非接觸性共培養BMSCs和髓核細胞時間差異性的實驗研究_《中國修復重建外科雜志》

作者：

陳雷 ¹ , 胡秀年 ¹ , 傅冷西 ² , 葉君健 ¹

福建醫科大學附屬第一醫院（福州，350004） 1 骨科，2中心實驗室;

關鍵詞：

BMSCs 髓核細胞非接觸性共培養兔

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目的探討在非接觸性共培養環境下，BMSCs定向分化為類髓核細胞在共培養時間上的差異性，尋找適合體內移植的最佳時間。方法取6只8周齡健康新西蘭大白兔（體重1.5～2.0 kg）骨髓及椎間盤髓核，分離、培養BMSCs和髓核細胞并進行免疫細胞化學鑒定。取原代髓核細胞和第2代生長良好的BMSCs體外建立非接觸性共培養模型。觀察共培養后第1、3、5代BMSCs的形態學變化并繪制生長曲線；RT-PCR檢測共培養5、10、15 d BMSCsⅡ型膠原和蛋白聚糖mRNA表達；Western blot檢測共培養5、10、15、20、25、30 d BMSCs Ⅱ型膠原和蛋白聚糖蛋白的表達。結果BMSCs相對特異性標記物CD44、CD90表達陽性，造血細胞表面標記物CD34、CD45表達陰性。髓核細胞Ⅱ型膠原、蛋白聚糖表達陽性。共培養后2周BMSCs形態發生明顯變化，呈多角形、不規則形；共培養后3代內，BMSCs生長速度無明顯差異，隨著傳代次數增加，細胞增殖明顯減慢。RT-PCR檢測示共培養后10、15 d BMSCs蛋白聚糖和Ⅱ型膠原mRNA表達明顯高于5 d時（P lt; 0.05），而10 d與15 d時差異無統計學意義（P gt; 0.05）。Western blot檢測示共培養后隨時間延長細胞表達Ⅱ型膠原和蛋白聚糖蛋白逐漸增加，5、10、15 d間差異有統計學意義（P lt; 0.05），15 d后各時間點間比較差異無統計學意義（P gt; 0.05）。結論在非接觸性共培養環境下，BMSCs在髓核細胞誘導下可向類髓核細胞分化，表達Ⅱ型膠原和蛋白聚糖，在共培養15 d時達到相對穩定，此時較適合進行體內移植。

引用本文： 陳雷,胡秀年,傅冷西,葉君健. 非接觸性共培養BMSCs和髓核細胞時間差異性的實驗研究. 中國修復重建外科雜志, 2012, 26(11): 1369-1374. doi: 復制

