BMP-14轉染脂肪來源干細胞后與軟骨細胞共培養的實驗研究_《中國修復重建外科雜志》

作者：

袁虹 ¹ , 張解元 ² , 張榮明 ¹

遼寧醫學院附屬第一醫院（遼寧錦州，121001）1整形燒傷科，2骨關節外科;

關鍵詞：

軟骨組織工程脂肪來源干細胞 BMP-14 軟骨細胞共培養兔

DOI：

10.7507/1002-1892.20130080

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目的研究BMP-14與軟骨細胞共同誘導脂肪來源干細胞（adipose-derived stem cells，ADSCs）向軟骨細胞分化是否具有協同作用，優化軟骨組織工程種子細胞來源。方法取2只雄性新西蘭大白兔（體重1.5、2.0 kg）關節軟骨和皮下脂肪組織，分離、培養軟骨細胞和ADSCs，取第3代細胞進行實驗。對ADSCs行成骨（茜素紅染色）、成軟骨（阿利新藍染色）和成脂（油紅O染色）誘導鑒定。測定Ad-巨細胞病毒-BMP-14-內部核糖體進入位點-人源化海腎綠色熒光蛋白1轉染的最佳感染復數（multiplicity of infection，MOI）并以其轉染ADSCs。實驗分為5組：A組，采用Transwell小室將BMP-14轉染的ADSCs與軟骨細胞共培養（1∶1）；B組，采用Transwell小室將未轉染的ADSCs與軟骨細胞共培養（1∶1）；C組，單純BMP-14轉染的ADSCs培養；D組，單純軟骨細胞培養；E組，單純ADSCs培養。培養3周后，取細胞采用阿利新藍法檢測糖胺聚糖（glycosaminoglycan，GAG）含量，Western blot檢測BMP-14和Ⅱ型膠原蛋白表達，RT-PCR檢測Sox-9基因表達。結果培養細胞經鑒定提示為ADSCs。倒置熒光顯微鏡觀察示，MOI值為150時轉染效果最好。A、C組GAG含量、BMP-14和Ⅱ型膠原蛋白表達以及Sox-9基因表達均明顯高于其余3組，A組高于C組，差異均有統計學意義（P lt; 0.05）；B、D組均明顯高于E組（P lt; 0.05），B、D組間差異均無統計學意義（P gt; 0.05）。結論BMP-14與軟骨細胞能共同誘導并促進兔ADSCs向軟骨細胞分化，兩者之間具有協同作用。

引用本文： 袁虹,張解元,張榮明. BMP-14轉染脂肪來源干細胞后與軟骨細胞共培養的實驗研究. 中國修復重建外科雜志, 2013, 27(3): 353-357. doi: 10.7507/1002-1892.20130080 復制

