獲得可以在強(qiáng)力霉素誘導(dǎo)條件下高效表達(dá)hBMP-2的人臍帶間充質(zhì)干細(xì)胞,為下一步可誘導(dǎo)表達(dá)hBMP-2的人臍帶間充質(zhì)干細(xì)胞移植治療骨壞死奠定堅(jiān)實(shí)的實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:Objective to construct a lentivirus vector that can induce the expression of human bone morphogenetic protein-2hBMP-2 gene and obtain the corresponding viral particles by transfection into human embryonic renal epithelial cell line 293FT. Human umbilical cord mesenchymal stem cells expressing hBMP-2 efficiently under doxycycline induction condition were infected and obtained, which laid a solid experimental foundation for further transplantation of human umbilical cord mesenchymal stem cells which can induce the expression of hBMP-2 in the treatment of osteonecrosis. Methods the full-length sequence of human bone morphogenetic protein -2hBMP-2 with attB recombinant site was amplified by polymerase chain reaction using pcDNA-hBMP-2 as template. Using Gateway vector construction technique, the amplified product was cloned into pDown vector by BP reaction. After sequencing correctly, pDown-hBMP-2, pUp-TRE and pLV/Des2-Neo were recombined with LR recombinant enzyme to construct lentivirus vector pLV-EXPN2-Neo-TRE-hBMP-2. PLV/EXPN2-Neo-TRE-hBMP-2 was mixed with pLV- helper-SL3 / pLV-helper-SL4 / pLV-helper-SL5), then the 293FT cells were co-transfected with liposome, and the lentivirus particles carrying hBMP-2 gene were packaged and infected with human umbilical cord mesenchymal stem cells. The expression of hBMP-2 in the target cells was detected by ELISA induced by doxycycline. Results by restriction endonuclease digestion, polymerase chain reaction and sequencing, hBMP-2 eukaryotic expression lentivirus vector was successfully constructed and co-transfected with packaging plasmid into 293FT cells. Lentivirus particles carrying hBMP-2 gene were successfully obtained. Then infected with human umbilical cord mesenchymal stem cells, under the condition of doxycycline induction, hBMP-2 was highly expressed in human umbilical cord mesenchymal stem cells. Conclusion the lentivirus vector which can induce the expression of hBMP-2 gene was successfully constructed, and the concentrated lentiviral-hBMP-2 virus solution was obtained. On this basis, the human umbilical cord mesenchymal stem cells which could efficiently express hBMP-2 under doxycycline induction were obtained. It provides a solid experimental basis for the next step to induce the transplantation of human umbilical cord mesenchymal stem cells expressing hBMP-2 in the treatment of osteonecrosis.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 宋純理,黨耕町,賈弘n\,郭昭慶,馬慶軍;辛伐他汀促進(jìn)骨髓基質(zhì)細(xì)胞的成骨分化[J];北京大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2003年05期

2 王琪;茍三懷;湯亭亭;劉巖;歐陽(yáng)躍平;席焱海;韓慶林;;臍血間充質(zhì)干細(xì)胞體外培養(yǎng)方法的改良及其生物學(xué)特性研究[J];第二軍醫(yī)大學(xué)學(xué)報(bào);2007年01期

3 劉杰;吳雪暉;羅飛;何清義;代飛;謝肇;許建中;;自體間充質(zhì)干細(xì)胞構(gòu)建組織工程骨修復(fù)人長(zhǎng)骨缺損的臨床觀(guān)察[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2008年09期

4 李建軍,楊紹娟,卜麗莎,高申,張文嵐,崔亞南,王宏,劉建國(guó),徐莘香;BMP-2基因轉(zhuǎn)染的人骨髓基質(zhì)干細(xì)胞復(fù)合PLA/PCL支架體外構(gòu)建組織工程骨[J];骨與關(guān)節(jié)損傷雜志;2004年03期

5 遲作華;張洹;陸琰;;臍血間充質(zhì)干細(xì)胞的分離鑒定及其向脂肪和成骨的分化[J];基礎(chǔ)醫(yī)學(xué)與臨床;2006年03期

6 姚天華;湯曉雨;楊壯群;李昂;屠軍波;馬建明;;體外誘導(dǎo)人臍血間充質(zhì)干細(xì)胞向軟骨細(xì)胞分化的初步研究[J];中國(guó)美容醫(yī)學(xué);2007年04期

7 張秀麗;蔣欣泉;;腺病毒介導(dǎo)的骨形成蛋白-2基因修飾促進(jìn)羊骨髓基質(zhì)細(xì)胞成骨分化的實(shí)驗(yàn)研究[J];上�？谇会t(yī)學(xué);2006年06期

8 侯玲玲,鄭敏,王冬梅,袁紅豐,李海民,陳琳,白慈賢,張涌,裴雪濤;人骨髓間充質(zhì)干細(xì)胞在成年大鼠腦內(nèi)的遷移及分化[J];生理學(xué)報(bào);2003年02期

9 黃永輝;夏青;沈鐵城;田進(jìn);許文榮;;人骨髓間充質(zhì)干細(xì)胞接種納米晶膠原骨構(gòu)建組織工程骨[J];生物骨科材料與臨床研究;2008年01期

10 梁敏,廖二元;孕酮對(duì)體外培養(yǎng)人成骨細(xì)胞增殖和分化的促進(jìn)作用[J];中國(guó)臨床康復(fù);2004年32期

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