1.	Zhang Y, An HS, Tannoury C, et al. Biological treatment for degenerative disc disease: implications for the field of physical medicine and rehabilitation. Am J Phys Med Rehabil, 2008, 87(9): 694-702.
2.	Yang X, Li X. Nucleus pulposus tissue engineering: a brief review. Eur Spine J , 2009, 18(11): 1564-1572.
3.	Cappello R, Bird JL, Pfeiffer D, et al. Notochordal cell produce and assemble extracellular matrix in a distinct manner, which may be tesponsible for the maintenance of healthy nucleus pulposus. Spine (Phila Pa 1976), 2006, 31(8): 873-883.
4.	Leung VY, Chan D, Cheung KM. Regeneration of intervertebral disc by mesenchymal stem cells: potentials, limitations, and future direction. Eur Spine J, 2006, 15 Suppl 3: S406-413.
5.	Iwashina T, Mochida J, Sskai D, et al. Feasibility of using a human nucleus pulposus cell line as a cell source in cell transplantation therapy for intervertebral disc degeneration. Spine (Phila Pa 1976), 2006, 31(11): 1177-1186.
6.	Strassburg S, Richardson SM, Freemont AJ, et al. Co-culture induces mesenchymal stem cell differentiation and modulation of the degenerate human nucleus pulposus cell phenotype. Regen Med, 2010, 5(5): 701-711.
7.	Risbud MV, Albert TJ, Guttapalli A, et al. Differentiation of mesenchymal stem cells towards a nucleus pulposus-like phenotype in vitro: implications for cell-based transplantation therapy. Spine (Phila Pa 1976), 2004, 29(23): 2627-2632.
8.	Steck E, Bettram H, Abel R, et al. Induction of intervertebral disc-like cells from adult mesenchymal stem cells. Stem Cells, 2005, 23(3): 403-411.
9.	Luo W, Xiong W, Qiu M, et al. Differentiation of mesenchymal stem cells towards a nucleus pulposus-like phenotype utilizing simulated microgravity In vitro. J Huazhong Univ Sci Technolog Med Sci, 2011, 31(2): 199-203.
10.	Sakai D, Mochida J, Iwashina T, et al. Regenerative effects of transplanting mesenchymal stem cells embedded in atelocollagen to the degenerated intervertebral disc. Biomaterials, 2006, 27(3): 335-345.
11.	Richardson SM, Hoyland JA. Stem cell regeneration of degenerated intervertebral discs: current status. Curr Pain Headache Rep, 2008, 12(2): 83-88.
12.	Noël D, Gazit D, Bouquet C, et al. Short-term BMP-2 expression is sufficient for in vivo osteochondral differentiation of mesenchymal stem cells. Stem Cells, 2004, 22(1): 74-85.
13.	Yang M, Ma QJ, Dang GT, et al. In vitro and in vivo induction of bone formation based on ex vivo gene therapy using rat adipose-derived adult. stem cells expressing BMP-7. Cytotherapy, 2005, 7(3): 273-281.
14.	Li X, Lee JP, Balian G, et al. Modulation of chondrocytic properties of fat-derived mesenchymal cells in co-cultures with nucleus pulposus. Connect Tissue Res, 2005, 46(2): 75-82.
15.	Richardson SM, Walker RV, Parker S, et al. Intervertebral disc cell-mediated mesenchymal stem cell differentiation. Stem Cells, 2006, 24(3): 707-716.
16.	Leung VY, Chan D, Cheung KM. Regeneration of intervertebral disc by mesenchymal stem cells: potentials, limitations, and future direction. Eur Spine J, 2006, 15 Suppl 3: S406-413.
17.	Strassburg S, Richardson SM, Freemont AJ, et al. Co-culture induces mesenchymal stem cell differentiation and modulation of the degenerate human nucleus pulposus cell phenotype. Regen Med, 2010, 5(5): 701-711.
18.	Pattison ST, Melrose J, Ghosh P, et al. Regulation of gelatinase-A (MMP-2) production by ovine intervertebral disc nucleus pulposus cells grown in alginate bead culture by transforming growth factor-β1 and insulin like growth factor-I. Cell Biol Int, 2001, 25(7): 679-689.
19.	Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining mμltipotent mesenchymal stromal cells. The International Society for Cellμlar Therapy position statement. Cytotherapy, 2006, 8(4): 315-317.
20.	Horwitz EM, Le Blanc K, Dominici M, et al. Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy, 2005, 7(5): 393-395.

