重組腺病毒Ad-人基質金屬蛋白酶1體外轉染大鼠BMSCs研究_《中國修復重建外科雜志》

作者：

杜超 ¹ , 蔣明德 ² , 曾維政 ² , 鄭淑梅 ² , 魏曉龍 ²

1第三軍醫大學研究生管理大隊（重慶，400038）;;
2成都軍區總醫院消化內科;

關鍵詞：

BMSCs 重組腺病毒基質金屬蛋白酶1 基因治療大鼠

DOI：

10.7507/1002-1892.20130119

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目的利用重組腺病毒Ad-人基質金屬蛋白酶1（human matrix metalloproteinase 1，hMMP-1）體外轉染大鼠BMSCs，為BMSCs聯合hMMP-1基因移植治療肝纖維化奠定實驗基礎。方法取2～3周齡SD大鼠骨髓采用全骨髓貼壁法分離培養BMSCs，傳至第3代并進行鑒定；用攜帶增強綠色熒光蛋白（enhanced green fluorescent protein，EGFP）標記的重組腺病毒Ad-hMMP-1-EGFP體外轉染第3代BMSCs，熒光顯微鏡下觀察轉染情況，流式細胞儀檢測轉染效率并確定最佳感染復數（multiplicity of infection，MOI）。實驗分為未轉染BMSCs組（A組）、Ad-EGFP轉染BMSCs組（B組）及Ad-hMMP-1-EGFP轉染BMSCs組（C組），采用MTT法檢測轉染后細胞增殖情況，RT-PCR及Western blot分別檢測轉染后各組細胞內hMMP-1基因和蛋白表達情況，ELISA法檢測上清中蛋白hMMP-1分泌水平，hMMP-1酶活性熒光定量試劑盒測定其活性。結果重組腺病毒轉染BMSCs后24 h可見綠色熒光表達，72 h時熒光強度最高，最佳MOI為200。MTT檢測示3組細胞均于培養3 d進入對數生長期，6 d后進入平臺期；各時間點3組間細胞吸光度（A）值比較，差異均無統計學意義（P gt; 0.05）。RT-PCR、Western blot及ELISA檢測示，A、B組均無hMMP-1基因和蛋白表達，C組hMMP-1基因和蛋白均高效表達。熒光定量試劑盒測定A、B組均未檢測到hMMP-1酶活性，C組hMMP-1酶活性為1.24 nmol/（mg·min）。結論利用重組腺病毒Ad-hMMP-1成功將外源基因導入大鼠BMSCs并高效表達，為BMSCs聯合hMMP-1基因移植治療肝纖維化奠定了實驗基礎。

引用本文： 杜超,蔣明德,曾維政,鄭淑梅,魏曉龍. 重組腺病毒Ad-人基質金屬蛋白酶1體外轉染大鼠BMSCs研究. 中國修復重建外科雜志, 2013, 27(5): 529-534. doi: 10.7507/1002-1892.20130119 復制

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.	楊長青, 胡國齡, 譚德明. 基質金屬蛋白酶-1、反義金屬蛋白酶組織抑制因子-1表達質粒對大鼠肝纖維化的影響. 中華傳染病雜志, 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. 楊長青, 胡國齡, 譚德明. 基質金屬蛋白酶-1、反義金屬蛋白酶組織抑制因子-1表達質粒對大鼠肝纖維化的影響. 中華傳染病雜志, 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.

《中國修復重建外科雜志》

重組腺病毒Ad-人基質金屬蛋白酶1體外轉染大鼠BMSCs研究

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《中國修復重建外科雜志》

重組腺病毒Ad-人基質金屬蛋白酶1體外轉染大鼠BMSCs研究

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