RECOMBINANT ADENOVIRUS Ad-HUMAN MATRIX METALLOPROTEINASE 1 TRANSFECTING BONE MARROW MESENCHYMAL STEM CELLS OF RATS IN VITRO_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

DU Chao ¹ , JIANG Mingde ² , ZENG Weizheng ² , ZHENG Shumei ² , WEI Xiaolong ²

1Department of Postgraduate, Third Military Medical University, Chongqing, 400038, P.R.China;;
2Department of Gastroenterology, General Hospital of Chengdu Military Command. Corresponding author: JIANG Mingde, E-mail: jiangmd88@yahoo.com.cn;

Corresponding author：

Keywords：

Bone marrow mesenchymal stem cells; Recombinant adenovirus; Matrix metalloproteinase 1; Gene therapy; Rat

DOI：

10.7507/1002-1892.20130119

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Objective To transfect bone marrow mesenchymal stem cells (BMSCs) of rats by recombinant adenovirus Ad-human matrix metalloproteinase 1 (hMMP-1) in vitro so as to lay the experimental foundation for the treatment of liver fibrosis with a combination of BMSCs and hMMP-1 gene transplantation. Methods BMSCs were isolated from bone marrow of 2-3 weeks old Sprague Dawley rats by whole bone marrow adherence method and identified, then transfected by recombinant adenovirus Ad-hMMP-1 carrying enhanced green fluorescent protein (EGFP) marker in vitro. The green fluorescent expression was observed by fluorescence microscope and the transfection efficiency was detected by flow cytometry to determine the optimum multiplicity of infection (MOI). BMSCs at passage 3 were divided into 3 groups: untransfected BMSCs group (group A), Ad-EGFP transfected BMSCs group (group B), and Ad-hMMP-1-EGFP transfected BMSCs group (group C); the gene and intracellular protein of hMMP-1 were detected by RT-PCR and Western blot; the ELISA assay was used to detect the supernatant protein expression, and the hMMP-1 activity was measured by fluorescent quantification kit. Results The green fluorescent was observed in BMSCs transfected by recombinant adenovirus at 24 hours after transfection; the fluorescence intensity was highest at 72 hours; and the optimum MOI was 200. The cells of 3 groups entered the logarithmic growth phase on the 3rd day and reached plateau phase on the 6th day by MTT assay; no significant difference was found in the cell proliferation rate among 3 groups (P gt; 0.05). RT-PCR, Western blot, and ELISA assay showed high expressions of the hMMP-1 gene and protein in group C, but no expression in groups A and B. The hMMP-1 activity was 1.24 nmol/(mg · min) in group C, but hMMP-1 activity was not detectable in groups A and B. Conclusion The exogenous hMMP-1 gene is successfully transfected into BMSCs of rats via recombinant adenovirus and can highly express, which lays the experimental foundation for the treatment of liver fibrosis with a combination of BMSCs and hMMP-1 gene transplantation.

Citation： DU Chao,JIANG Mingde,ZENG Weizheng,ZHENG Shumei,WEI Xiaolong. RECOMBINANT ADENOVIRUS Ad-HUMAN MATRIX METALLOPROTEINASE 1 TRANSFECTING BONE MARROW MESENCHYMAL STEM CELLS OF RATS IN VITRO. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(5): 529-534. doi: 10.7507/1002-1892.20130119 Copy