1.	15 Wang C, Ruan DK, Zhang C, et al. Effects of adeno-associated virus-2-mediated human BMP-7 gene transfection on the phenotype of nucleus pulposus cells. J Orthop Res, 2011, 29(6): 838-845. 16 Jung MR, Shim IK, Chung HJ, et al. Local BMP-7 release from a PLGA scaffolding-matrix for the repair of osteochondral defects in rabbits. J Control Release, 2012, 162(3): 485-491.
2.	17 Coleman CM, Loredo GA, Lo CW, et al. Correlation of GDF5 and connexin 43 mRNA expression during embryonic development. Anat Rec A Discov Mol Cell Evol Biol, 2003, 275(2): 1117-1121. 18 Meech R, Edelman DB, Jones FS, et al. The homeobox transcription factor Barx2 regulates chondrogenesis during limb development. Development, 2005, 132(9): 2135-2146.
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6.	Dong SW, Ying DJ, Duan XJ, et al. Bone regeneration using an acellular extracellular matrix and bone marrow mesenchymal stem cells expressing Cbfa1. Biosci Biotechnol Biochem, 2009, 73(10): 2226-2233.
7.	Raghunath J, Salacinski HJ, Sales KM, et al. Advancing cartilage tissue engineering: the application of stem cell technology. Curr Opin Biotechnol, 2005, 16(5): 503-509.
8.	任宇, 吳海青, 馬玉珍, 等. 牛脂肪間充質干細胞的分離、培養與鑒定. 生物工程學報, 2010, 26(12): 1645-1651.
9.	劉振寧, 賈長青, 韓長旭, 等. 生長分化因子5誘導兔脂肪干細胞成軟骨細胞分化的實驗研究. 中國修復重建外科雜志, 2009, 23(4): 483-489.
10.	張勇, 趙建寧, 韓寧波. 脂肪干細胞與軟骨細胞的共培養. 中國組織工程研究與臨床康復, 2009, 13(24): 4632-4636.
11.	張解元, 袁虹, 李諶, 等. 腺病毒介導BMP-2聯合低氧誘導因子1α突變型與單基因BMP-2分別轉染BMSCs后成骨效應的比較研究. 中國修復重建外科雜志, 2012, 26(9): 1102-1106.
12.	孫磊, 綦惠, 陳磊, 等. 兔軟骨細胞與骨髓基質細胞的平面共培養: 兩者培養比例篩選. 中國組織工程研究與臨床康復, 2010, 14(27): 4955-4659.
13.	English A, Jones EA, Corscadden D, et al. A comparative assessment of cartilage and joint fat pad as a potential source of cells for autologous therapy development in knee osteoarthritis. Rheumatology (Oxford), 2007, 46(11): 1676-1683.
14.	Bajek A, Olkowska J, Drewa T. Mesenchymal stem cells as a therapeutic tool in tissue and organ regeneration. Postepy Hig Med Dosw (Online), 2011, 65: 124-132.
15.	袁啟龍, 曾曉勇, 陳亮, 等. 兔不同部位脂肪來源干細胞體外成肌誘導能力的比較. 中國修復重建外科雜志, 2010, 24(10): 1228-1232.
16.	鄧春華, 孫祥宙, 高勇, 等. 大鼠脂肪組織來源干細胞的分離、培養和生物學特性的研究. 中華男科學雜志, 2008, 14(2): 99-105.
17.	Dorman LJ, Tucci M, Benghuzzi H. In vitro effects of bmp-2, bmp-7, and bmp-13 on proliferation and differentation of mouse mesenchymal stem cells. Biomed Sci Instrum, 2012, 48: 81-87.
18.	Bobinac D, Spanjol J, Marinovi? M, et al. Expression of bone morphogenetic proteins, cartilage-derived morphogenetic proteins and related receptors in normal and osteoarthritic human articular cartilage. Coll Antropol, 2008, 32 Suppl 2: 83-87.
19.	Vats A, Bielby RC, Tolley N, et al. Chondrogenic differentiation of human embryonic stem cells: the effect of the micro-environment. Tissue Eng, 2006, 12(6): 1687-1697.
20.	Takagi M, Umetsu Y, Fujiwara M, et al. High inoculation cell density could accelerate the differentiation of human bone marrow mesenchymal stem cells to chondrocyte cells. J Biosci Bioeng, 2007, 103(1): 98-100.
21.	Hillel AT, Taube JM, Cornish TC, et al. Characterization of human mesenchymal stem cell-engineered cartilage: analysis of its ultrastructure, cell density and chondrocyte phenotype compared to native adult and fetal cartilage. Cells Tissues Organs, 2010, 191(1): 12-20.
22.	Erickson IE, Huang AH, Chung C, et al. Differential maturation and structure-function relationships in mesenchymal stem cell- and chondrocyte-seeded hydrogels. Tissue Eng Part A, 2009, 15(5): 1041-1052.
23.	Wang L, Lin F, Wu J, et al. High efficiency adenovirus-mediated expression of truncated N-terminal huntingtin fragment (htt552) in primary rat astrocytes. Acta Biochim Biophys Sin (Shanghai), 2009, 41(4): 325-334.
24.	范先群, 肖彩雯, 周慧芳, 等. 基因修飾的組織工程骨修復眶骨缺損的實驗研究. 中華眼科雜志, 2009, 45(1): 66-72.

