細胞共培養法誘導兔脂肪來源干細胞向軟骨細胞分化的實驗研究_《中國修復重建外科雜志》

作者：

許海委 ¹ , 徐寶山 ¹ , 楊強 ¹ , 李秀蘭 ² , 張楊 ² , 張春秋 ³ , 伍耀宏 ¹ , 崔麗 ²

天津醫院（天津，300211）1脊柱外科，2骨科研究所細胞工程室;;
3天津理工大學機械工程學院;

關鍵詞：

脂肪來源干細胞軟骨細胞共培養誘導分化兔

DOI：

10.7507/1002-1892.20130043

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目的利用軟骨細胞與脂肪來源干細胞（adipose-derived stem cells，ADSCs）共培養，觀察ADSCs是否能向軟骨細胞定向分化。方法4月齡健康新西蘭大白兔8只，雌雄不限，體重2.2～2.7 kg，分離、培養兔軟骨細胞和ADSCs，取第2代細胞用于實驗。將實驗分為兩組，實驗組各取將1 mL細胞密度為2 × 104個/mL的軟骨細胞和ADSCs分別接種至Transwell小室6孔板上、下層共同培養，對照組單獨培養ADSCs。倒置相差顯微鏡觀察兩組ADSCs形態變化；培養14 d后行甲苯胺藍染色、Ⅱ型膠原免疫組織化學染色，實時熒光定量PCR檢測兩組ADSCs Ⅱ型膠原、蛋白聚糖、SOX9 mRNA表達。結果倒置相差顯微鏡觀察示，隨培養時間延長，實驗組ADSCs逐漸變為軟骨樣細胞形態，呈圓形；對照組ADSCs仍以梭形為主，呈束狀或漩渦狀生長。培養14 d，實驗組甲苯胺藍染色、Ⅱ型膠原免疫組織化學染色均為陽性；對照組均為陰性；實時熒光定量PCR檢測示，實驗組Ⅱ型膠原、蛋白聚糖、SOX9 mRNA相對表達量分別為1.43 ± 0.07、2.13 ± 0.08、1.08 ± 0.08，顯著高于對照組（0.04 ± 0.03、0.13 ± 0.04、0.10 ± 0.02），差異均有統計學意義（P lt; 0.05）。結論通過與軟骨細胞共同培養，ADSCs可定向分化為軟骨細胞。

引用本文： 許海委,徐寶山,楊強,李秀蘭,張楊,張春秋,伍耀宏,崔麗. 細胞共培養法誘導兔脂肪來源干細胞向軟骨細胞分化的實驗研究. 中國修復重建外科雜志, 2013, 27(2): 193-198. doi: 10.7507/1002-1892.20130043 復制

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2.	Khan WS, Tew SR, Adesida AB, et al. Human infrapatellar fat pad-derived stem cells express the pericyte marker 3G5 and show enhanced chondrogenesis after expansion in fibroblast growth factor-2. Arthritis Res Ther, 2008, 10(4): R74.
3.	Merceron C, Vinatier C, Portron S, et al. Differential effects of hypoxia on osteochondrogenic potential of human adipose-derived stem cells. Am J Physiol Cell Physiol, 2010, 298(2): C355-364.
4.	Hamid AA, Idrus RB, Saim AB, et al. Characterization of human adipose-derived stem cells and expression of chondrogenic genes during induction of cartilage differentiation. Clinics (Sao Paulo), 2012, 67(2): 99-106.
5.	Diekman BO, Rowland CR, Lennon DP, et al. Chondrogenesis of adult stem cells from adipose tissue and bone marrow: induction by growth factors and cartilage-derived matrix. Tissue Eng Part A, 2010, 16(2): 523-533.
6.	Li JW, Guo XL, He CL, et al. In vitro chondrogenesis of the goat bone marrow mesenchymal stem cells directed by chondrocytes in monolayer and 3-dimetional indirect co-culture system. Chin Med J (Engl), 2011, 124(19): 3080-3086.
7.	Fan J, Gong Y, Ren L, et al. In vitro engineered cartilage using synovium-derived mesenchymal stem cells with injectable gellan hydrogels. Acta Biomater, 2010, 6(3): 1178-1185.
8.	Jiang J, Li J, Hao X, et al. Use of autologous chondrocytes and bioinert perforated chambers to tissue engineer cartilage in vivo. J Surg Res, 2012, 173(1): e27-32.
9.	Vasiliadis HS, Lindahl A, Georgoulis AD, et al. Malalignment and cartilage lesions in the patellofemoral joint treated with autologous chondrocyte implantation. Knee Surg Sports Traumatol Arthrosc, 2011, 19(3): 452-457.
10.	Dai J, Wang X, Shen G. Cotransplantation of autologous bone marrow stromal cells and chondrocytes as a novel therapy for reconstruction of condylar cartilage. Med Hypotheses, 2011, 77(1): 132-133.
11.	楊強, 彭江, 盧世璧, 等. 軟骨脫細胞基質多孔支架與骨髓基質干細胞體外構建組織工程軟骨的研究. 中華醫學雜志, 2011, 91(17): 1161-1166.
12.	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.
13.	聶緒強, 陳懷紅, 唐寧, 等. 脂肪源性干細胞研究及其應用進展. 中國修復重建外科雜志, 2011, 25(7): 854-858.
14.	Hildner F, Albrecht C, Gabriel C, et al. State of the art and future perspectives of articular cartilage regeneration: a focus on adipose-derived stem cells and platelet-derived products. J Tissue Eng Regen Med, 2011, 5(4): e36-51.
15.	張明, 周哲, 周娟, 等. 人脂肪干細胞與豬尿路上皮細胞隔離共培養后向尿路上皮樣細胞分化. 中華臨床醫師雜志(電子版), 2012, 6(8): 109-112.
16.	Chao KC, Chao KF, Fu YS, et al. Islet-like clusters derived from mesenchymal stem cells in Wharton’s Jelly of the human umbilical cord for transplantation to control type 1 diabetes. PLoS One, 2008, 3(1): e1451.
17.	Li TZ, Kim JH, Cho HH, et al. Therapeutic potential of bone-marrow-derived mesenchymal stem cells differentiated with growth-factor-free coculture method in liver-injured rats. Tissue Eng Part A, 2010, 16(8): 2649-2659.
18.	于鵬. 兔骨髓間充質干細胞與髓核細胞共培養體系的實驗研究. 武漢: 華中科技大學, 2010.
19.	徐志剛. 脂肪間充質干細胞與自體髓核細胞共培養中相互作用分子機制初步研究. 武漢: 華中科技大學, 2010.
20.	張潔, 蔣海越, 何樂仁, 等. 殘耳軟骨細胞與脂肪干細胞共培養體內構建軟骨的實驗研究. 組織工程與重建外科雜志, 2011, 7(2): 75-79.