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2.	杨长青, 胡国龄, 谭德明. 基质金属蛋白酶-1、反义金属蛋白酶组织抑制因子-1表达质粒对大鼠肝纤维化的影响. 中华传染病杂志, 2000, 14(1): 28-31．.
3.	Kossakowska AE, Edwards DR, Lee SS, et al. Altered balancebetween matrix metalloproteinases and their inhibitors in experimental biliary fibrosis. Am J Pathol, 1998, 153(3): 1895-1902.
4.	Pardo A, Selman M. MMP-1: the elder of the family. Int J Biochem Cell Biol, 2005, 37(1): 283-288.
5.	Cantz T, Manns MP, Ott M. Stem cells in liver regeneration and therapy. Cell Tissue Res, 2008, 331(1): 271-282.
6.	Baron F, Gothot A. Mesenchymal stem cells: a new versatile therapeutic option. Rev Med Liege, 2007, 62(1): 9-14.
7.	Minamide A, Yoshida M, Kawakami M, et al. The effects of bone morphogenetic protein and basic fibroblast growth factor on cultured mesenchymal stem cells for spine fusion. Spine (Phila Pa 1976), 2007, 32(10): 1067-1071.
8.	Franz RW, Shah KJ, Johnson JD, et al. Short-tomid-term results using autologus bone-marrow mononuclear cell implantation therapy as a limb salvage procedure in patients with severe peripheral arterial disease. Vasc Endovasular Surg, 2011, 45(5): 398-406.
9.	Kalka C, Baumgartner I. Gene and stem cell therapy in peripheral arterial occlusive disease. Vasc Med, 2008, 13(2): 157-172.
10.	Ushitora M, Sakurai F, Yamaguchi T, et al. Prevention of hepatic ischemia-reperfusion injury by pre-administration of catalase-expressing adenovirus vectors. J Control Release, 2010, 142(3): 431-437.
11.	MacLeod SH, Elgadi MM, Bossi G, et al. HER3 targeting of adenovirus by fiber modification increases infection of breast cancer cells in vitro, but not following intratumoral injection in mice. Cancer Gene Ther, 2012, 19(12): 888-898.
12.	Xu F, Li S, Li XL, et al. PhaseⅠand biodistribution study of recombinant adenovirus vector-mediated herpes simplex virus thymidine kinase gene and ganciclovir administration in patients with head and neck cancer and other malignant tumors. Cancer Gene Ther, 2009, 16(9): 723-730.
13.	Fu Y, Zhang Z, Zhang G, et al. Adenovirus-mediated gene transfer of tissue factor pathway inhibitor induces apoptosis in vascular smooth muscle cells. Apoptosis, 2008, 13(5): 634-640.
14.	Yamamoto N, Terai S, Ohata S, et al. A subpopulation of bone marrow cells depleted by a novel antibody, anti-Liv8, is useful for cell therapy to repair damaged liver. Biochem Biophys Res Commun, 2004, 313(4): 1110-1118.
15.	Sato Y, Araki H, Kato J, et al. Human mesenchymal stem cells xenografted directly to rat liver are differentiated into human hepatocytes without fusion. Blood, 2005, 106(2): 756-763.
16.	Petersen BE, Bowen WC, Patrene KD, et al. Bone marrow as a potential source of hepatic ovalcells. Science, 1999, 284(5417): 1168-1670.
17.	Luk JM, Wang PP, Lee CK, et al. Hepatic potential of bone marrow stromal cells: Development of in vitro co-culture and intra-portal transplantation models. J Immunol Methods, 2005, 305(1): 39-47.
18.	Yu Y, Lu L, Qian X, et al. Antifibrotic effect of hepatocyte growth factor-expressing mesenchymal stem cells in small-for-size liver transplant rats. Stem Cells Dev, 2010, 19(6): 903-914.
19.	Hu JJ, Sun C, Lan L, et al. Therapeutic effect of transplanting beta2m-/Thy1+ bone marrow-derived hepatocyte stem cells transduced with lentiviral-mediated HGF gene into CCl4- injured rats. J Gene Med, 2010, 12(3): 244-254.
20.	Iimuro Y, Nishio T, Morimoto T, et al. Delivery of matrix metalloproteinase-1 attenuates established liver fibrosis in the rat. Gastroenterology, 2003, 124(2): 445-458.
21.	Harting MT, Jimenez F, Cox CS Jr. Isolation of mesenchymal stem cells (MSCs) from green fluorescent protein positive (GFP+) transgenic rodents: the grass is not always green(er). Stem Cells and Development, 2009, 18(1): 127-135.

