CHONDROGENESIS OF BONE MARROW MESENCHYMAL STEM CELLS INDUCED BY TRANSFORMING GROWTH FACTOR β<sub>3</sub> GENE IN DIANNAN SMALL-EAR PIGS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WANGHuijian ,  LIYanlin , CHENJianming , WANGXin , ZHAOFengkai , CAOShuhai

Department of Sports Medicine, the First Affiliated Hospital of Kunming Medical University, Kunming Yunnan, 650032, P. R. China;

Corresponding?author：

LIYanlin, Email: 852387873@qq.com

Keywords：

Cartilage tissue engineering; Transforming group factor β₃; Bone marrow mesenchymal stem cells; Cartilage differentiation; Gene transfection; Diannan small-ear pig

DOI：

10.7507/1002-1892.20140034

Video：

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Objective To observe transforming growth factor β₃ (TGF-β₃) gene expression and the chondrogenesis of bone marrow mesenchymal stem cells (BMSCs) after TGF-β₃ gene is transfected into BMSCs of Diannan small-ear pig. Methods Recombinant adenovirus 5 (rAd5) was extracted as gene vector and packed into recombinant adenovirus rAd5-TGF-β₃, double enzyme digestion and PCR identification were performed. BMSCs were isolated and cultured from bone marrow of 2-month-old Diannan small-ear pigs (weighing, 12-15 kg), and the 2nd generation of BMSCs were harvested for experiments. The experiments were divided into 3 groups. BMSCs were transfected with rAd5-TGF-β₃ as experimental group and with empty vector as control group, and non-transfected BMSCs were used as blank control group. The transfection efficiency of exogenous gene was identified by flow cytometry, TGF-β₃ protein expression by immunofluorescence and Western blot. The cell morphology of experimental group was observed by inverted phase contrast microscope, and the expression of collagen type II in each group was detected by Western blot. Results The rAd5-TGF-β₃ recombinant adenovirus was successfully constructed and transfected into BMSCs. Green fluorescence was observed by immunofluorescence microscope. Flow cytometry test showed the best transfection at 72 hours (transfection efficiency of 84.86%). Immunofluorescence staining showed that the expression of TGF-β₃ protein was obvious at 72 hours; Western blot showed that there was a TGF-β₃ positive band with a relative molecular mass of 30×10³, while the control group and blank control group had no positive band. Obvious chondrogenic differentiation was observed in the experimental group after transfection in vitro, while the control group and blank control group had no obvious chondrogenic differentiation. Western blot showed that there was collagen type II positive band with a relative molecular mass of 130×10³ at 21 days after culture, while the control group and blank control group had no positive band. Conclusion rAd5-TGF-β₃ gene can be successfully transfected into BMSCs via adenovirus vectors, and stable expression of TGF-β₃ protein can be observed, enhancing BMSCs differentiation into chondrocytes, which may provide an experimental basis for gene therapy of joint cartilage defects.

Citation： WANGHuijian, LIYanlin, CHENJianming, WANGXin, ZHAOFengkai, CAOShuhai. CHONDROGENESIS OF BONE MARROW MESENCHYMAL STEM CELLS INDUCED BY TRANSFORMING GROWTH FACTOR β₃ GENE IN DIANNAN SMALL-EAR PIGS. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(2): 149-154. doi: 10.7507/1002-1892.20140034 Copy

