人低氧诱导因子-1α和LIM矿化蛋白-1联合表达重组腺病毒构建及其成骨作用

Construction of recombinant adenovirus expressing combined human hypoxia inducible factor -1α and LIM mineralization protein -1 and its osteogenic effect

目的利用内部核糖体进入位点（IRES）连接低氧诱导因子-1α（HIF-1α）与LIM矿化蛋白-1（LMP-1），构建同时表达LMP-1和HIF-1α的腺病毒载体，检测其在体外培养大鼠骨髓基质干细胞（BMSCs）的成骨作用。方法应用聚合酶链反应（PCR）法扩增出人HIF-1α（2 481 bp）和LMP-1（1 374 bp）及IRES（564 bp）序列，依次插入pShuttle-IRES-hrGFP-1中构建腺病毒穿梭质粒pS-HIF-LMP-1-GFP；将其与质粒pAdEasy-1在BJ5183细胞中同源重组，得到重组腺病毒质粒（pAd-HIF-LMP-1-GFP）。经Pac I酶切转染AD293细胞进行包装得到重组腺病毒Ad-HIF-LMP-1-GFP。以腺病毒为载体，将人HIF-1α和LMP-1联合基因转染大鼠BMSCs细胞，分别观察转染后实验组与对照组碱性磷酸酶活性、钙形成以及骨钙素和核心结合蛋白因子2（Runx2）的表达变化，探讨HIF-1α与LMP-1联合表达的成骨作用。结果成功获取联合表达HIF-1α与LMP-1的重组腺病毒。HIF-1α和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，差异有统计学意义（P=0.000），活性分别为0.145、0.160、0.170、0.167（0.160±0.011） ng/μg蛋白和0.045、0.057、0.056、0.054（0.053±0.005） ng/μg蛋白；钙沉积明显增多，与对照组比较，差异有统计学意义（P=0.001），钙形成定量分别为0.636、0.544、0.591、0.515（0.570±0.050）和0.121、0.150、0.144、0.167（0.145±0.019）；骨钙素表达显著提高，与对照组比较，差异有统计学意义（P=0.017），分别为2.688、2.997、2.777（2.820±0.159）和2.009、1.916、1.944（1.956±0.047）；Runx2的表达明显增强，与对照组比较，差异有统计学意义（P=0.000），分别为0.440、0.465、0.456（0.454±0.010）和0.096、0.108、0.117（0.107±0.010）。结论成功利用IRES序列连接人HIF-1α和LMP-1基因构建重组腺病毒载体，Ad-HIF-LMP-1-GFP转染BMSCs后，能明显促进BMSCs向成骨细胞分化。

ObjectiveThis study was designed to construct a recombinant adenovirus vector expressing both hypoxia inducible factor-1α （HIF-1α） and LIM mineralization protein-1 （LMP-1） gene linked by internal ribozyme entry site （IRES）, and to detect the osteogenesis in rat bone marrow stromal stem cells.MethodsThe full-length of human HIF-1α （2 481 bp） and LMP-1 （1 374 bp） genes and IRES （564 bp） sequence were acquired by polymerase chain reaction （PCR） and inserted into pShuttle-IRES-hrGFP-1 to construct recombinant adenovirus shuttle plasmid pS-HIF-LMP-1-GFP. Recombinant adenovirus vector pAd-HIF-LMP-1-GFP was obtained from homologous recombination in BJ5183 cells by pS-HIF-LMP-1-GFP and pAdEasy-1. Recombinant adenovirus Ad-HIF-LMP-1-GFP was obtained by packaging in AD293 cells. Ad-HIF-LMP-1-GFP was transduced to the rat bone marrow mesenchymal stem cells （BMSCs）. The expression of HIF-1α and LMP-1 in BMSCs was observed by reverse transcription-polymerase chain reaction （RT-PCR）. The alkaline phosphatase （ALP） activity, calcium formation, osteocalcin and related transcription factor-2 （Runx2） expression changes between experimental group and control group were detected to evaluate the osteogenitic capacity of combined HIF-1α and LMP-1.ResultsThe recombinant adenovirus expressing combined HIF-1α and LMP-1 was successfully acquired. The expression of HIF-1α and LMP-1 was detected successfully in BMSCs. The ALP activity increased significantly （P=0.000）. ALP activity was 0.145, 0.160, 0.170, 0.167 （0.160±0.011） ng/μg protein in experimental group and that was 0.045, 0.057, 0.056, 0.054 （0.053±0.005） ng/μg protein in control group, respectively. The calcium formation increased significantly （P=0.001）. The quantification was 0.636, 0.544, 0.591, 0.515 （0.570±0.050） in experimental group and that was 0.121, 0.150, 0.144, 0.167 （0.145±0.019） in control group, respectively. The expression of osteocalcin and Runx2 was enhanced significantly （P=0.017 and 0.000）. The expression of osteocalcin was 2.688, 2.997, 2.777 （2.820±0.159） in experimental group and that was 2.009, 1.916, 1.944 （1.956±0.047） in control group, respectively. The expression of Runx2 was 0.440, 0.465, 0.456 （0.454±0.010） in experimental group and that was 0.096, 0.108, 0.117 （0.107±0.010） in control group, respectively.ConclusionThe recombinant adenovirus vector was successfully constructed by using IRES sequence to connect human HIF-1 alpha and LMP-1 gene. Combined HIF-1α and LMP-1 gene can promote the osteoblastic differentiation of BMSCs.