腺病毒介導BMP-2聯合低氧誘導因子1α突變型與單基因BMP-2分別轉染BMSCs后成骨效應的比較研究_《中國修復重建外科雜志》

作者：

張解元 ¹ , 袁虹 ² , 李諶 ¹ , 李全營 ¹ , 郭威 ¹ , 劉丹平 ¹

遼寧醫學院附屬第一醫院（遼寧錦州，121001）1骨關節外科，2整形燒傷科;

關鍵詞：

低氧誘導因子1α突變型 BMP-2 BMSCs 腺病毒基因轉染兔

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目的比較腺病毒載體Ad-BMP-2-內部核糖體進入位點（internal ribosome entry site，IRES）-低氧誘導因子1α突變型（hypoxia inducible factor 1αmu，HIF-1αmu）與Ad-巨細胞病毒（cytomegalovirus，CMV）-BMP-2-IRES-人源化海腎綠色熒光蛋白1（human renilla reniformis green fluorescent protein 1，hrGFP-1）分別轉染BMSCs后的成骨效應，優化成骨細胞種子來源。方法取1月齡新西蘭大白兔骨髓分離培養BMSCs。取第3代BMSCs進行病毒液轉染，根據轉染病毒液不同將實驗分為4組，A、B、C組分別采用感染復數（multiplicity of infection，MOI）為50、100、150和200的Ad-BMP-2-IRES-HIF-1αmu、Ad-CMV-BMP-2-IRES-hrGFP-1、Ad-CMV-IRES-hrGFP-1（空腺病毒載體）轉染細胞，D組為未被轉染的BMSCs。選擇最佳MOI值進行觀察。轉染后采用免疫組織化學染色檢測BMP-2表達，Western blot檢測BMP-2和HIF-1α蛋白表達，ALP活性測定和鈣結節茜素紅染色檢測細胞成骨分化能力。結果A、B、C組最佳MOI值分別為200、150和100。免疫組織化學染色示，A、B組BMP-2染色呈陽性，C、D組呈陰性；A組陽性細胞數明顯多于B組（P lt; 0.05）。Western blot檢測示，A、B組BMP-2蛋白表達明顯高于C、D組（P lt; 0.05），A組高于B組（P lt; 0.05）；A組HIF-1α蛋白表達明顯高于其余3組（P lt; 0.05），其余3組間差異無統計學意義（P gt; 0.05）。A、B組ALP活性明顯高于C、D組（P lt; 0.05），A組高于B組（P lt; 0.05）。茜素紅染色示，A、B組可見明顯鈣結節，C、D組未見鈣結節形成；且A組鈣結節數量多于B組（P lt; 0.05）。結論單載體雙基因Ad-BMP-2-IRES-HIF-1αmu與單載體單基因Ad-CMV-BMP-2-IRES-hrGFP-1分別轉染兔BMSCs后，前者BMP-2和成骨效應表達均高于后者。

引用本文： 張解元,袁虹,李諶,李全營,郭威,劉丹平. 腺病毒介導BMP-2聯合低氧誘導因子1α突變型與單基因BMP-2分別轉染BMSCs后成骨效應的比較研究. 中國修復重建外科雜志, 2012, 26(9): 1102-1106. doi: 復制

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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.
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. 基因修飾的組織工程骨修復眶骨缺損的實驗研究. 中華眼科雜志, 2009, 45(1): 66-72.
12.	林在俊, 朱振安, 湯婷婷, 等. HA 復合rhBMP-2 轉染的BMSCs 對羊脛骨延長骨愈合的影響. 中國修復重建外科雜志, 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.	張翠萍, 付小兵, 李淑云, 等. 低氧誘導因子-1的病毒載體制備及感染效果. 中華實驗外科雜志, 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.	李諶, 靳云龍, 劉丹平, 等. 突變型低氧誘導因子-1α腺病毒載體的構建及常氧表達. 中華實驗外科雜志, 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. 中國組織工程研究與臨床康復, 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. 基因修飾的組織工程骨修復眶骨缺損的實驗研究. 中華眼科雜志, 2009, 45(1): 66-72.
12. 林在俊, 朱振安, 湯婷婷, 等. HA 復合rhBMP-2 轉染的BMSCs 對羊脛骨延長骨愈合的影響. 中國修復重建外科雜志, 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. 張翠萍, 付小兵, 李淑云, 等. 低氧誘導因子-1的病毒載體制備及感染效果. 中華實驗外科雜志, 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. 李諶, 靳云龍, 劉丹平, 等. 突變型低氧誘導因子-1α腺病毒載體的構建及常氧表達. 中華實驗外科雜志, 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. 中國組織工程研究與臨床康復, 2010, 14(20): 3787-3792.

《中國修復重建外科雜志》

腺病毒介導BMP-2聯合低氧誘導因子1α突變型與單基因BMP-2分別轉染BMSCs后成骨效應的比較研究

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《中國修復重建外科雜志》

腺病毒介導BMP-2聯合低氧誘導因子1α突變型與單基因BMP-2分別轉染BMSCs后成骨效應的比較研究

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