1. Zhang Y, An HS, Tannoury C, et al. Biological treatment for degenerative disc disease: implications for the field of physical medicine and rehabilitation. Am J Phys Med Rehabil, 2008, 87(9): 694-702.
2. Yang X, Li X. Nucleus pulposus tissue engineering: a brief review. Eur Spine J , 2009, 18(11): 1564-1572.
3. Cappello R, Bird JL, Pfeiffer D, et al. Notochordal cell produce and assemble extracellular matrix in a distinct manner, which may be tesponsible for the maintenance of healthy nucleus pulposus. Spine (Phila Pa 1976), 2006, 31(8): 873-883.
4. Leung VY, Chan D, Cheung KM. Regeneration of intervertebral disc by mesenchymal stem cells: potentials, limitations, and future direction. Eur Spine J, 2006, 15 Suppl 3: S406-413.
5. Iwashina T, Mochida J, Sskai D, et al. Feasibility of using a human nucleus pulposus cell line as a cell source in cell transplantation therapy for intervertebral disc degeneration. Spine (Phila Pa 1976), 2006, 31(11): 1177-1186.
6. Strassburg S, Richardson SM, Freemont AJ, et al. Co-culture induces mesenchymal stem cell differentiation and modulation of the degenerate human nucleus pulposus cell phenotype. Regen Med, 2010, 5(5): 701-711.
7. Risbud MV, Albert TJ, Guttapalli A, et al. Differentiation of mesenchymal stem cells towards a nucleus pulposus-like phenotype in vitro: implications for cell-based transplantation therapy. Spine (Phila Pa 1976), 2004, 29(23): 2627-2632.
8. Steck E, Bettram H, Abel R, et al. Induction of intervertebral disc-like cells from adult mesenchymal stem cells. Stem Cells, 2005, 23(3): 403-411.
9. Luo W, Xiong W, Qiu M, et al. Differentiation of mesenchymal stem cells towards a nucleus pulposus-like phenotype utilizing simulated microgravity In vitro. J Huazhong Univ Sci Technolog Med Sci, 2011, 31(2): 199-203.
10. Sakai D, Mochida J, Iwashina T, et al. Regenerative effects of transplanting mesenchymal stem cells embedded in atelocollagen to the degenerated intervertebral disc. Biomaterials, 2006, 27(3): 335-345.
11. Richardson SM, Hoyland JA. Stem cell regeneration of degenerated intervertebral discs: current status. Curr Pain Headache Rep, 2008, 12(2): 83-88.
12. Noël D, Gazit D, Bouquet C, et al. Short-term BMP-2 expression is sufficient for in vivo osteochondral differentiation of mesenchymal stem cells. Stem Cells, 2004, 22(1): 74-85.
13. Yang M, Ma QJ, Dang GT, et al. In vitro and in vivo induction of bone formation based on ex vivo gene therapy using rat adipose-derived adult. stem cells expressing BMP-7. Cytotherapy, 2005, 7(3): 273-281.
14. Li X, Lee JP, Balian G, et al. Modulation of chondrocytic properties of fat-derived mesenchymal cells in co-cultures with nucleus pulposus. Connect Tissue Res, 2005, 46(2): 75-82.
15. Richardson SM, Walker RV, Parker S, et al. Intervertebral disc cell-mediated mesenchymal stem cell differentiation. Stem Cells, 2006, 24(3): 707-716.
16. Leung VY, Chan D, Cheung KM. Regeneration of intervertebral disc by mesenchymal stem cells: potentials, limitations, and future direction. Eur Spine J, 2006, 15 Suppl 3: S406-413.
17. Strassburg S, Richardson SM, Freemont AJ, et al. Co-culture induces mesenchymal stem cell differentiation and modulation of the degenerate human nucleus pulposus cell phenotype. Regen Med, 2010, 5(5): 701-711.
18. Pattison ST, Melrose J, Ghosh P, et al. Regulation of gelatinase-A (MMP-2) production by ovine intervertebral disc nucleus pulposus cells grown in alginate bead culture by transforming growth factor-β1 and insulin like growth factor-I. Cell Biol Int, 2001, 25(7): 679-689.
19. Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining mμltipotent mesenchymal stromal cells. The International Society for Cellμlar Therapy position statement. Cytotherapy, 2006, 8(4): 315-317.
20. Horwitz EM, Le Blanc K, Dominici M, et al. Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy, 2005, 7(5): 393-395.

《中國修復重建外科雜志》

非接觸性共培養BMSCs和髓核細胞時間差異性的實驗研究

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《中國修復重建外科雜志》

非接觸性共培養BMSCs和髓核細胞時間差異性的實驗研究

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

Format

Content

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