BMP-2基因重组慢病毒载体转染猪BMSCs成骨分化研究_《中国修复重建外科杂志》

作者：

许胜贵 ¹ , 林建华 ² , 刘蔚楠 ³ , 吴朝阳 ²

1 福建医科大学附属闽东医院骨科（福建宁德，355000）;;
2 福建医科大学附属第一医院骨科;;
3 福建中医药大学附属人民医院骨科;

关键词：

骨软骨组织工程 BMSCs BMP-2 慢病毒载体成骨分化猪

DOI：

10.7507/1002-1892.20130299

视频：

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摘要 全文 图表 视频 参考文献 施引文献 补充材料

目的构建 BMP-2基因重组慢病毒载体，并检测其转染猪BMSCs 后BMP-2表达活性及诱导BMSCs成骨分化情况，为进一步构建组织工程骨软骨奠定基础。方法取2只2月龄巴马香猪（体重约15 kg）髂骨骨髓，采用密度梯度离心法分离培养BMSCs，并传代。利用基因重组技术构建 BMP-2基因重组慢病毒载体，并以感染复数（multiplicity of infection，MOI）10、25、50、100、200转染第2代BMSCs，通过荧光显微镜及倒置相差显微镜观察确定最佳MOI值。以最佳 MOI 值感染 BMSCs作为实验组，空载体转染的 BMSCs（空载体组）及未转染的 BMSCs（空白组）作为对照，通过 RT-PCR、免疫组织化学染色、Western blot检测 BMP-2 mRNA及蛋白在 BMSCs 中的表达，并应用ALP染色、ALP活性检测及茜素红钙结节染色检测其成骨分化情况。结果经RT-PCR基因测序鉴定 BMP-2基因重组慢病毒载体构建成功，转染 BMSCs后荧光显微镜下可见绿色荧光表达，且MOI值为 100 时为最佳表达。RT-PCR 提示 BMP-2 基因成功转染至 BMSCs 中；免疫组织化学染色及 Western blot检测示转染后BMSCs并可持续、稳定、高效表达 BMP-2蛋白；培养后2周BMSCs可见ALP表达及钙结节形成。结论成功构建 BMP-2基因重组慢病毒载体并转染猪 BMSCs，BMP-2 基因转染入 BMSCs 后能持续、稳定、高效表达 BMP-2 基因和蛋白，并成功诱导 BMSCs 向成骨细胞分化。

引用本文： 许胜贵,林建华,刘蔚楠,吴朝阳. BMP-2基因重组慢病毒载体转染猪BMSCs成骨分化研究. 中国修复重建外科杂志, 2013, 27(11): 1380-1385. doi: 10.7507/1002-1892.20130299 复制

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11.	刘明, 项舟, 裴福兴, 等. BMSCs 种植双相复合支架修复兔关节软骨及软骨下骨缺损. 中国修复重建外科杂志, 2010, 24(1): 87-93.
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15.	Grgurevic L, Macek B, Mercep M, et al. Bone morphogenetic protein (BMP)1-3 enhances bone repair. Biochem Biophys Res Commun, 2011, 408(1): 25-31.
16.	Yazici C, Takahata M, Reynolds DG, et al. Self-complementary AAV2.5-BMP-2 -coated femoral allografts mediated superior bone healing versus live autografts in mice with equivalent biomechanics to unfractured femur. Mol Ther, 2011, 19(8): 1416-1425.
17.	Guillot PV, De Bari C, Dell’Accio F, et al. Comparative osteogenic transcription profiling of various fetal and adult mesenchymal stem cell sources. Differentiation, 2008, 76(9): 946-957．.
18.	Handschel J, Berr K, Depprich RA, et al. Induction of osteogenic markers in differentially treated cultures of embryonic stem cells. Head Face Med, 2008, 4: 10．.
19.	Maegawa N, Kawamura K, Hirose M, et al. Enhancement of osteoblastic differentiation of mesenchymal stromal cells cultured by selective combination of bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2). J Tissue Eng Regen Med, 2007, 1(4): 306-313.
20.	Verkey M, Kllcharski C, Haque T, et al. In vitro osteogenic response of rat bone marrow cells to bFGF and BMP-2 treatments. Clin Orthop Relat Res, 2006, (443): 113-123.
21.	Wyatt LE, Chung CY, Carlsen B, et al. Bone morphogenetic protein-2 (BMP-2) and transforming growth factor-β1 (TGF-β1) alter connexin 43 phosphorylation in MC3T3-E1 cells. BMC Cell Biol, 2001, 2: 14.
22.	Cockrell AS, Kafri T. Gene delivery by lentivirus vectors. Mol Biotechnol, 2007, 36(3): 184-204.
23.	Gould DJ, Favorov P. Vectors for the treatment of autoimmune diseases. Gene Therapy, 2003, 10(10): 912-927.
24.	D’Costa J, Mansfield SG, Humeau LM. Lentiviral vectors in clinical trials: Current status. Curr Opin Mol Ther, 2009, 11(5): 554-564.
25.	张慧, 王贵超, 韩兴龙, 等. 去分化间充质干细胞成骨再分化潜能的实验研究. 南京医科大学学报: 自然科学版, 2013, 33(8): 1044-1048.

