CONSTRUCTION AND IDENTIFICATION OF ADENOVIRUS VECTOR EXPRESSING BONE MORPHOGENETIC PROTEIN 2 AND TRANSFORMING GROWTH FACTOR β<sub>3</sub> GENES AND THEIR EXPRESSION IN BONE MARROW MESENCHYMAL STEM CELLS OF DIANNAN SMALL-EAR PIGS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WANGXin ¹ ,  LIYanlin ¹ , JINYaofeng ² , CHENJianming ³ , WANGHuijian ¹ , HEChuan ¹ , CAOShuhai ¹ , ZHAOFengkai ¹

1. Department of Sports Medicine, the First Affiliated Hospital of Kunming Medical University, Kunming Yunnan, 650032, P. R. China;
2. Department of Orthopaedics, the People's Hospital of Jiaxing City;
3. Department of Orthopaedics, the Second People's Hospital of Hunan Province;

Corresponding?author：

LIYanlin, Email: 852387873@qq.com

Keywords：

Cartilage tissue engineering; Adenovirus; Bone marrow mesenchymal stem cells; Gene transfection; Bone morphogenetic protein 2; Transforming growth factor β₃; Pig

DOI：

10.7507/1002-1892.20140197

Video：

Export PDF Favorites Scan Get Citation

Abstract Full text Figures/Tables Video References Cited by

Objective To construct and identify the recombinant adenovirus vector expressing bone morphogenetic protein 2(BMP-2) and transforming growth factor β₃(TGF-β₃) genes,to observe the expressions of BMP-2 and TGF-β₃ after transfected into bone marrow mesenchymal stem cells (BMSCs) of the Diannan small-ear pigs. Methods BMP-2 cDNA and TGF-β₃ cDNA were amplified by PCR,and were subcloned into the pEC3.1(+) plasmid to obtain pEC-GIE 3.1-BMP-2 and pEC-GIE3.1-TGF-β₃ plasmid respectively.They were subcloned into pGSadeno vector by homologous recombination reaction and HEK293 cells were transfected after linearization to obtain Ad-BMP-2 and Ad-TGF-β₃.The BMSCs were isolated from the bone marrow of Diannan small-ear pig and cultured.The 3rd passage BMSCs were transfered with Ad-BMP-2(group A),Ad-TGF-β₃(group B),Ad-BMP-2+Ad-TGF-β₃(group C),and untransfected cells served as a control (group D).The expressions of BMP-2 and TGF-β₃ genes and proteins were detected by PCR,immunofluorescence,and Western blot.The chondrogenic differentiation of BMSCs was evaluated by immunohistochemical of collagen type Ⅱ. Results The Ad-BMP-2 and Ad-TGF-β₃ were constructed successfully and confirmed by PCR and sequencing.The expression clones of Ad-BMP-2 and Ad-TGF-β₃ were packaged into maturated adenovirus successfully,the titer was 5.6×10⁸ and 1.6×10⁸ pfu/mL respectively.The PCR results showed a light band at 310 bp in group A and at 114 bp in group B,and both 310 bp and 114 bp bands in group C,but no band in group D.The image of immunofluorescence showed that there were red fluorescence and green fluorescence expressions in the cytoplasm of BMSCs at 72 hours after transfection in groups A and B,respectively;in group C,both red and green fluorescence expressions were detected,and no red or green fluorescence was detected in group D.The results of Western blot showed that there was a light band at 18×10³ in group A and at 50×10³ in group B;both 18×10³ and 50×10³ bands were detected in group C;but no band was detected in group D.The cells were positive for collagen type Ⅱ in groups A,B,and C;group C acquired strong collagen type Ⅱ staining when compared with group A and group B;in group D,the cells were negative for collagen type Ⅱ staining. Conclusion The recombinant adenovirus vector expressing BMP-2 and TGF-β₃ are constructed successfully.The BMP-2 and TGF-β₃ genes could be expressed effectively in BMSCs of Diannan small-ear pig after transfection,which could afford modified seeding cells for cartilage tissue engineering.

