ACM  >> Vol. 4 No. 2 (June 2014)

    骨活素基因转染的兔骨髓基质干细胞复合多孔丝素蛋白支架体外构建组织工程骨
    Construction of Tissue Engineering Bone with Osteoactivin Gene Transfected Rabbit Bone Marrow Stromal Cells and Porous Silk Fibroin Scaffold in Vitro

  • 全文下载: PDF(466KB) HTML    PP.27-33   DOI: 10.12677/ACM.2014.42006  
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作者:  

王涵,杨征毅,程峰,潘广嗣,易 晓辉,孙晋,曹依娜,袁林:广州医科大学附属第一医院口腔科,广州

关键词:
骨活素骨髓基质干细胞基因治疗组织工程骨Osteoactivin Bone Marrow Stromal Cells Gene Therapy Tissue Engineering Bone

摘要:

目的:用腺病毒表达载体将骨活素基因转染到兔骨髓基质干细(BMSCs),复合多孔丝素蛋白支架体外构建组织工程骨。方法:用表达骨活素基因的腺病毒载体转染体外培养的兔BMSC,免疫组化、原位杂交染色和蛋白印迹方法检测细胞骨活素的表达,并通过流式细胞仪和ALP活性检测分析其对细胞增殖、分化的影响。然后将转染后细胞接种到多孔丝素蛋白支架上,扫描电镜观察细胞贴附、生长状况。结果:转染后,骨活素基因在mRNA水平和蛋白水平均有表达;S期细胞比例和ALP活性明显增高。扫描电镜见转染细胞分布均匀,伸展良好。结论骨活素基因可高效转染兔BMSC,且促进细胞增殖及成骨转化。转染后细胞在多孔丝素蛋白支架上生长良好,骨活素基因治疗的组织工程骨构建成功。

Objective: Rabbit bone marrow stromal cells (BMSCs) infected by a recombinant adenoviral vector carrying the osteoactivin gene (Ad-OA) were seeded into porous silk fibroin scaffold to construct tissue engineering bone in vitro. Method: Ad-OA infected RBM SC cultured in vitro and the expression of OA in these cells after infection were determined by in situ hybridization and immune ohistochemical analysis. OA productions were confirmed by western blot analysis of the supernatant collected from the cells. The changes of cellular proliferation and differentiation in the cells were observed by flowcytometry and ALP activity analysis. OA transduced cells were then seeded into porous silk fibroin scaffolds. The attachment and growth of the cells on the scaffold were examined using SEM. Results: The expression of OA was confirmed in mRNA and protein levels in the cells after infection and the presence of OA was detected in the supernatant of the cells. In addition, cellular proportion in S period and ALP activity obviously increased in the cells. SEM examination revealed extensive cellular attachment and growth on the porous silk fibroin scaffolds composite in 1 day. Conclusion: Ad-OA could infect RBMSC with high efficiency and promote cellular proliferation and osteoblast conversion. The cells after infection grew well on a porous silk fibroin scaffold. Tissue engineering bone used to regional gene therapy is constructed successfully.

文章引用:
王涵, 杨征毅, 程峰, 潘广嗣, 易晓辉, 孙晋, 曹依娜, 袁林. 骨活素基因转染的兔骨髓基质干细胞复合多孔丝素蛋白支架体外构建组织工程骨 [J]. 临床医学进展, 2014, 4(2): 27-33. http://dx.doi.org/10.12677/ACM.2014.42006

参考文献

[1] Abdelmagid, S.M., Barbe, M.F., Rico, M.C., Salihoglu, S., et al. (2008) Osteoactivin, an anabolic factor that regulates osteoblast differentiation and function. Experimental Cell Research, 314, 2334-2351.
[2] Chiarini, A., Petrini, P., Bozzini, S., Dal Pra, I. and Armato, U. (2003) Silk fibroin/poly(carbonate)-urethane as a substrate for cell growth: In vitro interactions with human cells. Biomaterials, 24, 789-799.
[3] Jin, H.J., Chen, J.S., Karageorgiou, V., et al. (2004) Human bone marrow stromal cell responses on electrospun silk fibroin mats. Biomaterials, 25, 1039-1047.
[4] Deng, J., She, R., Huang, W., Dong, Z., Mo, G. and Liu, B. (2013) A silk fibroin/chitosan scaffold in combination with bone marrow-derived mesenchymal stem cells to repair cartilage defects in the rabbit knee. Journal of Materials Science: Materials in Medicine, 24, 2037-2046.
[5] Wang, G., Yang, H., Li, M., Lu, S., Chen, X. and Cai, X. (2010) The use of silk fibroin/hydroxyapatite composite cocultured with Rabbit Bone-Marrow Stromal cells in the healing of a segmental bone defect. The Journal of Bone & Joint Surgery, 92, 320-325.
[6] Zhang, Y., Fan, W., Nothdurft, L., Wu, C., Zhou, Y., Crawford, R. and Xiao, Y. (2011) In vitro and in vivo evaluation of adenovirus combined silk fibroin scaffolds for bone morphogenetic protein-7 gene delivery. Tissue Engineering Part C: Methods, 17, 789-797.
[7] Owen, T.A., Smock, S.L., Prakash, S., et al. (2003) Identification and characterization of the genes encoding human and mouse osteoactivin. Critical Reviews in Eukaryotic Gene Expression, 13, 205-220.
[8] Schaffner, P. and Dard, M.M. (2003) Structure and function of RGD peptides involved in bone biology. Cellular and Molecular Life Sciences, 60, 119-132.
[9] Susan, J., Mclarthy, A., Kaplan, L., et al. (2000) Functionalized silk protein biomaterials for regeneration. Proceeding in Sixth World Biomaterials Congress Transactions, Hawaii, 2000, 1206.
[10] Abdelmagid, S.M., Barbe, M.F., Arango-Hisijara, I., Owen, T.A., Popoff, S.N. and Safadi, F.F. (2007) Osteoactivin acts as downstream mediator of BMP-2 effects on osteoblast function. Journal of Cellular Physiology, 210, 26-37.
[11] Steinbrech, D.S., Mehrara, B.J., Rower, N.M., et al. (2000) Gene expression of TGF-beta, TRF-beta receptor and extracellular matrix proteins during membranous bone healing in rats. Plastic and Reconstructive Surgery, 105, 2028- 2038.
[12] 李建军, 刘建国, 韩东, 孙洪斌,卜丽莎, 杨绍娟, 张文岚, 徐莘香 (2003) 人骨形态发生蛋白2腺病毒表达载体转染人骨髓基质干细胞及对其增殖的影响. 吉林大学学报(医学版), 4, 421-423.