rhBMP-2、bFGF双基因共转染兔骨髓间充质干细胞复合nHA/RHLC/PLA支架及其体内外成骨分化研究  

Osteogenesis of a novel bone bioengineering biocomposite after cotransfection of rhBMP-2 and bFGF into BMSCs and combination with nHA/RHLC/PLA scaffold

作　　者：郝伟[1] 姜明[2] 周东生[3] 王新[1] 刘洪智[1] 周路纲[1] 孙煜杰[1] 

机构地区：[1]青岛大学医学院附属烟台毓璜顶医院创伤骨科,烟台264000 [2]济南军区总医院口腔科,济南250031 [3]山东大学附属省立医院创伤骨科,济南250021

出　　处：《中国矫形外科杂志》2011年第13期1130-1134,共5页Orthopedic Journal of China

摘　　要：[目的]探讨rhBMP-2、bFGF双基因共转染兔骨髓间充质干细胞(bone marrow stem cells,BMSCs)复合nHA/RHLC/PLA支架及其体内异位成骨研究。[方法]构建携带rhBMP-2及bFGF基因片段的双基因表达载体prh-BMP-2-IRES-bFGF并转染兔BMSCs,免疫细胞化学检测目的基因表达,进而与nHA/RHLC/PLA支架复合构建骨组织工程复合体。rhBMP-2及bFGF单独转染组、未干预BMSCs组设为对照。实时定量RT-PCR检测成骨相关基因(I型胶原、骨连蛋白、骨桥蛋白)表达,扫描电镜观察细胞/材料复合情况。体外培养7 d后埋于自体股部肌袋,4周后取材,放射学及组织学方法检测成骨情况。[结果]转染后BMSCs的rhBMP-2及bFGF蛋白表达均呈阳性。复合材料后,其成骨相关基因表达明显高于rhBMP-2及bFGF单独转染组、未干预BMSCs组(P<0.05,n=3),放射学及组织学检测也证实其异位成骨明显。[结论]采用rhBMP-2及bFGF双基因转染技术能够有效促进BMSCs成骨分化,与新型材料nHA/RHLC/PLA支架复合所构建的骨组织工程复合体,体现出良好的体内外成骨活力,进一步研究将探讨其针对大段骨缺损的修复效能。[ Objective ] To investigate the osteogenesis of a novel bone bioengineering biocomposite by combining nHA/ RHLC/PLA scaffold with BMSCs after cotransfection with rhBMP - 2 and bFGF. [ Methods ] PrhBMP - 2 - IRES - bFGF was firstly fabricated and transfected into BMSCs （ rhBMP - 2 - bFGF/BMSCs） . Expression of rhBMP - 2 and bFGF was identified by immunocytochemistry. The pIRES2 - EGFP - rhBMP - 2 or pCI - neo - bFGF transfected and untreated BMSCs were seined as controls. Then the BMSCs were combined with nHA/RHLC/PLA scaffold. Real - time RT - PCR analysis of osteogenic specific genes including collagen I, osteonectin, osteopontin and SEM observation were undertaken. After 7 days, the biocomposites had been transplanted into autologous muscular intervals for 4 weeks and then explanted for radiologic and histologic analyses. [ Results] In rhBMP- 2 -bFGF/BMSCs group, rhBMP -2 and bFGF was positive expressed, expression of osteogenic specific genes was quantatively higher than those in control groups after combination with scaffold, new bone tissue was formed histologi- cally and radiographically （ P 〈 0. 05, n = 3 ） . [ Conclusion ] Double - transfection of rhBMP - 2 and bFGF can promote osteogenic differentiation of BMSCs. Noticeable osteogenesis of the biocomposite by combination rhBMP - 2 and bFGF transfected BMSCs with nHA/HLC/PLA scaffold has been achieved and will be applied into the large bone defect repair process.

关 键 词：RHBMP-2 BFGF 骨髓间充质干细胞 nHA/RHLC/PLA 成骨 

分 类 号：R318.08[医药卫生—生物医学工程]