1. Khan Y, Yaszemski MJ, Mikos AG, et al. Tissue engineering of bone: material and matrix considerations. J Bone Joint Surg (Am), 2008, 90 Suppl 1: 36-42.
2. Bessa PC, Casal M, Reis RL. Bone morphogenetic proteins in tissue engineering: the road from the laboratory to the clinic, part I (basic concepts). J Tissue Eng Regn Med, 2008, 2(1): 1-13.
3. Yoon ST, Boden SD. Osteoinductive molecules in orthopaedics: basic science and preclinical studies. Clin Orthop Relat Res, 2002, (395): 33-43.
4. 林建华, 赖建明, 林文平, 等. 胶原-壳聚糖/胶原-纳米羟基磷灰石仿生支架材料的制备及其生物相容性检测. 中国修复重建外科杂志, 2012, 26(8): 1001-1006.
5. 赖建明, 林建华, 林文平, 等. 猪TGF-β1基因重组慢病毒载体的构建及其在BMSCs中的表达. 中国修复重建外科杂志, 2012, 26(7): 849-854.
6. 林朝贵, 范林, 陈良龙. 利用In-Fusion技术构建存活素-增强型绿色荧光蛋白融合基因重组慢病毒表达载体. 心血管康复医学杂志, 2010, 19(1): 10-14.
7. Rudert M. Histological evaluation of osteochondral defects: consideration of animal models with emphasis on the rabbit, experimental setup, follow-up and applied methods. Cells Tissues Organs, 2002, 171(4): 229-240.
8. Frosch KH, Drengk A, Krause P, et al. Stem cell-coated titanium implants for the partial joint resurfacing of the knee. Biomaterials, 2006, 27(12): 2542-2549.
9. Mithoefer K, McAdams T, Williams RJ, et al. Clinical efficacy of the microfracture technique for articular cartilage repair in the knee: an evidence-based systematic analysis. Am J Sport Med, 2009, 37(10): 2053-2063.
10. Asheesh B, Williams RJ III. Cartilage resurfacing using synthetic composite plugs. Techniques in Knee Surgery, 2009, 8(1): 29-36.
11. 刘明, 项舟, 裴福兴, 等. BMSCs 种植双相复合支架修复兔关节软骨及软骨下骨缺损. 中国修复重建外科杂志, 2010, 24(1): 87-93.
12. Caplan AI. Mesenchymal stem cells: cell-based reconstructive therapy in orthopedics. Tissue Eng, 2005, 11(7-8): 1198-2211.
13. Yamaguchi A, Ishizuya T, Kintou N, et al. Effects of BMP-2, BMP- 4, and BMP-6 on osteoblastic differentiation of bone marrowderived stromal cell lines, ST2 and MC3T3-G2/PA6. Biochem Biophys Res Commun, 1996, 220(2): 366-371.
14. Xie G, Sun J, Zhong G, et al. Hydroxyapatite nanoparticles as a controlled-release carrier of BMP-2: absorption and release kinetics in vitro. J Mater Sci Mater Med, 2010, 21(6): 1875-1880.
15. Grgurevic L, Macek B, Mercep M, et al. Bone morphogenetic protein (BMP)1-3 enhances bone repair. Biochem Biophys Res Commun, 2011, 408(1): 25-31.
16. Yazici C, Takahata M, Reynolds DG, et al. Self-complementary AAV2.5-BMP-2 -coated femoral allografts mediated superior bone healing versus live autografts in mice with equivalent biomechanics to unfractured femur. Mol Ther, 2011, 19(8): 1416-1425.
17. Guillot PV, De Bari C, Dell’Accio F, et al. Comparative osteogenic transcription profiling of various fetal and adult mesenchymal stem cell sources. Differentiation, 2008, 76(9): 946-957．.
18. Handschel J, Berr K, Depprich RA, et al. Induction of osteogenic markers in differentially treated cultures of embryonic stem cells. Head Face Med, 2008, 4: 10．.
19. Maegawa N, Kawamura K, Hirose M, et al. Enhancement of osteoblastic differentiation of mesenchymal stromal cells cultured by selective combination of bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2). J Tissue Eng Regen Med, 2007, 1(4): 306-313.
20. Verkey M, Kllcharski C, Haque T, et al. In vitro osteogenic response of rat bone marrow cells to bFGF and BMP-2 treatments. Clin Orthop Relat Res, 2006, (443): 113-123.
21. Wyatt LE, Chung CY, Carlsen B, et al. Bone morphogenetic protein-2 (BMP-2) and transforming growth factor-β1 (TGF-β1) alter connexin 43 phosphorylation in MC3T3-E1 cells. BMC Cell Biol, 2001, 2: 14.
22. Cockrell AS, Kafri T. Gene delivery by lentivirus vectors. Mol Biotechnol, 2007, 36(3): 184-204.
23. Gould DJ, Favorov P. Vectors for the treatment of autoimmune diseases. Gene Therapy, 2003, 10(10): 912-927.
24. D’Costa J, Mansfield SG, Humeau LM. Lentiviral vectors in clinical trials: Current status. Curr Opin Mol Ther, 2009, 11(5): 554-564.
25. 张慧, 王贵超, 韩兴龙, 等. 去分化间充质干细胞成骨再分化潜能的实验研究. 南京医科大学学报: 自然科学版, 2013, 33(8): 1044-1048.

《中国修复重建外科杂志》

BMP-2基因重组慢病毒载体转染猪BMSCs成骨分化研究

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《中国修复重建外科杂志》

BMP-2基因重组慢病毒载体转染猪BMSCs成骨分化研究

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