Citation： WANGXin, LIYanlin, JINYaofeng, CHENJianming, WANGHuijian, HEChuan, CAOShuhai, ZHAOFengkai. CONSTRUCTION AND IDENTIFICATION OF ADENOVIRUS VECTOR EXPRESSING BONE MORPHOGENETIC PROTEIN 2 AND TRANSFORMING GROWTH FACTOR β₃ GENES AND THEIR EXPRESSION IN BONE MARROW MESENCHYMAL STEM CELLS OF DIANNAN SMALL-EAR PIGS. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(7): 896-902. doi: 10.7507/1002-1892.20140197 Copy

1.	Wang W,Li B,Yang JZ,et al.The restoration of full-thickness cartilage defects with BMSCs and TGF-beta 1 loaded PLGA/fibrin gel constructs.Biomaterials,2010,31(34):8964-8973.
2.	Christensen BB,Foldager CB,Hansen OM,et al.A novel nano-structured porous polycaprolactone scaffold improves hyaline cartilage repair in a rabbit model compared to a collagen typeI/Ⅲ scaffold:in vitro and in vivo studies.Knee Surg Sports Traumatol Arthrosc,2012,20(6):1192-1204.
3.	Tan Wen-cheng,Zha Zhen-gang,Zhang Jia-qing,et al.Animal-origin osteochondral scaffold combined with bone marrow mesenchymal stem cells/chondrocytes for repair if composite osteochondral defects in rabbit knee joints.中國組織工程研究與臨床康復,2011,15(12):2265-2269.
4.	Abdallah BM,Kassem M.Human mesenchymal stem cells:from basic biology to clinical applications.Gene Ther,2008,15(2):109-116.
5.	Zhu L,Wu Y,Jiang H,et al.Engineered cartilage with internal porous high-density polyethylene support from bone marrow stromal cells:A preliminary study in nude mice.Br J Oral Maxillofac Surg,2010,48(6):462-465.
6.	謝在春,郭衛真,盧東榮,等.BMP-2誘導C3H10T1/2細胞成軟骨分化的分子機制研究.廣州醫學院學報,2012,40(2):11-15.
7.	Li Z,Kupcsik L,Yao SJ,et al.Mechanical load modulates chondrogenesis of human mesenchymal stem cells through the TGF-beta pathway.J Cell Mol Med,2010,14(6A):1338-1346.
8.	Bhang SH,Jeon JY,La WG,et al.Enhanced chondrogenic marker expression of human mesenchymal stem cells by interaction with both TGF-β3 and hyaluronic acid.Biotechnol Appl Biochem,2011,58(4):271-276.
9.	Fan H,Tao H,Wu Y,et al.TGF-β₃ immobilized PLGA-gelatin/chondroitin sulfate/hyaluronic acid hybrid scaffold for cartilage regeneration.J Biomed Mater Res A,2010,95(4):982-992.
10.	Redman SN,Oldfield SF,Archer CW,et al.Current strategies for articular cartilage repair.Eur Cell Mater,2005,9:23-32.
11.	Muller S,Breederveld RS,Tuinehreijer WE.Results of osteochondral autologous transplantation in the knee.Open Orthop J,2010,4:111-114.
12.	王彥明,余家闊,于長隆,等.Pridie鉆孔術修復膝關節軟骨全層缺損的臨床療效觀察.中國微創外科雜志,2006,6(11):861-863.
13.	Becher C,Driessen A,Hess T,et al.Microfracture for chondral defects of the talus:maintenance of early results at midterm follow-up.Knee Surg Sports Traumatol Arthrosc,2010,18(5):656-663.
14.	O'Driscoll SW.Preclinical cartilage repair:current status and future perspectives.Clin Orthop Relat Res,2011,(391 Suppl):S391-401.
15.	Jin XB,Sun YS,Zhang K,et al.Tissue engineered cartilage from hTGF beta2 transduced human adipose derived stem cells seeded in PLGA/alginate compound in vitro and in vivo.J Biomed Mater Res A,2008,86(4):1077-1087.
16.	許運,陳亮,史勇,等.SOX9促進人臍帶間充質干細胞聚集增強其向軟骨分化的潛能.中華醫學雜志,2012,92(29):2050-2054.
17.	張清林,呂惠成,吳一民,等.轉化生長因子β₁聯合骨形態發生蛋白2誘導骨髓間充質干細胞體外向軟骨細胞的分化.中國組織工程研究與臨床康復,2010,14(24):4371-4375.
18.	龍華,袁華,馬保安,等.腺病毒介導TGF-β₁及BMP-7基因共表達感染骨髓基質干細胞修復兔關節軟骨缺損.中國骨與關節損傷雜志,2008,23(6):471-473.
19.	Stevens MM,Marini RP,Martin I,et al.FGF-2 enhances TGF-beta l-induced periosteal chondrogenesis.J Orthop Res,2004,22(5):1114-1119.
20.	Yoon BS,Ovchinnikov DA,Yoshii I,et al.Bmpr1a and Bmpr1b have overlapping functions and are essential for chondrogenesis in vivo.Proc Natl Acad Sci U S A,2005,102(14):5062-5067.
21.	Partridge KA,Oreffo RO.Gene delivery in bone tissue engineering:progress and prospects using viral and nonviral strategies.Tissue Eng,2004,10(1-2):295-307.
22.	Hidaka C,Goodrich LR,Chen CT,et al.Acceleration of eartilage repair by genetically modified ehondrocytes over expressing bone morphogenetie protein-7.J Orthop Res,2003,21(4):573-583.
23.	Sakimura K,Matsumoto T,Miyamoto C,et al.Effects of insulin like growth factor I on transforming growth factor betal induced chondrogenesis of synovium-derived mesenchymal stem cells cultured in a polyglycolic acid scaffold.Cells Tissues Organs,2006,183(2):55-61.
24.	Song JJ,Aswad R,Kanaan RA,et al.Connective tissue growth factor (CTGF) acts as a downstream mediator of TGF betal to induce mesenchymal cell condensation.J Cell Physiol,2007,210(2):398-410.
25.	Mueller MB,Fischer M,Zellner J,et al.Hypertrophy in mesenchymal stem cell chondrogenesis:effect of TGF-beta isoforms and chondrogenic conditioning.Cells Tissues Organs,2010,192(3):158-166.

