COMPARATIVE STUDY ON OSTEOGENIC EFFECT OF BONE MARROW MESENCHYMAL STEM CELLS TRANSFECTED BY ADENOVIRUS-BONE MORPHOGENETIC PROTEIN 2-INTERNAL RIBOSOME ENTRY SITE-HYPOXIA INDUCIBLE FACTOR 1αmu AND BY BONE MORPHOGENETIC PROTEIN 2 SINGLE GENE_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHANG Jieyuan ¹ , YUAN Hong ² , LI Chen ¹ , LI Quanying ¹ , GUO Wei ¹ , LIU Danping ¹

1Department of Orthopaedic and Joint Surgery, 2Department of Burn and Plastic Surgery, the First Affiliated Hospital of Liaoning Medical College, Jinzhou Liaoning, 121001, P.R.China. Corresponding author: LIU Danping, E-mail: liudanping2009@sohu.com;

Corresponding author：

Keywords：

Hypoxia inducible factor 1αmu; Bone morphogenetic protein 2; Bone marrow mesenchymal stem cells; Adenovirus; Gene transfection; Rabbit

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ObjectiveTo compare the osteogenic effect of bone marrow mesenchymal stem cells (BMSCs) transfected by adenovirus-bone morphogenetic protein 2-internal ribosome entry site-hypoxia inducible factor 1αmu (Ad-BMP-2-IRES-HIF-1αmu) and by Ad-cytomegalovirus (CMV)-BMP-2-IRES-human renilla reniformis green fluorescent protein 1 (hrGFP-1) single gene so as to optimize the source of osteoblasts. MethodsBMSCs were separated and cultured from 1-month-old New Zealand white rabbit. The BMSCs at passage 3 were transfected by virus. The experiment was divided into 4 groups (groups A, B, C, and D) according to different virus: BMSCs were transfected by Ad-BMP-2-IRES-HIF-1αmu in group A, by Ad-CMV-BMP-2-IRES-hrGFP-1 in group B, by Ad-CMV-IRES-hrGFP-1 in group C, and BMSCs were not transfected in group D. The optimum multiplicity of infection (MOI) (50, 100, 150, and 200) was calculated and then the cells were transfected by the optimum MOI, respectively. The expression of BMP-2 gene was detected by immunohistochemistry staining after transfected, the expressions of BMP-2 protein and HIF-1α protein were detected by Western blot method. The osteogenic differentiation potential was detected by alkaline phosphatase (ALP) activity and Alizarin red staining. ResultsThe optimum MOI of groups A, B, and C was 200, 150, and 100, respectively. The expression of BMP-2 was positive in groups A and B, and was negative in groups C and D by immunohistochemistry staining; the number of positive cells in group A was more than that in group B (P ﹤ 0.05). The expression of BMP-2 protein in groups A and B was significantly higher than that in groups C and D (P ﹤ 0.05), group A was higher than group B (P ﹤ 0.05). The expression of HIF-1α protein in group A was significantly higher than those in the other 3 groups (P ﹤ 0.05), no significant difference was found among the other 3 groups (P ﹥ 0.05). ALP activity in groups A and B was significantly higher than that in groups C and D (P ﹤ 0.05), group A was higher than group B (P ﹤ 0.05). Calcium nodules could be seen in groups A and B, but not in groups C and D; the number of calcium nodules in group A was higher than that in group B (P ﹤ 0.05). ConclusionThe expression of BMP-2 and osteogenic effect of BMSCs transfected by Ad-BMP-2-IRES-HIF-1αmu (double genes in single carrier) are higher than those of BMSCs transfected by Ad-CMV-BMP-2-IRES-hrGFP-1 (one gene in single carrier).

Citation： ZHANG Jieyuan,YUAN Hong,LI Chen,LI Quanying,GUO Wei,LIU Danping. COMPARATIVE STUDY ON OSTEOGENIC EFFECT OF BONE MARROW MESENCHYMAL STEM CELLS TRANSFECTED BY ADENOVIRUS-BONE MORPHOGENETIC PROTEIN 2-INTERNAL RIBOSOME ENTRY SITE-HYPOXIA INDUCIBLE FACTOR 1αmu AND BY BONE MORPHOGENETIC PROTEIN 2 SINGLE GENE. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(9): 1102-1106. doi: Copy

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2.	Nishimura R, Okuda K. Hypoxia is important for establishing vascularization during corpus luteum formation in cattle. J Reprod Dev, 2010, 56(1): 110-116.
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11.	范先群, 肖彩雯, 周慧芳, 等. 基因修饰的组织工程骨修复眶骨缺损的实验研究. 中华眼科杂志, 2009, 45(1): 66-72.
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14.	Liu X, Zeng B, Zhang C. Osteogenic and angiogenic effects of mesenchymal stromal cells with co-transfected human Ang-1 gene and BMP2 gene. Biotechnol Lett, 2011, 33(10): 1933-1938.
15.	张翠萍, 付小兵, 李淑云, 等. 低氧诱导因子-1的病毒载体制备及感染效果. 中华实验外科杂志, 2008, 25(12): 1587-1588.
16.	Wang JS, Liu X, Xue ZY, et al. Effects of aging on time course of neovascularization-related gene expression following acute hindlimb ischemia in mice. Chin Med J (Engl), 2011, 124(7): 1075-1081.
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19.	李谌, 靳云龙, 刘丹平, 等. 突变型低氧诱导因子-1α腺病毒载体的构建及常氧表达. 中华实验外科杂志, 2011, 28(9): 1539-1540.
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21.	Geng H, Harvey CT, Pittsenbarger J, et al. HDAC4 protein regulates HIF1α protein lysine acetylation and cancer cell response to hypoxia. J Biol Chem, 2011, 286(44): 38095-38102.
22.	Liu DP, Wang GX, Hu L, et al. Construction of adenovirus-mediated eukaryoti cexpression vector co-expressing mutant hypoxia-inducible factor-1 alpha target protein and humanized Renilla reniformis green fluorescent protein reporter molecule under normoxic conditions. 中国组织工程研究与临床康复, 2010, 14(20): 3787-3792.