1. Iredale JP, Pickering J, Pick J, et al. Mechanisms of spontaneous resolution of rat liver fibrosis: Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors. J Clin Invest, 1998, 102(3): 538-549．.
2. 杨长青, 胡国龄, 谭德明. 基质金属蛋白酶-1、反义金属蛋白酶组织抑制因子-1表达质粒对大鼠肝纤维化的影响. 中华传染病杂志, 2000, 14(1): 28-31．.
3. Kossakowska AE, Edwards DR, Lee SS, et al. Altered balancebetween matrix metalloproteinases and their inhibitors in experimental biliary fibrosis. Am J Pathol, 1998, 153(3): 1895-1902.
4. Pardo A, Selman M. MMP-1: the elder of the family. Int J Biochem Cell Biol, 2005, 37(1): 283-288.
5. Cantz T, Manns MP, Ott M. Stem cells in liver regeneration and therapy. Cell Tissue Res, 2008, 331(1): 271-282.
6. Baron F, Gothot A. Mesenchymal stem cells: a new versatile therapeutic option. Rev Med Liege, 2007, 62(1): 9-14.
7. Minamide A, Yoshida M, Kawakami M, et al. The effects of bone morphogenetic protein and basic fibroblast growth factor on cultured mesenchymal stem cells for spine fusion. Spine (Phila Pa 1976), 2007, 32(10): 1067-1071.
8. Franz RW, Shah KJ, Johnson JD, et al. Short-tomid-term results using autologus bone-marrow mononuclear cell implantation therapy as a limb salvage procedure in patients with severe peripheral arterial disease. Vasc Endovasular Surg, 2011, 45(5): 398-406.
9. Kalka C, Baumgartner I. Gene and stem cell therapy in peripheral arterial occlusive disease. Vasc Med, 2008, 13(2): 157-172.
10. Ushitora M, Sakurai F, Yamaguchi T, et al. Prevention of hepatic ischemia-reperfusion injury by pre-administration of catalase-expressing adenovirus vectors. J Control Release, 2010, 142(3): 431-437.
11. MacLeod SH, Elgadi MM, Bossi G, et al. HER3 targeting of adenovirus by fiber modification increases infection of breast cancer cells in vitro, but not following intratumoral injection in mice. Cancer Gene Ther, 2012, 19(12): 888-898.
12. Xu F, Li S, Li XL, et al. PhaseⅠand biodistribution study of recombinant adenovirus vector-mediated herpes simplex virus thymidine kinase gene and ganciclovir administration in patients with head and neck cancer and other malignant tumors. Cancer Gene Ther, 2009, 16(9): 723-730.
13. Fu Y, Zhang Z, Zhang G, et al. Adenovirus-mediated gene transfer of tissue factor pathway inhibitor induces apoptosis in vascular smooth muscle cells. Apoptosis, 2008, 13(5): 634-640.
14. Yamamoto N, Terai S, Ohata S, et al. A subpopulation of bone marrow cells depleted by a novel antibody, anti-Liv8, is useful for cell therapy to repair damaged liver. Biochem Biophys Res Commun, 2004, 313(4): 1110-1118.
15. Sato Y, Araki H, Kato J, et al. Human mesenchymal stem cells xenografted directly to rat liver are differentiated into human hepatocytes without fusion. Blood, 2005, 106(2): 756-763.
16. Petersen BE, Bowen WC, Patrene KD, et al. Bone marrow as a potential source of hepatic ovalcells. Science, 1999, 284(5417): 1168-1670.
17. Luk JM, Wang PP, Lee CK, et al. Hepatic potential of bone marrow stromal cells: Development of in vitro co-culture and intra-portal transplantation models. J Immunol Methods, 2005, 305(1): 39-47.
18. Yu Y, Lu L, Qian X, et al. Antifibrotic effect of hepatocyte growth factor-expressing mesenchymal stem cells in small-for-size liver transplant rats. Stem Cells Dev, 2010, 19(6): 903-914.
19. Hu JJ, Sun C, Lan L, et al. Therapeutic effect of transplanting beta2m-/Thy1+ bone marrow-derived hepatocyte stem cells transduced with lentiviral-mediated HGF gene into CCl4- injured rats. J Gene Med, 2010, 12(3): 244-254.
20. Iimuro Y, Nishio T, Morimoto T, et al. Delivery of matrix metalloproteinase-1 attenuates established liver fibrosis in the rat. Gastroenterology, 2003, 124(2): 445-458.
21. Harting MT, Jimenez F, Cox CS Jr. Isolation of mesenchymal stem cells (MSCs) from green fluorescent protein positive (GFP+) transgenic rodents: the grass is not always green(er). Stem Cells and Development, 2009, 18(1): 127-135.

Chinese Journal of Reparative and Reconstructive Surgery

RECOMBINANT ADENOVIRUS Ad-HUMAN MATRIX METALLOPROTEINASE 1 TRANSFECTING BONE MARROW MESENCHYMAL STEM CELLS OF RATS IN VITRO

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