腺病毒介导BMP-2/7共表达转染兔滑膜MSCs体外向纤维软骨样细胞分化的研究_《中国修复重建外科杂志》

作者：

符培亮 ¹ , 张雷 ¹ , 吴海山 ¹ , 丛锐军 ¹ , 陈松 ¹ , 丁喆如 ¹ , 胡凯猛 ²

1 上海长征医院关节外科（上海，200003）;;
2第二军医大学组织胚胎学教研室 △为共同第一作者;

关键词：

腺病毒载体 BMP-2/7 滑膜MSCs 纤维软骨样细胞兔

DOI：

10.7507/1002-1892.20130079

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

目的通过使用腺病毒介导BMP-2/7共表达载体转染兔滑膜MSCs（synovial-derived MSCs，SMSCs），观察其在体外向纤维软骨样细胞分化的可行性。方法取3月龄新西兰大白兔6只，雌雄不限，体重（2.1 ± 0.3） kg；取滑膜组织分离纯化SMSCs后，进行形态学观察及免疫细胞荧光染色、流式细胞仪、细胞周期鉴定，并检测其成脂、成骨、成软骨多向分化潜能。同时包装pAdTrack-BMP-2-内部核糖体进入位点（internal ribosome entry site，IRES）-BMP-7腺病毒载体，转染SMSCs后进行增殖动力学、核型分析、流式细胞仪检测细胞DNA含量、致瘤性分析等安全性鉴定。体外在不完全成软骨培养基中培养，观察其分化状态，并行RT-PCR检测、免疫荧光染色及甲苯胺蓝染色检测。结果从兔滑膜组织中分离出的SMSCs，其细胞形态、表面标记、多向分化潜能等均符合MSCs的特点。pAdTrack-BMP-2-IRES-BMP-7转染SMSCs的转染率约为70%。安全性鉴定结果显示转染后的SMSCs倍增时间、染色体数目及DNA含量均正常，且无致瘤性。体外不完全成软骨培养基诱导培养21 d后，RT-PCR检测示SMSCs 的Ⅰ、Ⅱ型胶原表达明显增加，以Ⅱ型胶原为主；软骨分化信号分子RhoA与Sox-9的表达经诱导后也明显增加。免疫荧光染色及甲苯胺蓝染色结果亦显示转染后的SMSCs向纤维软骨样细胞分化。结论使用腺病毒载体安全有效，可在体外定向诱导兔SMSCs向纤维软骨样细胞分化。

引用本文： 符培亮,张雷,吴海山,丛锐军,陈松,丁喆如,胡凯猛. 腺病毒介导BMP-2/7共表达转染兔滑膜MSCs体外向纤维软骨样细胞分化的研究. 中国修复重建外科杂志, 2013, 27(3): 345-352. doi: 10.7507/1002-1892.20130079 复制