1. 15 Wang C, Ruan DK, Zhang C, et al. Effects of adeno-associated virus-2-mediated human BMP-7 gene transfection on the phenotype of nucleus pulposus cells. J Orthop Res, 2011, 29(6): 838-845. 16 Jung MR, Shim IK, Chung HJ, et al. Local BMP-7 release from a PLGA scaffolding-matrix for the repair of osteochondral defects in rabbits. J Control Release, 2012, 162(3): 485-491.
2. 17 Coleman CM, Loredo GA, Lo CW, et al. Correlation of GDF5 and connexin 43 mRNA expression during embryonic development. Anat Rec A Discov Mol Cell Evol Biol, 2003, 275(2): 1117-1121. 18 Meech R, Edelman DB, Jones FS, et al. The homeobox transcription factor Barx2 regulates chondrogenesis during limb development. Development, 2005, 132(9): 2135-2146.
3.
4.
5. Khaled EG, Saleh M, Hindocha S, et al. Tissue engineering for bone production-stem cells, gene therapy and scaffolds. Open Orthop J, 2011, 5 Suppl 2: 289-295.
6. Dong SW, Ying DJ, Duan XJ, et al. Bone regeneration using an acellular extracellular matrix and bone marrow mesenchymal stem cells expressing Cbfa1. Biosci Biotechnol Biochem, 2009, 73(10): 2226-2233.
7. Raghunath J, Salacinski HJ, Sales KM, et al. Advancing cartilage tissue engineering: the application of stem cell technology. Curr Opin Biotechnol, 2005, 16(5): 503-509.
8. 任宇, 吳海青, 馬玉珍, 等. 牛脂肪間充質干細胞的分離、培養與鑒定. 生物工程學報, 2010, 26(12): 1645-1651.
9. 劉振寧, 賈長青, 韓長旭, 等. 生長分化因子5誘導兔脂肪干細胞成軟骨細胞分化的實驗研究. 中國修復重建外科雜志, 2009, 23(4): 483-489.
10. 張勇, 趙建寧, 韓寧波. 脂肪干細胞與軟骨細胞的共培養. 中國組織工程研究與臨床康復, 2009, 13(24): 4632-4636.
11. 張解元, 袁虹, 李諶, 等. 腺病毒介導BMP-2聯合低氧誘導因子1α突變型與單基因BMP-2分別轉染BMSCs后成骨效應的比較研究. 中國修復重建外科雜志, 2012, 26(9): 1102-1106.
12. 孫磊, 綦惠, 陳磊, 等. 兔軟骨細胞與骨髓基質細胞的平面共培養: 兩者培養比例篩選. 中國組織工程研究與臨床康復, 2010, 14(27): 4955-4659.
13. English A, Jones EA, Corscadden D, et al. A comparative assessment of cartilage and joint fat pad as a potential source of cells for autologous therapy development in knee osteoarthritis. Rheumatology (Oxford), 2007, 46(11): 1676-1683.
14. Bajek A, Olkowska J, Drewa T. Mesenchymal stem cells as a therapeutic tool in tissue and organ regeneration. Postepy Hig Med Dosw (Online), 2011, 65: 124-132.
15. 袁啟龍, 曾曉勇, 陳亮, 等. 兔不同部位脂肪來源干細胞體外成肌誘導能力的比較. 中國修復重建外科雜志, 2010, 24(10): 1228-1232.
16. 鄧春華, 孫祥宙, 高勇, 等. 大鼠脂肪組織來源干細胞的分離、培養和生物學特性的研究. 中華男科學雜志, 2008, 14(2): 99-105.
17. Dorman LJ, Tucci M, Benghuzzi H. In vitro effects of bmp-2, bmp-7, and bmp-13 on proliferation and differentation of mouse mesenchymal stem cells. Biomed Sci Instrum, 2012, 48: 81-87.
18. Bobinac D, Spanjol J, Marinovi? M, et al. Expression of bone morphogenetic proteins, cartilage-derived morphogenetic proteins and related receptors in normal and osteoarthritic human articular cartilage. Coll Antropol, 2008, 32 Suppl 2: 83-87.
19. Vats A, Bielby RC, Tolley N, et al. Chondrogenic differentiation of human embryonic stem cells: the effect of the micro-environment. Tissue Eng, 2006, 12(6): 1687-1697.
20. Takagi M, Umetsu Y, Fujiwara M, et al. High inoculation cell density could accelerate the differentiation of human bone marrow mesenchymal stem cells to chondrocyte cells. J Biosci Bioeng, 2007, 103(1): 98-100.
21. Hillel AT, Taube JM, Cornish TC, et al. Characterization of human mesenchymal stem cell-engineered cartilage: analysis of its ultrastructure, cell density and chondrocyte phenotype compared to native adult and fetal cartilage. Cells Tissues Organs, 2010, 191(1): 12-20.
22. Erickson IE, Huang AH, Chung C, et al. Differential maturation and structure-function relationships in mesenchymal stem cell- and chondrocyte-seeded hydrogels. Tissue Eng Part A, 2009, 15(5): 1041-1052.
23. Wang L, Lin F, Wu J, et al. High efficiency adenovirus-mediated expression of truncated N-terminal huntingtin fragment (htt552) in primary rat astrocytes. Acta Biochim Biophys Sin (Shanghai), 2009, 41(4): 325-334.
24. 范先群, 肖彩雯, 周慧芳, 等. 基因修飾的組織工程骨修復眶骨缺損的實驗研究. 中華眼科雜志, 2009, 45(1): 66-72.

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《中國修復重建外科雜志》

BMP-14轉染脂肪來源干細胞后與軟骨細胞共培養的實驗研究

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