BMP-2基因重組慢病毒載體轉染豬BMSCs成骨分化研究_《中國修復重建外科雜志》

作者：

許勝貴 ¹ , 林建華 ² , 劉蔚楠 ³ , 吳朝陽 ²

1 福建醫科大學附屬閩東醫院骨科（福建寧德，355000）;;
2 福建醫科大學附屬第一醫院骨科;;
3 福建中醫藥大學附屬人民醫院骨科;

關鍵詞：

骨軟骨組織工程 BMSCs BMP-2 慢病毒載體成骨分化豬

DOI：

10.7507/1002-1892.20130299

視頻：

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目的構建 BMP-2基因重組慢病毒載體，并檢測其轉染豬BMSCs 后BMP-2表達活性及誘導BMSCs成骨分化情況，為進一步構建組織工程骨軟骨奠定基礎。方法取2只2月齡巴馬香豬（體重約15 kg）髂骨骨髓，采用密度梯度離心法分離培養BMSCs，并傳代。利用基因重組技術構建 BMP-2基因重組慢病毒載體，并以感染復數（multiplicity of infection，MOI）10、25、50、100、200轉染第2代BMSCs，通過熒光顯微鏡及倒置相差顯微鏡觀察確定最佳MOI值。以最佳 MOI 值感染 BMSCs作為實驗組，空載體轉染的 BMSCs（空載體組）及未轉染的 BMSCs（空白組）作為對照，通過 RT-PCR、免疫組織化學染色、Western blot檢測 BMP-2 mRNA及蛋白在 BMSCs 中的表達，并應用ALP染色、ALP活性檢測及茜素紅鈣結節染色檢測其成骨分化情況。結果經RT-PCR基因測序鑒定 BMP-2基因重組慢病毒載體構建成功，轉染 BMSCs后熒光顯微鏡下可見綠色熒光表達，且MOI值為 100 時為最佳表達。RT-PCR 提示 BMP-2 基因成功轉染至 BMSCs 中；免疫組織化學染色及 Western blot檢測示轉染后BMSCs并可持續、穩定、高效表達 BMP-2蛋白；培養后2周BMSCs可見ALP表達及鈣結節形成。結論成功構建 BMP-2基因重組慢病毒載體并轉染豬 BMSCs，BMP-2 基因轉染入 BMSCs 后能持續、穩定、高效表達 BMP-2 基因和蛋白，并成功誘導 BMSCs 向成骨細胞分化。

引用本文： 許勝貴,林建華,劉蔚楠,吳朝陽. BMP-2基因重組慢病毒載體轉染豬BMSCs成骨分化研究. 中國修復重建外科雜志, 2013, 27(11): 1380-1385. doi: 10.7507/1002-1892.20130299 復制

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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．.
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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.	張慧, 王貴超, 韓興龍, 等. 去分化間充質干細胞成骨再分化潛能的實驗研究. 南京醫科大學學報: 自然科學版, 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. 林建華, 賴建明, 林文平, 等. 膠原-殼聚糖/膠原-納米羥基磷灰石仿生支架材料的制備及其生物相容性檢測. 中國修復重建外科雜志, 2012, 26(8): 1001-1006.
5. 賴建明, 林建華, 林文平, 等. 豬TGF-β1基因重組慢病毒載體的構建及其在BMSCs中的表達. 中國修復重建外科雜志, 2012, 26(7): 849-854.
6. 林朝貴, 范林, 陳良龍. 利用In-Fusion技術構建存活素-增強型綠色熒光蛋白融合基因重組慢病毒表達載體. 心血管康復醫學雜志, 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. 劉明, 項舟, 裴福興, 等. BMSCs 種植雙相復合支架修復兔關節軟骨及軟骨下骨缺損. 中國修復重建外科雜志, 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. 張慧, 王貴超, 韓興龍, 等. 去分化間充質干細胞成骨再分化潛能的實驗研究. 南京醫科大學學報: 自然科學版, 2013, 33(8): 1044-1048.

《中國修復重建外科雜志》

BMP-2基因重組慢病毒載體轉染豬BMSCs成骨分化研究

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《中國修復重建外科雜志》

BMP-2基因重組慢病毒載體轉染豬BMSCs成骨分化研究

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