1.	赵文君, 邢国胜, 于顺禄. 滑膜间充质干细胞及其在组织工程中的应用. 中国修复重建外科杂志, 2011, 25(12): 1504-1507.
2.	De Bari C, Dell’Accio F, Tylzanowski P, et al. Multipotent mesenchymal stem cells from adult human synovial membrane. Arthritis Rheum, 2001, 44(8): 1928-1942.
3.	Djouad F, Bony C, Haupl T, et al. Transcriptional profiles discriminate bone marrow-derived and synovium-derived mesenchymal stem cells. Arthritis Res Ther, 2005, 7(6): 1304-1315.
4.	Jo CH, Ahn HJ, Kim HJ, et al. Surface characterization and chondrogenic differentiation of mesenchymal stromal cells derived from synovium. Cytotherapy, 2007, 9(4): 316-327.
5.	Morito T, Muneta T, Hara K, et al. Synovial fluid-derived mesenchymal stem cells increase after intra-articular ligament injury in humans. Rheumatology (Oxford), 2008, 47(8): 1137-1143.
6.	Zhang S, Muneta T, Morito T, et al. Autologous synovial fluid enhances migration of mesenchymal stem cells from synovium of osteoarthritis patients in tissue culture system. J Orthop Res, 2008, 26(10): 1413-1418.
7.	Horie M, Sekiya I, Muneta T, et al. Intra-articular injected synovial stem cells differentiate into meniscal cells directly and promote meniscal regeneration without mobilization to distant organs in rat massive meniscal defect. Stem Cells, 2009, 27(4): 878-887.
8.	Sakaguchi Y, Sekiya I, Yagishita K, et al. Comparison of human stem cells derived from various mesenchymal tissues. Arthritis Rheum, 2005, 52(8): 2521-2529.
9.	Shirasawa S, Sekiya I, Sakaguchi Y, et al. In vitro chondrogenesis of human synovium-derived mesenchynlal stem cells: optimal condition and cmnparison with bone morrow-derived cells. J Cell Biochem, 2006, 97(1): 84-97.
10.	Park Y, Suginoto M, Watrin A, et al. BMP-2 induces the expression of chondrocyte-specific genes in bovine synovium-derived progenitor cells cultured in three-dimensional alginate hydrogel. Osteoarthritis Cartilage, 2005, 13(6): 527-536.
11.	Kurth T, Hedbom E, Shintani N, et al. Chondrogenic potential of human synovial mesenchymal stem cells in alginate. Osteparthritis Cartilage, 2007, 15(10): 1178-1189.
12.	Miyamoto C, Matsumoto T, Sakimura K, et al. Osteogenic protein-1 with transforming growth factor-beta 1: potent inducer of chondrogenesis of synovial mesenchymal stem cells in vitro. J Orthop Sci, 2007, 12(6): 555-561.
13.	张正政, 李卫平, 杨睿, 等. 滑膜间充质干细胞分离培养、免疫表型鉴定及在混合淋巴反应体系中的抑制效应. 中国组织工程研究, 2012, 16(19): 3531-3519.
14.	Shi Y, Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell, 2003, 113(6): 685-700.
15.	Nagase T, Muneta T, Ju YJ, et al. Analysis of the chondrogenic potential of human synovial stem cells according to harvest site and culture parameters in knees with medial compartment osteoarthritis. Arthritis Rheum, 2008, 58(5): 1389-1398.
16.	Maniatopiulos C, Sidek J, Melcher AH. Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats. Cell Tissue Res, 1988, 254(2): 317-330.
17.	Wan M, Cao X. BMP signaling in skeletal development. Biochem Biophys Res Commun, 2005, 328(3): 651-657.
18.	Bi W, Huang W, Whitworth DJ, et al. Haploinsufficiency of Sox9 results in defective cartilage primordia and premature skeletal mineralization. Proc Natl Acad Sci U S A, 2001, 98(12): 6698-6703.
19.	Nishimura K, Solchaga LA, Caplan AI, et al. Chondroprogenitor cells of synovial tissue. Arthritis Rheum, 1999, 42(12): 2631-2637.
20.	Dell’Accio F, Vanlauwe J, Bellemans J, et al. Expanded phenotypically stable chondrocytes persisit in the repair tissue and contribute to cartilage matrix formation and structural integration in a goat model of autologous chondrocyte implantation. J Orthop Res, 2003, 21(1): 123-131.
21.	Brittberg M, Peterson L, Sjogren-Jansson E, et al. Articular cartilage engineering with autologous chondrocyte transplantation. A review of recent developments. J Bone Joint Surg (Am), 2003, 85-A Suppl 3: 109-115.
22.	Johnstone B, Hering TM, Caplan AI, et al. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res, 1998, 238(1): 265-272.
23.	Sekiya I, Larson BL, Vuoristo JT, et al. Comparison of effect of BMP-2, -4, and -6 on in vitro cartilage formation of human adult stem cells from bone marrow stroma. Cell Tissue Res, 2005, 320(2): 269-276.
24.	Yokoyama A, Sekiya I, Miyazaki K, et al. In vitro cartilage formation of composites of synovium-derived mesenchymal stem cells with collagen gel. Cell Tissue Res, 2005, 322(2): 289-298.
25.	Wu L, Ishikawa Y, Genge B, et al. Effect of osteogenic protein-1 on the development and mineralization of primary cultures of avian growth plate chondrocytes: modulation by retinoic acid. J Cell Biochem, 1997, 67(4): 498-513.
26.	Jones BA, Pei M. Synovium-derived stem cells: a tissue-specific stem cell for cartilage engineering and regeneration. Tissue Eng Part B Rev, 2012, 18(4): 301-311.
27.	Krüger JP, Endres M, Neumann K, et al. Chondrogenic differentiation of human subchondral progenitor cells is affected by synovial fluid from donors with osteoarthritis or rheumatoid arthritis. J Orthop Surg Res, 2012, 7: 10.
28.	Dhinsa BS, Adesida AB. Current clinical therapies for cartilage repair, their limitation and the role of stem cells. Curr Stem Cell Res Ther, 2012, 7(2): 143-148.

