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

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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.中国组织工程研究与临床康复,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.	谢在春,郭卫真,卢东荣,等.BMP-2诱导C3H10T1/2细胞成软骨分化的分子机制研究.广州医学院学报,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.
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11.	Muller S,Breederveld RS,Tuinehreijer WE.Results of osteochondral autologous transplantation in the knee.Open Orthop J,2010,4:111-114.
12.	王彦明,余家阔,于长隆,等.Pridie钻孔术修复膝关节软骨全层缺损的临床疗效观察.中国微创外科杂志,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.	许运,陈亮,史勇,等.SOX9促进人脐带间充质干细胞聚集增强其向软骨分化的潜能.中华医学杂志,2012,92(29):2050-2054.
17.	张清林,吕惠成,吴一民,等.转化生长因子β₁联合骨形态发生蛋白2诱导骨髓间充质干细胞体外向软骨细胞的分化.中国组织工程研究与临床康复,2010,14(24):4371-4375.
18.	龙华,袁华,马保安,等.腺病毒介导TGF-β₁及BMP-7基因共表达感染骨髓基质干细胞修复兔关节软骨缺损.中国骨与关节损伤杂志,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.中国组织工程研究与临床康复,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. 谢在春,郭卫真,卢东荣,等.BMP-2诱导C3H10T1/2细胞成软骨分化的分子机制研究.广州医学院学报,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. 王彦明,余家阔,于长隆,等.Pridie钻孔术修复膝关节软骨全层缺损的临床疗效观察.中国微创外科杂志,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. 许运,陈亮,史勇,等.SOX9促进人脐带间充质干细胞聚集增强其向软骨分化的潜能.中华医学杂志,2012,92(29):2050-2054.
17. 张清林,吕惠成,吴一民,等.转化生长因子β₁联合骨形态发生蛋白2诱导骨髓间充质干细胞体外向软骨细胞的分化.中国组织工程研究与临床康复,2010,14(24):4371-4375.
18. 龙华,袁华,马保安,等.腺病毒介导TGF-β₁及BMP-7基因共表达感染骨髓基质干细胞修复兔关节软骨缺损.中国骨与关节损伤杂志,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

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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

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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