1. Aghaloo T, Cowan CM, Zhang X, et al. The effect of NELL1 and bone morphogenetic protein-2 on calvarial bone regeneration. J Oral Maxillofac Surg, 2010, 68(2): 300-308.
2. Nishimura R, Okuda K. Hypoxia is important for establishing vascularization during corpus luteum formation in cattle. J Reprod Dev, 2010, 56(1): 110-116.
3. Grgurevic L, Macek B, Mercep M, et al. Bone morphogenetic protein (BMP)1-3 enhances bone repair. Biochem Biophys Res Commun, 2011, 408(1): 25-31.
4. Ziane S, Schlaubitz S, Miraux S, et al. A thermosensitive low molecular weight hydrogel as scaffold for tissue engineering. Eur Cell Mater, 2012, 23: 147-160.
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. Li JH, Liu DY, Zhang FM, et al. Human dental pulp stem cell is a promising autologous seed cell for bone tissue engineering. Chin Med J (Engl), 2011, 124(23): 4022-4028.
8. 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.
9. Jackson WM, Lozito TP, Djouad F, et al. Differentiation and regeneration potential of mesenchymal progenitor cells derived from traumatized muscle tissue. J Cell Mol Med, 2011, 15(11): 2377-2388.
10. 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.
11. 范先群, 肖彩雯, 周慧芳, 等. 基因修饰的组织工程骨修复眶骨缺损的实验研究. 中华眼科杂志, 2009, 45(1): 66-72.
12. 林在俊, 朱振安, 汤婷婷, 等. HA 复合rhBMP-2 转染的BMSCs 对羊胫骨延长骨愈合的影响. 中国修复重建外科杂志, 2008, 22(2): 134-138.
13. Song X, Liu S, Qu X, et al. BMP2 and VEGF promote angiogenesis but retard terminal differentiation of osteoblasts in bone regeneration by up-regulating Id1. Acta Biochim Biophys Sin (Shanghai), 2011, 43(10): 796-804.
14. Liu X, Zeng B, Zhang C. Osteogenic and angiogenic effects of mesenchymal stromal cells with co-transfected human Ang-1 gene and BMP2 gene. Biotechnol Lett, 2011, 33(10): 1933-1938.
15. 张翠萍, 付小兵, 李淑云, 等. 低氧诱导因子-1的病毒载体制备及感染效果. 中华实验外科杂志, 2008, 25(12): 1587-1588.
16. Wang JS, Liu X, Xue ZY, et al. Effects of aging on time course of neovascularization-related gene expression following acute hindlimb ischemia in mice. Chin Med J (Engl), 2011, 124(7): 1075-1081.
17. Metzen E. Enzyme substrate recognition in oxygen sensing: how the HIF trap snaps. Biochem J, 2007, 408(2): e5-6.
18. Semenza GL. Evaluation of HIF-1 inhibitors as anticancer agents. Drug Discov Today, 2007, 12(19-20): 853-859.
19. 李谌, 靳云龙, 刘丹平, 等. 突变型低氧诱导因子-1α腺病毒载体的构建及常氧表达. 中华实验外科杂志, 2011, 28(9): 1539-1540.
20. Tseng WP, Yang SN, Lai CH, et al. Hypoxia induces BMP-2 expression via ILK, Akt, mTOR, and HIF-1 pathways in osteoblasts. J Cell Physiol, 2010, 223(3): 810-818.
21. Geng H, Harvey CT, Pittsenbarger J, et al. HDAC4 protein regulates HIF1α protein lysine acetylation and cancer cell response to hypoxia. J Biol Chem, 2011, 286(44): 38095-38102.
22. Liu DP, Wang GX, Hu L, et al. Construction of adenovirus-mediated eukaryoti cexpression vector co-expressing mutant hypoxia-inducible factor-1 alpha target protein and humanized Renilla reniformis green fluorescent protein reporter molecule under normoxic conditions. 中国组织工程研究与临床康复, 2010, 14(20): 3787-3792.

Chinese Journal of Reparative and Reconstructive Surgery

COMPARATIVE STUDY ON OSTEOGENIC EFFECT OF BONE MARROW MESENCHYMAL STEM CELLS TRANSFECTED BY ADENOVIRUS-BONE MORPHOGENETIC PROTEIN 2-INTERNAL RIBOSOME ENTRY SITE-HYPOXIA INDUCIBLE FACTOR 1αmu AND BY BONE MORPHOGENETIC PROTEIN 2 SINGLE GENE

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