蔡震 1 , 潘博 2 , 林琳 2 , 蔣海越 2 , 莊洪興 2 , 游曉波 1 , 傅榮 1
  • 1 四川省醫(yī)學(xué)科學(xué)院·四川省人民醫(yī)院整形外科（成都，610072）;;
  • 2 中國(guó)醫(yī)學(xué)科學(xué)院 北京協(xié)和醫(yī)學(xué)院整形外科醫(yī)院整形七科;

目的 探討殘耳軟骨細(xì)胞能否在體外模擬軟骨誘導(dǎo)微環(huán)境，促進(jìn)脂肪來源干細(xì)胞（adipose derived stem cells，ADSCs）向軟骨分化并形成軟骨樣組織。方法 取外耳再造術(shù)中廢棄的先天性小耳畸形患者殘耳軟骨組織與皮下脂肪組織分離培養(yǎng)，分別收集第2代殘耳軟骨細(xì)胞與第3代ADSCs，以3∶7比例混合培養(yǎng)作為實(shí)驗(yàn)組（A組），單純殘耳軟骨細(xì)胞和單純ADSCs作為對(duì)照（分別為B組和C組）。取各組細(xì)胞1.0 × 106個(gè)離心培養(yǎng)獲得細(xì)胞球后，體外培養(yǎng)28 d，大體觀察各組細(xì)胞球取材時(shí)的外觀變化，測(cè)量濕重；阿利辛藍(lán)比色法檢測(cè)糖胺多糖（glycosaminoglycan，GAG）含量；RT-PCR檢測(cè)各組標(biāo)本的Ⅱ型膠原、蛋白聚糖（Aggrecan）mRNA表達(dá)；并行HE、甲苯胺藍(lán)、番紅O組織學(xué)觀察及Ⅱ型膠原免疫組織化學(xué)檢測(cè)。結(jié)果 體外培養(yǎng)28 d后，A、B組標(biāo)本形成白色半透明圓盤狀組織塊，表面光滑，彈性可；C組標(biāo)本組織塊有明顯收縮，呈黃色球狀，無彈性。A、B組標(biāo)本濕重及GAG含量顯著高于C組（P  lt; 0.05）；A、B組間差異無統(tǒng)計(jì)學(xué)意義（t=1.820 3，P=0.068 7；t=1.861 4，P=0.062 7）。RT-PCR檢測(cè)示A、B組標(biāo)本均可見Ⅱ型膠原與Aggrecan mRNA條帶清晰表達(dá)，C組未見明顯表達(dá)；A、B組Ⅱ型膠原與Aggrecan mRNA表達(dá)均顯著高于C組（P  lt; 0.05），A、B組間差異無統(tǒng)計(jì)學(xué)意義（t=1.457 6，P=0.144 9；t=1.519 5，P=0.128 6）。組織學(xué)觀察示：A組與B組細(xì)胞球標(biāo)本形成了大量軟骨陷窩樣結(jié)構(gòu)，細(xì)胞外基質(zhì)均有不同程度染色；C組細(xì)胞球標(biāo)本組織內(nèi)主要為纖維性成分，未見軟骨陷窩，細(xì)胞外基質(zhì)染色陰性。A、B組可見在軟骨陷窩周圍有不同程度棕黃色沉淀即Ⅱ型膠原表達(dá)，C組未見明顯表達(dá)；A、B組灰度值顯著低于C組（P  lt; 0.01），A、B組間差異無統(tǒng)計(jì)學(xué)意義（t=1.661 5，P=0.097 0）。結(jié)論 殘耳軟骨細(xì)胞可在體外獨(dú)立模擬軟骨誘導(dǎo)微環(huán)境，促進(jìn)ADSCs軟骨定向分化并形成軟骨組織。

引用本文： 蔡震,潘博,林琳,蔣海越,莊洪興,游曉波,傅榮. 殘耳軟骨細(xì)胞誘導(dǎo)脂肪來源干細(xì)胞體外軟骨形成實(shí)驗(yàn)研究. 中國(guó)修復(fù)重建外科雜志, 2013, 27(1): 83-88. doi: 復(fù)制

1. 蔡震, 潘博, 林琳, 等. 先天性小耳畸形殘耳軟骨細(xì)胞的體外生物學(xué)行為的實(shí)驗(yàn)研究. 中國(guó)美容整形外科雜志, 2010, 21(9): 553-556.
2. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng, 2001, 7(2): 211-226.
3. Awad HA, Wickham MQ, Leddy HA, et al. Chondrogenic differentiation of adipose-derived adult stem cells in agarose, alginate, and gelatin scaffolds. Biomaterials, 2004, 25(16): 3211-3222.
4. Hennig T, Lorenz H, Thiel A. Reduced chondrogenic potential of adipose tissue derived stromal cells correlates with an altered TGFbeta receptor and BMP profile and is overcome by BMP-6. J Cell Physiol, 2007, 211(3): 682-691.
5. Wei Y, Hu Y, Lv R, et al. Regulation of adipose-derived adult stem cells differentiating into chondrocytes with the use of rhBMP-2. Cytotherapy, 2006, 8(6): 570-579.
6. Estes BT, Wu AW, Guilak F. Potent induction of chondrocytic differentiation of human adipose-derived adult stem cells by bone morphogenetic protein 6. Arthritis Rheum, 2006, 54(4): 1222-1232.
7. 蔡震, 潘博, 林琳, 等. 脂肪來源干細(xì)胞的生物學(xué)特性及細(xì)胞表型. 中國(guó)組織工程研究與臨床康復(fù), 2010, 14(3): 6685-6688.
8. Denker AE, Nicoll SB, Tuan RS. Formation of cartilage-like spheroids by micromass cultures of murine C3H10T1/2 cells upon treatment with transforming growth factor-beta 1. Differentiation, 1995, 59(1): 25-34.
9. Lv X, Zhou G, Liu X, et al. Chondrogenesis by co-culture of adipose-derived stromal cells and chondrocytes in vitro. Connect Tissue Res, 2012, 24(6): 2-6.
10. Heng BC, Liu H, Cao T. Potential benefits of co-transplanting autologous adult stem cells together with human embryonic stem cells or their differentiated derivatives. Ann Clin Lab Sci, 2005, 35(1): 3-6.
11. Heng BC, Cao T, Lee EH. Directing stem cell differentiation into the chondrogenic lineage in vitro. Stem Cells, 2004, 22(7): 1152-1167.
12. Spalazzi JP, Dionisio KL, Jiang J, et al. Osteoblast and chondrocyte interactions during coculture on scaffolds. IEEE Eng Med Biol Mag, 2003, 22(5): 27-34.
13. 周廣東, 苗春雷, 王曉云, 等. 軟骨細(xì)胞與骨髓基質(zhì)細(xì)胞共培養(yǎng)體外軟骨形成的實(shí)驗(yàn)研究. 中華醫(yī)學(xué)雜志, 2004, 84(20): 1716-1720.
14. 苗春雷, 周廣東, 劉天一, 等. 軟骨細(xì)胞與骨髓基質(zhì)細(xì)胞共培養(yǎng)體外構(gòu)建軟骨的初步研究. 上海第二醫(yī)科大學(xué)學(xué)報(bào), 2004, 24(4): 246-249.
15. Dunker N, Schmitt K, Krieglstein K, et al. TGF-beta is required for programmed cell death in interdigital webs of the developing mouse limb. Mech Dev, 2002, 113(2): 111-120.
16. Grimaud E, Heymann D, Redini F, et al. Recent advances in TGF-beta effects on chondrocyte metabolism. Potential therapeutic roles of TGF-beta in cartilage disorders. Cytokine Growth Factor Rev, 2002, 13(3): 241-257.
17. Horner A, Kemp P, Summers C, et al. Expression and distribution of transforming growth factor-beta isoforms and their signaling receptors in growing human bone. Bone, 1998, 23(2): 95-102.
18. Goessler UR, Hormann K, Riedel F, et al. Tissue engineering with chondrocytes and function of the extracellular matrix (Review). Int J Mol Med, 2004, 13(4): 505-513.
19. Xu M, Wani M, Dai YS, et al. Differentiation of bone marrow stromal cells into the cardiac phenotype requires intercellular communication with myocytes. Circulation, 2004, 110(17): 2658-2665.
20. Andersson H, van den Berg A. Microfabrication and microfluidics for tissue engineering: state of the art and future opportunities. Lab Chip, 2004, 4(2): 98-103.
  1. 1. 蔡震, 潘博, 林琳, 等. 先天性小耳畸形殘耳軟骨細(xì)胞的體外生物學(xué)行為的實(shí)驗(yàn)研究. 中國(guó)美容整形外科雜志, 2010, 21(9): 553-556.
