• 遼寧醫(yī)學(xué)院附屬第一醫(yī)院（遼寧錦州，121001）1骨關(guān)節(jié)外科，2整形燒傷科;

目的 比較腺病毒載體Ad-BMP-2-內(nèi)部核糖體進(jìn)入位點(diǎn)（internal ribosome entry site，IRES）-低氧誘導(dǎo)因子1α突變型（hypoxia inducible factor 1αmu，HIF-1αmu）與Ad-巨細(xì)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(zhuǎn)染BMSCs后的成骨效應(yīng)，優(yōu)化成骨細(xì)胞種子來(lái)源。 方法取1月齡新西蘭大白兔骨髓分離培養(yǎng)BMSCs。取第3代BMSCs進(jìn)行病毒液轉(zhuǎn)染，根據(jù)轉(zhuǎn)染病毒液不同將實(shí)驗(yàn)分為4組，A、B、C組分別采用感染復(fù)數(shù)（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(zhuǎn)染細(xì)胞，D組為未被轉(zhuǎn)染的BMSCs。選擇最佳MOI值進(jìn)行觀察。轉(zhuǎn)染后采用免疫組織化學(xué)染色檢測(cè)BMP-2表達(dá)，Western blot檢測(cè)BMP-2和HIF-1α蛋白表達(dá)，ALP活性測(cè)定和鈣結(jié)節(jié)茜素紅染色檢測(cè)細(xì)胞成骨分化能力。 結(jié)果A、B、C組最佳MOI值分別為200、150和100。免疫組織化學(xué)染色示，A、B組BMP-2染色呈陽(yáng)性，C、D組呈陰性；A組陽(yáng)性細(xì)胞數(shù)明顯多于B組（P  lt; 0.05）。Western blot檢測(cè)示，A、B組BMP-2蛋白表達(dá)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(dá)明顯高于其余3組（P  lt; 0.05），其余3組間差異無(wú)統(tǒng)計(jì)學(xué)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(jiàn)明顯鈣結(jié)節(jié)，C、D組未見(jiàn)鈣結(jié)節(jié)形成；且A組鈣結(jié)節(jié)數(shù)量多于B組（P  lt; 0.05）。 結(jié)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(zhuǎn)染兔BMSCs后，前者BMP-2和成骨效應(yīng)表達(dá)均高于后者。

引用本文： 張解元,袁虹,李諶,李全營(yíng),郭威,劉丹平. 腺病毒介導(dǎo)BMP-2聯(lián)合低氧誘導(dǎo)因子1α突變型與單基因BMP-2分別轉(zhuǎn)染BMSCs后成骨效應(yīng)的比較研究. 中國(guó)修復(fù)重建外科雜志, 2012, 26(9): 1102-1106. doi: 復(fù)制

版權(quán)信息： ?四川大學(xué)華西醫(yī)院華西期刊社《中國(guó)修復(fù)重建外科雜志》版權(quán)所有，未經(jīng)授權(quán)不得轉(zhuǎn)載、改編

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(fù)眶骨缺損的實(shí)驗(yàn)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(fù)合rhBMP-2 轉(zhuǎn)染的BMSCs 對(duì)羊脛骨延長(zhǎng)骨愈合的影響. 中國(guó)修復(fù)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(dǎo)因子-1的病毒載體制備及感染效果. 中華實(shí)驗(yàn)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(dǎo)因子-1α腺病毒載體的構(gòu)建及常氧表達(dá). 中華實(shí)驗(yàn)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(guó)組織工程研究與臨床康復(fù), 2010, 14(20): 3787-3792.
  1. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 11. 范先群, 肖彩雯, 周慧芳, 等. 基因修飾的組織工程骨修復(fù)眶骨缺損的實(shí)驗(yàn)研究. 中華眼科雜志, 2009, 45(1): 66-72.
  12. 12. 林在俊, 朱振安, 湯婷婷, 等. HA 復(fù)合rhBMP-2 轉(zhuǎn)染的BMSCs 對(duì)羊脛骨延長(zhǎng)骨愈合的影響. 中國(guó)修復(fù)重建外科雜志, 2008, 22(2): 134-138.
  13. 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. 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. 15. 張翠萍, 付小兵, 李淑云, 等. 低氧誘導(dǎo)因子-1的病毒載體制備及感染效果. 中華實(shí)驗(yàn)外科雜志, 2008, 25(12): 1587-1588.
  16. 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. 17. Metzen E. Enzyme substrate recognition in oxygen sensing: how the HIF trap snaps. Biochem J, 2007, 408(2): e5-6.
  18. 18. Semenza GL. Evaluation of HIF-1 inhibitors as anticancer agents. Drug Discov Today, 2007, 12(19-20): 853-859.
  19. 19. 李諶, 靳云龍, 劉丹平, 等. 突變型低氧誘導(dǎo)因子-1α腺病毒載體的構(gòu)建及常氧表達(dá). 中華實(shí)驗(yàn)外科雜志, 2011, 28(9): 1539-1540.
  20. 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. 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. 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(guó)組織工程研究與臨床康復(fù), 2010, 14(20): 3787-3792.