1. Feng G, Wan Y, Balian G, et al. Adenovirus-mediated expression of growth and differentiation factor-5 promotes chondrogenesis of adipose stem cells. Growth Factors, 2008, 26(3): 132-142.
2. Khan WS, Tew SR, Adesida AB, et al. Human infrapatellar fat pad-derived stem cells express the pericyte marker 3G5 and show enhanced chondrogenesis after expansion in fibroblast growth factor-2. Arthritis Res Ther, 2008, 10(4): R74.
3. Merceron C, Vinatier C, Portron S, et al. Differential effects of hypoxia on osteochondrogenic potential of human adipose-derived stem cells. Am J Physiol Cell Physiol, 2010, 298(2): C355-364.
4. Hamid AA, Idrus RB, Saim AB, et al. Characterization of human adipose-derived stem cells and expression of chondrogenic genes during induction of cartilage differentiation. Clinics (Sao Paulo), 2012, 67(2): 99-106.
5. Diekman BO, Rowland CR, Lennon DP, et al. Chondrogenesis of adult stem cells from adipose tissue and bone marrow: induction by growth factors and cartilage-derived matrix. Tissue Eng Part A, 2010, 16(2): 523-533.
6. Li JW, Guo XL, He CL, et al. In vitro chondrogenesis of the goat bone marrow mesenchymal stem cells directed by chondrocytes in monolayer and 3-dimetional indirect co-culture system. Chin Med J (Engl), 2011, 124(19): 3080-3086.
7. Fan J, Gong Y, Ren L, et al. In vitro engineered cartilage using synovium-derived mesenchymal stem cells with injectable gellan hydrogels. Acta Biomater, 2010, 6(3): 1178-1185.
8. Jiang J, Li J, Hao X, et al. Use of autologous chondrocytes and bioinert perforated chambers to tissue engineer cartilage in vivo. J Surg Res, 2012, 173(1): e27-32.
9. Vasiliadis HS, Lindahl A, Georgoulis AD, et al. Malalignment and cartilage lesions in the patellofemoral joint treated with autologous chondrocyte implantation. Knee Surg Sports Traumatol Arthrosc, 2011, 19(3): 452-457.
10. Dai J, Wang X, Shen G. Cotransplantation of autologous bone marrow stromal cells and chondrocytes as a novel therapy for reconstruction of condylar cartilage. Med Hypotheses, 2011, 77(1): 132-133.
11. 楊強, 彭江, 盧世璧, 等. 軟骨脫細胞基質多孔支架與骨髓基質干細胞體外構建組織工程軟骨的研究. 中華醫學雜志, 2011, 91(17): 1161-1166.
12. 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.
13. 聶緒強, 陳懷紅, 唐寧, 等. 脂肪源性干細胞研究及其應用進展. 中國修復重建外科雜志, 2011, 25(7): 854-858.
14. Hildner F, Albrecht C, Gabriel C, et al. State of the art and future perspectives of articular cartilage regeneration: a focus on adipose-derived stem cells and platelet-derived products. J Tissue Eng Regen Med, 2011, 5(4): e36-51.
15. 張明, 周哲, 周娟, 等. 人脂肪干細胞與豬尿路上皮細胞隔離共培養后向尿路上皮樣細胞分化. 中華臨床醫師雜志(電子版), 2012, 6(8): 109-112.
16. Chao KC, Chao KF, Fu YS, et al. Islet-like clusters derived from mesenchymal stem cells in Wharton’s Jelly of the human umbilical cord for transplantation to control type 1 diabetes. PLoS One, 2008, 3(1): e1451.
17. Li TZ, Kim JH, Cho HH, et al. Therapeutic potential of bone-marrow-derived mesenchymal stem cells differentiated with growth-factor-free coculture method in liver-injured rats. Tissue Eng Part A, 2010, 16(8): 2649-2659.
18. 于鵬. 兔骨髓間充質干細胞與髓核細胞共培養體系的實驗研究. 武漢: 華中科技大學, 2010.
19. 徐志剛. 脂肪間充質干細胞與自體髓核細胞共培養中相互作用分子機制初步研究. 武漢: 華中科技大學, 2010.
20. 張潔, 蔣海越, 何樂仁, 等. 殘耳軟骨細胞與脂肪干細胞共培養體內構建軟骨的實驗研究. 組織工程與重建外科雜志, 2011, 7(2): 75-79.

《中國修復重建外科雜志》

細胞共培養法誘導兔脂肪來源干細胞向軟骨細胞分化的實驗研究

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《中國修復重建外科雜志》

細胞共培養法誘導兔脂肪來源干細胞向軟骨細胞分化的實驗研究

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