• 福建醫(yī)科大學(xué)附屬第一醫(yī)院（福州，350004） 1 骨科，2中心實(shí)驗(yàn)室;

目的 探討在非接觸性共培養(yǎng)環(huán)境下，BMSCs定向分化為類(lèi)髓核細(xì)胞在共培養(yǎng)時(shí)間上的差異性，尋找適合體內(nèi)移植的最佳時(shí)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(pán)髓核，分離、培養(yǎng)BMSCs和髓核細(xì)胞并進(jìn)行免疫細(xì)胞化學(xué)鑒定。取原代髓核細(xì)胞和第2代生長(zhǎng)良好的BMSCs體外建立非接觸性共培養(yǎng)模型。觀(guān)察共培養(yǎng)后第1、3、5代BMSCs的形態(tài)學(xué)變化并繪制生長(zhǎng)曲線(xiàn)；RT-PCR檢測(cè)共培養(yǎng)5、10、15 d BMSCsⅡ型膠原和蛋白聚糖mRNA表達(dá)；Western blot檢測(cè)共培養(yǎng)5、10、15、20、25、30 d BMSCs Ⅱ型膠原和蛋白聚糖蛋白的表達(dá)。 結(jié)果BMSCs相對(duì)特異性標(biāo)記物CD44、CD90表達(dá)陽(yáng)性，造血細(xì)胞表面標(biāo)記物CD34、CD45表達(dá)陰性。髓核細(xì)胞Ⅱ型膠原、蛋白聚糖表達(dá)陽(yáng)性。共培養(yǎng)后2周BMSCs形態(tài)發(fā)生明顯變化，呈多角形、不規(guī)則形；共培養(yǎng)后3代內(nèi)，BMSCs生長(zhǎng)速度無(wú)明顯差異，隨著傳代次數(shù)增加，細(xì)胞增殖明顯減慢。RT-PCR檢測(cè)示共培養(yǎng)后10、15 d BMSCs蛋白聚糖和Ⅱ型膠原mRNA表達(dá)明顯高于5 d時(shí)（P  lt; 0.05），而10 d與15 d時(shí)差異無(wú)統(tǒng)計(jì)學(xué)意義（P  gt; 0.05）。Western blot檢測(cè)示共培養(yǎng)后隨時(shí)間延長(zhǎng)細(xì)胞表達(dá)Ⅱ型膠原和蛋白聚糖蛋白逐漸增加，5、10、15 d間差異有統(tǒng)計(jì)學(xué)意義（P  lt; 0.05），15 d后各時(shí)間點(diǎn)間比較差異無(wú)統(tǒng)計(jì)學(xué)意義（P  gt; 0.05）。 結(jié)論在非接觸性共培養(yǎng)環(huán)境下，BMSCs在髓核細(xì)胞誘導(dǎo)下可向類(lèi)髓核細(xì)胞分化，表達(dá)Ⅱ型膠原和蛋白聚糖，在共培養(yǎng)15 d時(shí)達(dá)到相對(duì)穩(wěn)定，此時(shí)較適合進(jìn)行體內(nèi)移植。

引用本文： 陳雷,胡秀年,傅冷西,葉君健. 非接觸性共培養(yǎng)BMSCs和髓核細(xì)胞時(shí)間差異性的實(shí)驗(yàn)研究. 中國(guó)修復(fù)重建外科雜志, 2012, 26(11): 1369-1374. doi: 復(fù)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. 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. 2. Yang X, Li X. Nucleus pulposus tissue engineering: a brief review. Eur Spine J , 2009, 18(11): 1564-1572.
  3. 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. 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. 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. 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. 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. 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. 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. 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. 11. Richardson SM, Hoyland JA. Stem cell regeneration of degenerated intervertebral discs: current status. Curr Pain Headache Rep, 2008, 12(2): 83-88.
  12. 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. 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. 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. 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. 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. 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. 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. 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. 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.