1. 赵文君, 邢国胜, 于顺禄. 滑膜间充质干细胞及其在组织工程中的应用. 中国修复重建外科杂志, 2011, 25(12): 1504-1507.
2. De Bari C, Dell’Accio F, Tylzanowski P, et al. Multipotent mesenchymal stem cells from adult human synovial membrane. Arthritis Rheum, 2001, 44(8): 1928-1942.
3. Djouad F, Bony C, Haupl T, et al. Transcriptional profiles discriminate bone marrow-derived and synovium-derived mesenchymal stem cells. Arthritis Res Ther, 2005, 7(6): 1304-1315.
4. Jo CH, Ahn HJ, Kim HJ, et al. Surface characterization and chondrogenic differentiation of mesenchymal stromal cells derived from synovium. Cytotherapy, 2007, 9(4): 316-327.
5. Morito T, Muneta T, Hara K, et al. Synovial fluid-derived mesenchymal stem cells increase after intra-articular ligament injury in humans. Rheumatology (Oxford), 2008, 47(8): 1137-1143.
6. Zhang S, Muneta T, Morito T, et al. Autologous synovial fluid enhances migration of mesenchymal stem cells from synovium of osteoarthritis patients in tissue culture system. J Orthop Res, 2008, 26(10): 1413-1418.
7. Horie M, Sekiya I, Muneta T, et al. Intra-articular injected synovial stem cells differentiate into meniscal cells directly and promote meniscal regeneration without mobilization to distant organs in rat massive meniscal defect. Stem Cells, 2009, 27(4): 878-887.
8. Sakaguchi Y, Sekiya I, Yagishita K, et al. Comparison of human stem cells derived from various mesenchymal tissues. Arthritis Rheum, 2005, 52(8): 2521-2529.
9. Shirasawa S, Sekiya I, Sakaguchi Y, et al. In vitro chondrogenesis of human synovium-derived mesenchynlal stem cells: optimal condition and cmnparison with bone morrow-derived cells. J Cell Biochem, 2006, 97(1): 84-97.
10. Park Y, Suginoto M, Watrin A, et al. BMP-2 induces the expression of chondrocyte-specific genes in bovine synovium-derived progenitor cells cultured in three-dimensional alginate hydrogel. Osteoarthritis Cartilage, 2005, 13(6): 527-536.
11. Kurth T, Hedbom E, Shintani N, et al. Chondrogenic potential of human synovial mesenchymal stem cells in alginate. Osteparthritis Cartilage, 2007, 15(10): 1178-1189.
12. Miyamoto C, Matsumoto T, Sakimura K, et al. Osteogenic protein-1 with transforming growth factor-beta 1: potent inducer of chondrogenesis of synovial mesenchymal stem cells in vitro. J Orthop Sci, 2007, 12(6): 555-561.
13. 张正政, 李卫平, 杨睿, 等. 滑膜间充质干细胞分离培养、免疫表型鉴定及在混合淋巴反应体系中的抑制效应. 中国组织工程研究, 2012, 16(19): 3531-3519.
14. Shi Y, Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell, 2003, 113(6): 685-700.
15. Nagase T, Muneta T, Ju YJ, et al. Analysis of the chondrogenic potential of human synovial stem cells according to harvest site and culture parameters in knees with medial compartment osteoarthritis. Arthritis Rheum, 2008, 58(5): 1389-1398.
16. Maniatopiulos C, Sidek J, Melcher AH. Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats. Cell Tissue Res, 1988, 254(2): 317-330.
17. Wan M, Cao X. BMP signaling in skeletal development. Biochem Biophys Res Commun, 2005, 328(3): 651-657.
18. Bi W, Huang W, Whitworth DJ, et al. Haploinsufficiency of Sox9 results in defective cartilage primordia and premature skeletal mineralization. Proc Natl Acad Sci U S A, 2001, 98(12): 6698-6703.
19. Nishimura K, Solchaga LA, Caplan AI, et al. Chondroprogenitor cells of synovial tissue. Arthritis Rheum, 1999, 42(12): 2631-2637.
20. Dell’Accio F, Vanlauwe J, Bellemans J, et al. Expanded phenotypically stable chondrocytes persisit in the repair tissue and contribute to cartilage matrix formation and structural integration in a goat model of autologous chondrocyte implantation. J Orthop Res, 2003, 21(1): 123-131.
21. Brittberg M, Peterson L, Sjogren-Jansson E, et al. Articular cartilage engineering with autologous chondrocyte transplantation. A review of recent developments. J Bone Joint Surg (Am), 2003, 85-A Suppl 3: 109-115.
22. Johnstone B, Hering TM, Caplan AI, et al. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res, 1998, 238(1): 265-272.
23. Sekiya I, Larson BL, Vuoristo JT, et al. Comparison of effect of BMP-2, -4, and -6 on in vitro cartilage formation of human adult stem cells from bone marrow stroma. Cell Tissue Res, 2005, 320(2): 269-276.
24. Yokoyama A, Sekiya I, Miyazaki K, et al. In vitro cartilage formation of composites of synovium-derived mesenchymal stem cells with collagen gel. Cell Tissue Res, 2005, 322(2): 289-298.
25. Wu L, Ishikawa Y, Genge B, et al. Effect of osteogenic protein-1 on the development and mineralization of primary cultures of avian growth plate chondrocytes: modulation by retinoic acid. J Cell Biochem, 1997, 67(4): 498-513.
26. Jones BA, Pei M. Synovium-derived stem cells: a tissue-specific stem cell for cartilage engineering and regeneration. Tissue Eng Part B Rev, 2012, 18(4): 301-311.
27. Krüger JP, Endres M, Neumann K, et al. Chondrogenic differentiation of human subchondral progenitor cells is affected by synovial fluid from donors with osteoarthritis or rheumatoid arthritis. J Orthop Surg Res, 2012, 7: 10.
28. Dhinsa BS, Adesida AB. Current clinical therapies for cartilage repair, their limitation and the role of stem cells. Curr Stem Cell Res Ther, 2012, 7(2): 143-148.

《中国修复重建外科杂志》

腺病毒介导BMP-2/7共表达转染兔滑膜MSCs体外向纤维软骨样细胞分化的研究

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《中国修复重建外科杂志》

腺病毒介导BMP-2/7共表达转染兔滑膜MSCs体外向纤维软骨样细胞分化的研究

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