腺病毒介導BMP-2/7共表達轉染兔滑膜MSCs體外向纖維軟骨樣細胞分化的研究_《中國修復重建外科雜志》

作者：

符培亮 ¹ , 張雷 ¹ , 吳海山 ¹ , 叢銳軍 ¹ , 陳松 ¹ , 丁喆如 ¹ , 胡凱猛 ²

1 上海長征醫院關節外科（上海，200003）;;
2第二軍醫大學組織胚胎學教研室 △為共同第一作者;

關鍵詞：

腺病毒載體 BMP-2/7 滑膜MSCs 纖維軟骨樣細胞兔

DOI：

10.7507/1002-1892.20130079

視頻：

導出 下載 收藏 掃碼 引用

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

目的通過使用腺病毒介導BMP-2/7共表達載體轉染兔滑膜MSCs（synovial-derived MSCs，SMSCs），觀察其在體外向纖維軟骨樣細胞分化的可行性。方法取3月齡新西蘭大白兔6只，雌雄不限，體重（2.1 ± 0.3） kg；取滑膜組織分離純化SMSCs后，進行形態學觀察及免疫細胞熒光染色、流式細胞儀、細胞周期鑒定，并檢測其成脂、成骨、成軟骨多向分化潛能。同時包裝pAdTrack-BMP-2-內部核糖體進入位點（internal ribosome entry site，IRES）-BMP-7腺病毒載體，轉染SMSCs后進行增殖動力學、核型分析、流式細胞儀檢測細胞DNA含量、致瘤性分析等安全性鑒定。體外在不完全成軟骨培養基中培養，觀察其分化狀態，并行RT-PCR檢測、免疫熒光染色及甲苯胺藍染色檢測。結果從兔滑膜組織中分離出的SMSCs，其細胞形態、表面標記、多向分化潛能等均符合MSCs的特點。pAdTrack-BMP-2-IRES-BMP-7轉染SMSCs的轉染率約為70%。安全性鑒定結果顯示轉染后的SMSCs倍增時間、染色體數目及DNA含量均正常，且無致瘤性。體外不完全成軟骨培養基誘導培養21 d后，RT-PCR檢測示SMSCs 的Ⅰ、Ⅱ型膠原表達明顯增加，以Ⅱ型膠原為主；軟骨分化信號分子RhoA與Sox-9的表達經誘導后也明顯增加。免疫熒光染色及甲苯胺藍染色結果亦顯示轉染后的SMSCs向纖維軟骨樣細胞分化。結論使用腺病毒載體安全有效，可在體外定向誘導兔SMSCs向纖維軟骨樣細胞分化。

引用本文： 符培亮,張雷,吳海山,叢銳軍,陳松,丁喆如,胡凱猛. 腺病毒介導BMP-2/7共表達轉染兔滑膜MSCs體外向纖維軟骨樣細胞分化的研究. 中國修復重建外科雜志, 2013, 27(3): 345-352. doi: 10.7507/1002-1892.20130079 復制

1.	趙文君, 邢國勝, 于順祿. 滑膜間充質干細胞及其在組織工程中的應用. 中國修復重建外科雜志, 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.	張正政, 李衛平, 楊睿, 等. 滑膜間充質干細胞分離培養、免疫表型鑒定及在混合淋巴反應體系中的抑制效應. 中國組織工程研究, 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. 趙文君, 邢國勝, 于順祿. 滑膜間充質干細胞及其在組織工程中的應用. 中國修復重建外科雜志, 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. 張正政, 李衛平, 楊睿, 等. 滑膜間充質干細胞分離培養、免疫表型鑒定及在混合淋巴反應體系中的抑制效應. 中國組織工程研究, 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.

《中國修復重建外科雜志》

腺病毒介導BMP-2/7共表達轉染兔滑膜MSCs體外向纖維軟骨樣細胞分化的研究

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

上一篇

下一篇

Format

Content

《中國修復重建外科雜志》

腺病毒介導BMP-2/7共表達轉染兔滑膜MSCs體外向纖維軟骨樣細胞分化的研究

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

上一篇

下一篇

Format

Content

摘要全文圖表視頻參考文獻施引文獻補充材料