殘耳軟骨細胞誘導脂肪來源干細胞體外軟骨形成實驗研究_《中國修復重建外科雜志》

作者：

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 四川省醫學科學院·四川省人民醫院整形外科（成都，610072）;;
2 中國醫學科學院北京協和醫學院整形外科醫院整形七科;

關鍵詞：

殘耳軟骨細胞脂肪來源干細胞細胞球共培養

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

目的探討殘耳軟骨細胞能否在體外模擬軟骨誘導微環境，促進脂肪來源干細胞（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，分別收集第2代殘耳軟骨細胞與第3代ADSCs，以3∶7比例混合培養作為實驗組（A組），單純殘耳軟骨細胞和單純ADSCs作為對照（分別為B組和C組）。取各組細胞1.0 × 106個離心培養獲得細胞球后，體外培養28 d，大體觀察各組細胞球取材時的外觀變化，測量濕重；阿利辛藍比色法檢測糖胺多糖（glycosaminoglycan，GAG）含量；RT-PCR檢測各組標本的Ⅱ型膠原、蛋白聚糖（Aggrecan）mRNA表達；并行HE、甲苯胺藍、番紅O組織學觀察及Ⅱ型膠原免疫組織化學檢測。結果體外培養28 d后，A、B組標本形成白色半透明圓盤狀組織塊，表面光滑，彈性可；C組標本組織塊有明顯收縮，呈黃色球狀，無彈性。A、B組標本濕重及GAG含量顯著高于C組（P lt; 0.05）；A、B組間差異無統計學意義（t=1.820 3，P=0.068 7；t=1.861 4，P=0.062 7）。RT-PCR檢測示A、B組標本均可見Ⅱ型膠原與Aggrecan mRNA條帶清晰表達，C組未見明顯表達；A、B組Ⅱ型膠原與Aggrecan mRNA表達均顯著高于C組（P lt; 0.05），A、B組間差異無統計學意義（t=1.457 6，P=0.144 9；t=1.519 5，P=0.128 6）。組織學觀察示：A組與B組細胞球標本形成了大量軟骨陷窩樣結構，細胞外基質均有不同程度染色；C組細胞球標本組織內主要為纖維性成分，未見軟骨陷窩，細胞外基質染色陰性。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，C組未見明顯表達；A、B組灰度值顯著低于C組（P lt; 0.01），A、B組間差異無統計學意義（t=1.661 5，P=0.097 0）。結論殘耳軟骨細胞可在體外獨立模擬軟骨誘導微環境，促進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. 殘耳軟骨細胞誘導脂肪來源干細胞體外軟骨形成實驗研究. 中國修復重建外科雜志, 2013, 27(1): 83-88. doi: 復制

1.	蔡震, 潘博, 林琳, 等. 先天性小耳畸形殘耳軟骨細胞的體外生物學行為的實驗研究. 中國美容整形外科雜志, 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. 脂肪來源干細胞的生物學特性及細胞表型. 中國組織工程研究與臨床康復, 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. 軟骨細胞與骨髓基質細胞共培養體外軟骨形成的實驗研究. 中華醫學雜志, 2004, 84(20): 1716-1720.
14.	苗春雷, 周廣東, 劉天一, 等. 軟骨細胞與骨髓基質細胞共培養體外構建軟骨的初步研究. 上海第二醫科大學學報, 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. 蔡震, 潘博, 林琳, 等. 先天性小耳畸形殘耳軟骨細胞的體外生物學行為的實驗研究. 中國美容整形外科雜志, 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. 脂肪來源干細胞的生物學特性及細胞表型. 中國組織工程研究與臨床康復, 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. 軟骨細胞與骨髓基質細胞共培養體外軟骨形成的實驗研究. 中華醫學雜志, 2004, 84(20): 1716-1720.
14. 苗春雷, 周廣東, 劉天一, 等. 軟骨細胞與骨髓基質細胞共培養體外構建軟骨的初步研究. 上海第二醫科大學學報, 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.

《中國修復重建外科雜志》

殘耳軟骨細胞誘導脂肪來源干細胞體外軟骨形成實驗研究

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

Format

Content

摘要全文圖表視頻參考文獻施引文獻補充材料