1. Wang W,Li B,Yang JZ,et al.The restoration of full-thickness cartilage defects with BMSCs and TGF-beta 1 loaded PLGA/fibrin gel constructs.Biomaterials,2010,31(34):8964-8973.
2. Christensen BB,Foldager CB,Hansen OM,et al.A novel nano-structured porous polycaprolactone scaffold improves hyaline cartilage repair in a rabbit model compared to a collagen typeI/Ⅲ scaffold:in vitro and in vivo studies.Knee Surg Sports Traumatol Arthrosc,2012,20(6):1192-1204.
3. Tan Wen-cheng,Zha Zhen-gang,Zhang Jia-qing,et al.Animal-origin osteochondral scaffold combined with bone marrow mesenchymal stem cells/chondrocytes for repair if composite osteochondral defects in rabbit knee joints.中國組織工程研究與臨床康復,2011,15(12):2265-2269.
4. Abdallah BM,Kassem M.Human mesenchymal stem cells:from basic biology to clinical applications.Gene Ther,2008,15(2):109-116.
5. Zhu L,Wu Y,Jiang H,et al.Engineered cartilage with internal porous high-density polyethylene support from bone marrow stromal cells:A preliminary study in nude mice.Br J Oral Maxillofac Surg,2010,48(6):462-465.
6. 謝在春,郭衛真,盧東榮,等.BMP-2誘導C3H10T1/2細胞成軟骨分化的分子機制研究.廣州醫學院學報,2012,40(2):11-15.
7. Li Z,Kupcsik L,Yao SJ,et al.Mechanical load modulates chondrogenesis of human mesenchymal stem cells through the TGF-beta pathway.J Cell Mol Med,2010,14(6A):1338-1346.
8. Bhang SH,Jeon JY,La WG,et al.Enhanced chondrogenic marker expression of human mesenchymal stem cells by interaction with both TGF-β3 and hyaluronic acid.Biotechnol Appl Biochem,2011,58(4):271-276.
9. Fan H,Tao H,Wu Y,et al.TGF-β₃ immobilized PLGA-gelatin/chondroitin sulfate/hyaluronic acid hybrid scaffold for cartilage regeneration.J Biomed Mater Res A,2010,95(4):982-992.
10. Redman SN,Oldfield SF,Archer CW,et al.Current strategies for articular cartilage repair.Eur Cell Mater,2005,9:23-32.
11. Muller S,Breederveld RS,Tuinehreijer WE.Results of osteochondral autologous transplantation in the knee.Open Orthop J,2010,4:111-114.
12. 王彥明,余家闊,于長隆,等.Pridie鉆孔術修復膝關節軟骨全層缺損的臨床療效觀察.中國微創外科雜志,2006,6(11):861-863.
13. Becher C,Driessen A,Hess T,et al.Microfracture for chondral defects of the talus:maintenance of early results at midterm follow-up.Knee Surg Sports Traumatol Arthrosc,2010,18(5):656-663.
14. O'Driscoll SW.Preclinical cartilage repair:current status and future perspectives.Clin Orthop Relat Res,2011,(391 Suppl):S391-401.
15. Jin XB,Sun YS,Zhang K,et al.Tissue engineered cartilage from hTGF beta2 transduced human adipose derived stem cells seeded in PLGA/alginate compound in vitro and in vivo.J Biomed Mater Res A,2008,86(4):1077-1087.
16. 許運,陳亮,史勇,等.SOX9促進人臍帶間充質干細胞聚集增強其向軟骨分化的潛能.中華醫學雜志,2012,92(29):2050-2054.
17. 張清林,呂惠成,吳一民,等.轉化生長因子β₁聯合骨形態發生蛋白2誘導骨髓間充質干細胞體外向軟骨細胞的分化.中國組織工程研究與臨床康復,2010,14(24):4371-4375.
18. 龍華,袁華,馬保安,等.腺病毒介導TGF-β₁及BMP-7基因共表達感染骨髓基質干細胞修復兔關節軟骨缺損.中國骨與關節損傷雜志,2008,23(6):471-473.
19. Stevens MM,Marini RP,Martin I,et al.FGF-2 enhances TGF-beta l-induced periosteal chondrogenesis.J Orthop Res,2004,22(5):1114-1119.
20. Yoon BS,Ovchinnikov DA,Yoshii I,et al.Bmpr1a and Bmpr1b have overlapping functions and are essential for chondrogenesis in vivo.Proc Natl Acad Sci U S A,2005,102(14):5062-5067.
21. Partridge KA,Oreffo RO.Gene delivery in bone tissue engineering:progress and prospects using viral and nonviral strategies.Tissue Eng,2004,10(1-2):295-307.
22. Hidaka C,Goodrich LR,Chen CT,et al.Acceleration of eartilage repair by genetically modified ehondrocytes over expressing bone morphogenetie protein-7.J Orthop Res,2003,21(4):573-583.
23. Sakimura K,Matsumoto T,Miyamoto C,et al.Effects of insulin like growth factor I on transforming growth factor betal induced chondrogenesis of synovium-derived mesenchymal stem cells cultured in a polyglycolic acid scaffold.Cells Tissues Organs,2006,183(2):55-61.
24. Song JJ,Aswad R,Kanaan RA,et al.Connective tissue growth factor (CTGF) acts as a downstream mediator of TGF betal to induce mesenchymal cell condensation.J Cell Physiol,2007,210(2):398-410.
25. Mueller MB,Fischer M,Zellner J,et al.Hypertrophy in mesenchymal stem cell chondrogenesis:effect of TGF-beta isoforms and chondrogenic conditioning.Cells Tissues Organs,2010,192(3):158-166.

