丁立峰;周振东;杨军;郑刚;李建军;

• 1:中国医科大学盛京医院脊柱创伤骨科

摘要(Abstract)：

[目的]构建聚乳酸-聚己内酯(PLA/PCL)吸附骨形态发生蛋白2(bone morphogenetic protein-2,BMP-2)基因的聚乙二醇(PEG)纳米复合物形成生物可降解仿生骨材料,接种兔骨髓间充质干细胞(rabbit bonemesenchymal stem cells,rBMSCs),检测BMP-2基因的转染情况及其对细胞增殖、分化的影响。[方法]通过溶液共混法制备PLA/PCL载基因仿生骨,接种rBMSCs细胞于仿生骨之上,培养48h;Real-time PCR检测转染后细胞BMP-2及骨钙素mRNA表达水平;应用Western Blot及免疫组化检测rBMSCs转染后BMP-2蛋白表达;免疫荧光检测rBMSCs细胞Ⅰ型胶原蛋白表达;ELISA检测转染后细胞培养上清中分泌BMP-2浓度,同时检测细胞内碱性磷酸酶活性,从而分析细胞分化情况;流式细胞检测转染BMP-2后细胞周期的变化。[结果]以未转染细胞作为对照,免疫组化表明转染后细胞内BMP-2表达量明显上调(P<0.05);Real-time PCR检测结果表明BMP-2与骨钙素mRNA表达显著增加(P<0.05);同时Western Blot结果同免疫组化结果相似,BMP-2也有明显上调(P<0.01);免疫荧光检测Ⅰ型胶原表达量显著上升(P<0.01);ELISA检测细胞培养上清中BMP-2分泌量也有增加(P<0.05);碱性磷酸酶较对照组相比活性显著增强(P<0.05),而流式细胞检测S期细胞无明显变化(P>0.05)。[结论]载基因仿生骨具有稳定而安全的转染性能,其转染后对rBMSCs细胞的分化效果是明显的,而对于细胞周期变化的影响并不显著。

关键词(KeyWords)： 骨形态发生蛋白-2;兔骨髓间充质干细胞;组织工程骨

Abstract:

Keywords:

基金项目(Foundation): 辽宁省自然科学基金资助项目(项目编号:20082118);; 辽宁省教育厅科学技术研究资助项目(项目编号:L2010652)

作者(Author): 丁立峰;周振东;杨军;郑刚;李建军;

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DOI:

参考文献(References)：

• [1]张明磊,常非,高忠礼,等.BMP-2重组质粒转染骨髓间充质干细胞及表达的检测[J].中国实验诊断学,2010,11:1742-1744.
• [2]郭洪刚,刘静,刘海峰,等.携载人重组骨形态形成蛋白2及脂肪基质细胞的仿生骨膜构建及强化兔桡骨的再生能力[J].中国组织工程研究与临床康复,2007,48:9734-9738.
• [3]Backly R,Cancedda R.Bone marrow stem cells in clinical applica-tion:harnessing paracrine roles and niche mechanisms[J].AdvBiochem Engin/Biotechnol,2010,123:265-292.
• [4]Chokalingam K,Hunter S,Gooch C,et al.Three-dimensional invitro effects of compression and time in culture on aggregate modulusand on gene expression and protein content of collagen type II in murinechondrocytes[J].Tissue Eng Part A,2009,15:2807-2816.
• [5]郝伟,胡蕴玉,姜明,等.基于脂肪干细胞Ⅰ型胶原凝胶PLGA-β-TCP支架的新型仿生骨组织工程复合体的构建及生物学性能[J].第四军医大学学报,2008,7:584-587.
• [6]Chen JH,Tang Q,Tian C,et al.Biocompatibility of novel bionicscaffold with nanostructure[J].J Clin Rehabil Tiss Eng Res,2009,29:5775-5778.
• [7]Jiang J,Fan CY,Zhang BF.Experimental construction of BMP2and VEGF gene modified tissue engineering bone in vitro[J].Int JMol Sci,2011,12,1744-1755.
• [8]柴岗,张艳,刘伟,等.组织工程骨在颅颌面骨缺损临床修复中的应用[J].中华医学杂志,2003,19:1676-1681.
• [9]Farokhzad OC,Langer R.Impact of nanotechnology on drug deliv-ery[J].ACS Nano,2009,1:16-20.
• [10]Chen XA,Zhang LJ,He ZJ,et al.Plasmid-encapsulated polyethy-lene glycol-grafted polyethylenimine nanoparticles for gene deliv-ery into rat mesenchymal stem cells[J].Int J Nanomed,2011,6:843-853.
• [11]Lissenberg-Thunnissen SN,Gorter D,Sier C,et al.Use and effi-cacy of bone morphogenetic proteins in fracture healing[J].Int Or-thop,2011,35:1271-1280.
• [12]Saito A,Suzuki Y,Ogata SI,et al.Accelerated bone repair with theuse of a synthetic BMP-2derived peptide and bone--marrowstromal cells[J].Biomed Mater Res Part A,2005,1:77-82.
• [13]李亚非,李青,马福成,等.兔骨折骨痂中骨形态发生蛋白和成纤维细胞生长因子基因的表达[J].中华医学杂志,1996,8:620-621.
• [14]Zhang SF,Cezary KA,Michael R,et al.Polyethylenimine-PEGcoated albumin nanoparticles for BMP-2 delivery[J].Biomaterials,2010,31:952-963.
• [15]Huang YC,Connell M,Park Y,et al.Fabrication and in vitro tes-ting of polymeric delivery system for condensed DNA[J].J BiomedMater Res A,2003,4:1384-1392.
• [16]Luu YK,Kim K,Hsiao BS,et al.Development of a nanostruc-tured DNA delivery scaffold via electrospinning of PLGA and PLA-PEG block copolymers[J].J Control Release,2003,2:341-353.
• [17]Fu CH,Sun XL,Liu DH,et al.Biodegradable tri-block copolymerpoly(lactic acid)-poly(ethylene glycol)-poly(L-lysine)(PLA-PEG-PLL)as a non-viral vector to enhance gene transfection[J].Int J Mol Sci,2011,2:1371-1388.
• [18]娄鸣,李晓红,饶国洲.BMP-2,BGP mRNA表达量及ALP活性的变化对大鼠骨髓间充质干细胞体外诱导成骨细胞的影响[J].现代检验医学杂志,2008,5:28-31.