反转录病毒介导基质金属蛋白酶特异性组织抑制剂1基因转染骨髓间充质干细胞的成骨能力

Retroviral-mediated tissue inhibitor of metalloproteinase-1 gene transfected human mandible bone marrow mesenchymal stem cells and their osteogenic potential

背景:间充质干细胞成骨分化早期是成骨性细胞外基质细胞外间质的堆积,其主要成分均是基质金属蛋白酶特异性组织抑制剂1(tissue Inhibitor of Metalloproteinases1,TIMP-1)作用的基质金属蛋白酶作用底物,通过增加TIMP-1的表达,是否可以抑制基质金属蛋白酶的活性,进而影响间充质干细胞的成骨分化能力?目的:观察反转录病毒介导TIMP-1基因转染骨髓间充质干细胞的成骨能力。方法:设计In-Fusion引物扩增TIMP-1基因片段,与酶切后线性化的pBABE-Puro反转录病毒表达载体进行位点同源重组,构建TIMP-1基因反转录病毒表达载体,RT-PCR和测序验证,并使用GP-293细胞进行包装。通过密度梯度法分离人下颌骨来源骨髓间充质干细胞,流式细胞仪验证其表面标记。将TIMP-1反转录病毒包装液感染骨髓间充质干细胞并筛选稳定表达TIMP-1细胞株,RT-PCR和蛋白电泳验证其表达,并使用成骨诱导液诱导10d,观察其对成骨能力的影响,RT-PCR验证其成骨相关基因的表达。结果与结论:经测序证实,RT-PCR所获得的TIMP-1基因cDNA序列与NM_003254.2(624bp)序列完全一致;通过表面标记鉴定证实成功分离了人骨髓间充质干细胞,并具良好的成骨、成脂能力;RT-PCR和Western Blot验证反转录病毒载体成功介导TIMP-1转染骨髓间充质干细胞,RT-PCR表明成骨诱导10d时,TIMP-1基因感染组成骨增加,RUNX-2和COL1mRNA表达增加。提示TIMP-1基因可通过反转录病毒成功转染人骨髓间充质干细胞,并能促进其成骨。

BACKGROUND： Extracellular matrix （ECM） accumulation of mesenchymal stem cells （MSCs） occurs at the early phase of osteogenesis.And the main components of ECM deposited in that process are specific substrates of matrix metalloproteinases （MMPs） suppressed by tissue inhibitor of matrix metalloproteinase-1 （TIMP-1）.Whether MSCs can inhibit the activity of MMPs and improve the ability of osteogenic differentiation by increasing the expression of TIMP-1？ OBJECTIVE： To investigate the osteogenic potential of human mandible bone marrow mesenchymal stem cells （hMBM-MSCs） modified by TIMP-1 gene mediated with retroviral transfection.METHODS： In-Fusion primers for TIMP-1 gene was designed,and reacted with digested linearized pBABE-Puro retroviral expression vector by homologous recombination sites and verified TIMP-1 expression with reverse transcription-polymerase chain reaction （RT-PCR） and sequencing methods,and then packaged with GP293 cell line.Bone marrow mesenchymal stem cells in mandibular surgery patients were separated by density gradient method,and their surface markers were verified by flow cytometry.TIMP-1 packaging liquid after retrovirus transfection infected and selected hMBM-MSCs cell lines which stably expressed TIMP-1 gene,and RT-PCR and protein electrophoresis validate its expression.HMBM-MSCs were induced using osteogenic induction medium for 10 days,and their osteogenic potential was observed.The expression of osteogenic related genes was verified by RT-PCR.RESULTS AND CONCLUSION： Confirmed by DNA sequencing,RT-PCR obtained cDNA sequences of TIMP-1 gene totally matched with NM_003254.2 （624 bp） identical sequences.HMBM-MSCs were successfully separated according to surface makers identification,and manifested a good osteogenic and adipogenic ability.RT -PCR and Western Blot were used to verify the successful expression of hMBM-MSCs by retroviral vector-mediated transfection.RT-PCR showed that when hMBM-MSCs were induced with osteogenic induction media for 10 days,TIMP-1 increased markedly the mRNA expression of RUNX-2 and COL1.TIMP-1 gene can be successfully transfected into hMBM-MSCs by retroviral transfection,and promote the effect of osteogenesis.