摘要
目的 利用pAdEasy腺病毒载体系统构建人LIM矿化蛋白-1（LMP-1）重组腺病毒,检测其在体外培养大鼠骨髓基质干细胞（BMSCs）的促成骨作用并分析其分子机制.方法 应用聚合酶链反应（PCR）法扩增出人LMP-1全长基因,插入pShuttle-IRES-hrGFP-1中构建成腺病毒穿梭质粒pS-LMP-1-GFP,转化DH5a大肠杆菌,挑选细菌单克隆进行酶切鉴定和DNA测序;通过pS-LMP-1-GFP与质粒pAdEasy-1在BJ5183细胞中同源重组,得到携带人LMP-1基因的重组腺病毒载体（pAdLMP-1-GFP）.经PacⅠ酶切暴露两侧长末端重复序列,脂质体介导线性化质粒转染AD293细胞进行包装得到重组腺病毒Ad-LMP-1-GFP.以腺病毒为载体,将 人LMP-1 基因体外转染于第3代大鼠BMSCs内,检测LMP-1基因在BMSCs的表达,分别观察转染后实验组与对照组碱性磷酸酶活性变化,Western blot检测骨钙素（OCN）和β-连环蛋白（β-catenin）的表达,评价LMP-1 基因的成骨能力及其分子机制.结果 成功获取人LMP-1基因,并包装成重组腺病毒.LMP-1基因能在BMSCs 中高效表达,转染后BMSCs的碱性磷酸酶活性增强,与对照组比较P=0.000,分别为0.096、0.116、0.118、0.119（0.110±0.011）和 0.045、0.057、0.056、0.054（0.053±0.005）;骨钙素的表达显著升高,与对照组比较P=0.001,分别为0.661、0.767、0.651（0.693±0.064）和0.177、0.290、0.222（0.229±0.050）;β-catenin的表达明显增强,与对照组比较P=0.002,分别为0.841、0.806、0.867（0.838±0.030）和0.185、0.236、0.266（0.229±0.040）.结论 成功利用pAdEasy系统构建人LMP-1重组腺病毒载体,包装获得重组腺病毒.Ad-LMP-1-GFP转染BMSCs后,LMP-1基因能促进BMSCs向成骨细胞分化,并且可能通过β-catenin信号分子发挥作用.
Objective To construct a recombinant adenovirus vector containing LIM mineralization protein-1 （LMP-1）, and investigate the osteoinductive activity of bone marrow mesenchymal stem cells （BMSCs） which were transduced with recombinant adenoviral vector carrying LMP-1 gene and the molecular mechanism.Methods LMP-1 gene was acquired by polymerase chain reaction （PCR）, and inserted into the pShuttle-IRES-hrGFP-1.The adenovirus Shuttle Plasmid pS-LMP-1-GFP was constructed and transformed into E.coli DH5a.Recombinant adenovirus vector carrying human LMP-1 gene （pAdLMP-1-GFP） was obtained from homologous recombination in BJ5183 cells by pS-LMP-1-GFP with plasmid pAdEasy-1.The Pac I was used to expose the long terminal repeats on both sides, and the plasmid was transfected into AD293 cells to get the adenovirus Ad-LMP-1-GFP.Ad-LMP-1-GFP was infected into the 3rd passage of BMSCs.The expression of LMP-1 was detected by RT-PCR.The osteogenic activity of BMSCs and the molecular mechanism were evaluated by the activity of alkaline phosphatase （ALP） and the expression of osteocalcin and β-catenin detected by Western blotting.Results The LMP-1 gene was successfully acquired and confirmed and packaged to recombinant adenovirus.The expression of LMP-1 was detected successfully in BMSCs.The ALP activity increased significantly,P=0.000 comparing to the control group, ALP activity were 0.096, 0.116, 0.118, 0.119 （0.110±0.011） and 0.045, 0.057, 0.056, 0.054 （0.053±0.005） respectively;the expression of osteocalcin and β-catenin enhanced significantly, P=0.001 and P=0.002 comparing to the control group, osteocalcin was 0.661, 0.767, 0.651 （0.693±0.064） and 0.177, 0.290, 0.222 （0.229±0.050）;β-catenin was 0.841, 0.806, 0.867 （0.838±0.030） and 0.185, 0.236, 0.266 （0.229±0.040） respectively.Conclusion We successfully construct the Ad-LMP-1 recombinant adenovirus vector and get a high transfection efficacy in BMSCs.LMP-1 gene can induce the osteoblast differentiation of BMSCs and improve
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2017年第3期452-455,共4页
Chinese Journal of Experimental Surgery
基金
河南省基础与前沿技术研究项目,河南省科技攻关项目,国家自然科学基金青年基金（81301555）
关键词
LIM-1矿化蛋白
间充质干细胞
成骨活性
基因转染
LIM mineralization protein-1
Mesenchymal stem cells
Osteogenic activity
Gene transfect