Chinese Journal of Reparative and Reconstructive Surgery

CONSTRUCTION AND IDENTIFICATION OF ADENOVIRUS VECTOR EXPRESSING BONE MORPHOGENETIC PROTEIN 2 AND TRANSFORMING GROWTH FACTOR β₃ GENES AND THEIR EXPRESSION IN BONE MARROW MESENCHYMAL STEM CELLS OF DIANNAN SMALL-EAR PIGS

Abstract Full text Figures/Tables Video References Cited by

Previous Article

Next Article

Format

Content

Chinese Journal of Reparative and Reconstructive Surgery

CONSTRUCTION AND IDENTIFICATION OF ADENOVIRUS VECTOR EXPRESSING BONE MORPHOGENETIC PROTEIN 2 AND TRANSFORMING GROWTH FACTOR β3 GENES AND THEIR EXPRESSION IN BONE MARROW MESENCHYMAL STEM CELLS OF DIANNAN SMALL-EAR PIGS

Abstract Full text Figures/Tables Video References Cited by

Previous Article

Next Article

Format

Content

CONSTRUCTION AND IDENTIFICATION OF ADENOVIRUS VECTOR EXPRESSING BONE MORPHOGENETIC PROTEIN 2 AND TRANSFORMING GROWTH FACTOR β₃ GENES AND THEIR EXPRESSION IN BONE MARROW MESENCHYMAL STEM CELLS OF DIANNAN SMALL-EAR PIGS