  2. 2. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng, 2001, 7(2): 211-226.
  3. 3. Awad HA, Wickham MQ, Leddy HA, et al. Chondrogenic differentiation of adipose-derived adult stem cells in agarose, alginate, and gelatin scaffolds. Biomaterials, 2004, 25(16): 3211-3222.
  4. 4. Hennig T, Lorenz H, Thiel A. Reduced chondrogenic potential of adipose tissue derived stromal cells correlates with an altered TGFbeta receptor and BMP profile and is overcome by BMP-6. J Cell Physiol, 2007, 211(3): 682-691.
  5. 5. Wei Y, Hu Y, Lv R, et al. Regulation of adipose-derived adult stem cells differentiating into chondrocytes with the use of rhBMP-2. Cytotherapy, 2006, 8(6): 570-579.
  6. 6. Estes BT, Wu AW, Guilak F. Potent induction of chondrocytic differentiation of human adipose-derived adult stem cells by bone morphogenetic protein 6. Arthritis Rheum, 2006, 54(4): 1222-1232.
  7. 7. 蔡震, 潘博, 林琳, 等. 脂肪來源干細(xì)胞的生物學(xué)特性及細(xì)胞表型. 中國(guó)組織工程研究與臨床康復(fù), 2010, 14(3): 6685-6688.
  8. 8. Denker AE, Nicoll SB, Tuan RS. Formation of cartilage-like spheroids by micromass cultures of murine C3H10T1/2 cells upon treatment with transforming growth factor-beta 1. Differentiation, 1995, 59(1): 25-34.
  9. 9. Lv X, Zhou G, Liu X, et al. Chondrogenesis by co-culture of adipose-derived stromal cells and chondrocytes in vitro. Connect Tissue Res, 2012, 24(6): 2-6.
  10. 10. Heng BC, Liu H, Cao T. Potential benefits of co-transplanting autologous adult stem cells together with human embryonic stem cells or their differentiated derivatives. Ann Clin Lab Sci, 2005, 35(1): 3-6.
  11. 11. Heng BC, Cao T, Lee EH. Directing stem cell differentiation into the chondrogenic lineage in vitro. Stem Cells, 2004, 22(7): 1152-1167.
  12. 12. Spalazzi JP, Dionisio KL, Jiang J, et al. Osteoblast and chondrocyte interactions during coculture on scaffolds. IEEE Eng Med Biol Mag, 2003, 22(5): 27-34.
  13. 13. 周廣東, 苗春雷, 王曉云, 等. 軟骨細(xì)胞與骨髓基質(zhì)細(xì)胞共培養(yǎng)體外軟骨形成的實(shí)驗(yàn)研究. 中華醫(yī)學(xué)雜志, 2004, 84(20): 1716-1720.
  14. 14. 苗春雷, 周廣東, 劉天一, 等. 軟骨細(xì)胞與骨髓基質(zhì)細(xì)胞共培養(yǎng)體外構(gòu)建軟骨的初步研究. 上海第二醫(yī)科大學(xué)學(xué)報(bào), 2004, 24(4): 246-249.
  15. 15. Dunker N, Schmitt K, Krieglstein K, et al. TGF-beta is required for programmed cell death in interdigital webs of the developing mouse limb. Mech Dev, 2002, 113(2): 111-120.
  16. 16. Grimaud E, Heymann D, Redini F, et al. Recent advances in TGF-beta effects on chondrocyte metabolism. Potential therapeutic roles of TGF-beta in cartilage disorders. Cytokine Growth Factor Rev, 2002, 13(3): 241-257.
  17. 17. Horner A, Kemp P, Summers C, et al. Expression and distribution of transforming growth factor-beta isoforms and their signaling receptors in growing human bone. Bone, 1998, 23(2): 95-102.
  18. 18. Goessler UR, Hormann K, Riedel F, et al. Tissue engineering with chondrocytes and function of the extracellular matrix (Review). Int J Mol Med, 2004, 13(4): 505-513.
  19. 19. Xu M, Wani M, Dai YS, et al. Differentiation of bone marrow stromal cells into the cardiac phenotype requires intercellular communication with myocytes. Circulation, 2004, 110(17): 2658-2665.
  20. 20. Andersson H, van den Berg A. Microfabrication and microfluidics for tissue engineering: state of the art and future opportunities. Lab Chip, 2004, 4(2): 98-103.