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13.	Battula VL,Treml S,Bareiss PM,et al.Isolation of functionally distinct mesenehymal stem cell subsets using antibodies against CD56,CD271,and mesenchymal stem cell antigen-1.Haematologica,2009,94(2):173-184.
14.	郭璇,霍然,呂仁榮,等.兔骨髓間充質干細胞培養及向成軟骨細胞的誘導分化.中國組織工程研究與臨床康復,2011,15(6):963-966.
15.	Gfimaud E,Heymann D,Rédini F.Recent advances in TGF-beta effeets on chondrocytem etabolism.Potential therapeutic roles of TGF-beta in cartilage disorders.Cytokine Growth Factor Rev,2002,13(3):241-257.
16.	Swieszkowski W,Tuan BH,Kurzydlowski KJ.Repair and regeneration of osteochondral defects in the articular joints.Biomol Eng,2007,24(5):489-495.
17.	Shi S,Mercer S,Eckert GJ,et al.Regulation of articular chondrocyte aggrecan and collagen gene expression by multiple growth factor gene transfer.J Orthop Res,2012,30(7):1026-1031.
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19.	Wang X,McManus M.Lentivirus production.J Vis Exp,2009,(32):1499.
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1. Beane OS,Darling EM.Isolation,characterization,and differentiation of stem cells for cartilage regeneration.Ann Biomed Eng,2012,40(10):2079-2097.
2. 胡彬,劉曉華,趙林.骨髓間充質干細胞的分離培養與鑒定.中國組織工程研究與臨床康復,2011,15(27):5108-5111.
3. Djouad F,Mrugala D,No?l D,et al.Engineered mesenchymal stem cells for cartilage repair.Regen Med,2006,1(4):529-537.
4. Yang W,Gomes RR,Brown AJ,et al.Chondrogenic differentiation on perlecan domain I,collagen Ⅱ,and bone morphogenetic protein 2-based matrices.Tissue Eng,2006,12(7):2009-2024.
5. 鄭東,楊述華,馮勇,等.工程化生長轉化因子β₃促進軟骨前體細胞向軟骨分化的實驗研究.南京醫科大學學報:自然科學版,2007,27(6):558-562.
6. Na K,Kim S,Woo DG,et al.Synergistic effect of TGFbeta-3 on chondrogenic differentiation of rabbit chondrocytes in thermo-reversible hydrogel constructs blended with hyaluronic acid by in vivo test.J Bietechnol,2007,128(2):412-422.
7. 熊懷林,鄧莉,高小青,等.膠質源性神經營養因子基因修飾骨髓間充質干細胞與缺氧復氧神經細胞的共培養.中國組織工程研究與臨床康復,2011,15(49):9141-9145.
8. Wang YT,Wu XT,Wang F.Regeneration potential and mechanism of bone marrow mesenchymal stem cell transplantation for treating intervertebral disc degeneration.Orthop Sci,2010,15(6):707-719.
9. Bobis S,Jarocha D,Majka M.Mesenchymal stem cells:characteristics and clinical applications.Folia Histochem Cytobiol,2006,44(4):215-230.
10. Battula VL,Treml S,Bareiss PM,et al.Isolation of functionally distinct mesenehymal stem cell subsets using antibodies against CD56,CD271,and mesenchymal stem cell antigen-1.Haematologica,2009,94(2):173-184.
11. 步宏,劉戟,李勝富,等.中國近交系豬的氟烷基因型研究.中國修復重建外科雜志,2000,14(5):311-314.
12. Cozzi E,Bosio E,Seveso M,et al.Xenotransplantation as a model of integrated,multidisciplinary research.Organogenesis,2009,5(1):288-296.
13. Battula VL,Treml S,Bareiss PM,et al.Isolation of functionally distinct mesenehymal stem cell subsets using antibodies against CD56,CD271,and mesenchymal stem cell antigen-1.Haematologica,2009,94(2):173-184.
14. 郭璇,霍然,呂仁榮,等.兔骨髓間充質干細胞培養及向成軟骨細胞的誘導分化.中國組織工程研究與臨床康復,2011,15(6):963-966.
15. Gfimaud E,Heymann D,Rédini F.Recent advances in TGF-beta effeets on chondrocytem etabolism.Potential therapeutic roles of TGF-beta in cartilage disorders.Cytokine Growth Factor Rev,2002,13(3):241-257.
16. Swieszkowski W,Tuan BH,Kurzydlowski KJ.Repair and regeneration of osteochondral defects in the articular joints.Biomol Eng,2007,24(5):489-495.
17. Shi S,Mercer S,Eckert GJ,et al.Regulation of articular chondrocyte aggrecan and collagen gene expression by multiple growth factor gene transfer.J Orthop Res,2012,30(7):1026-1031.
18. 楊慧菊,孫怡,趙國棟.腺病毒載體的新進展和在生物鎮痛中的應用.實用疼痛醫學雜志,2011,7(3):217-221.
19. Wang X,McManus M.Lentivirus production.J Vis Exp,2009,(32):1499.
20. Baltzer AW,Lattermann C,Whalen JD,et al.Genetic enhaneement of fracture repair:healing of an experimental segmental defect by adenoviral transfer of the BMP2.Gene Thera,2000,7(9):734-739.
21. Pacak CA,Conlon T,Mah CS,et al.Relative persistence of AAV serotype 1 vector genomes in dystrophic musele.Genet Vaccines Ther,2008,10(15):6-14.

Chinese Journal of Reparative and Reconstructive Surgery

CHONDROGENESIS OF BONE MARROW MESENCHYMAL STEM CELLS INDUCED BY TRANSFORMING GROWTH FACTOR β₃ GENE IN DIANNAN SMALL-EAR PIGS

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CHONDROGENESIS OF BONE MARROW MESENCHYMAL STEM CELLS INDUCED BY TRANSFORMING GROWTH FACTOR β₃ GENE IN DIANNAN SMALL-EAR PIGS