血管内皮生长因子-葡聚糖-聚乳酸-羟基乙酸缓释微球的治疗性血管再生实验研究
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摘要
目的探讨血管内皮生长因子-葡聚糖-聚乳酸-羟基乙酸缓释微球在促进大鼠缺血下肢血管生成的作用。方法制备血管内皮生长因子-葡聚糖-聚乳酸-羟基乙酸微球,扫描电镜观察其形态,采用酶联免疫吸附测定法检测其体外释药性能。将负载血管内皮生长因子-葡聚糖-聚乳酸-羟基乙酸微球纤维蛋白凝胶注射到大鼠缺血下肢肌肉内,1个月后进行组织学和免疫组织化学检测,评价血管再生情况。结果血管内皮生长因子-葡聚糖-聚乳酸-羟基乙酸微球体外释放血管内皮生成因子持续时间超过4周,可显著提高注射部位毛细血管和α-平滑肌肌动蛋白阳性血管的密度,并促进CD34、C-kit和血管内皮生长因子受体2阳性细胞的动员。结论肌肉注射负载血管内皮生长因子-葡聚糖-聚乳酸-羟基乙酸微球的纤维蛋白凝胶是一种较为理想的治疗性血管再生方法。
Aim To investigate the effect of recombinant human vascular endothelial growth factor-dextran-polylactic acid-glycolic acid(VEGF-dextran-PLGA) sustained-release microspheres on promoting angiogenesis in ischemic lower limbs of rats.Methods VEGF-dextran-PLGA microspheres were prepared and their morphology was observed by scanning electron microscopy.The drug release properties were determined by ELISA in vitro.The fibrin gel loaded with VEGF-dextran-PLGA microspheres was injected into the ischemic lower limb muscle of rats,and histological and immunohistochemical staining were performed 1 month later to evaluate the angiogenesis.Results VEGF-dextran-PLGA microspheres released VEGF in vitro for more than 4 weeks.They could significantly increase the density of capillaries andα-smooth muscle actin-positive blood vessels at the injection site,and promote the mobilization of CD34,C-kit and VEGFR-2 positive cells.Conclusion Intramuscular injection of fibrin gel loaded with VEGF-dextran-PLGA microspheres is an ideal therapeutic angiogenesis method.
Aim To investigate the effect of recombinant human vascular endothelial growth factor-dextran-polylactic acid-glycolic acid(VEGF-dextran-PLGA) sustained-release microspheres on promoting angiogenesis in ischemic lower limbs of rats.Methods VEGF-dextran-PLGA microspheres were prepared and their morphology was observed by scanning electron microscopy.The drug release properties were determined by ELISA in vitro.The fibrin gel loaded with VEGF-dextran-PLGA microspheres was injected into the ischemic lower limb muscle of rats,and histological and immunohistochemical staining were performed 1 month later to evaluate the angiogenesis.Results VEGF-dextran-PLGA microspheres released VEGF in vitro for more than 4 weeks.They could significantly increase the density of capillaries andα-smooth muscle actin-positive blood vessels at the injection site,and promote the mobilization of CD34,C-kit and VEGFR-2 positive cells.Conclusion Intramuscular injection of fibrin gel loaded with VEGF-dextran-PLGA microspheres is an ideal therapeutic angiogenesis method.
引文
[1]Bao H,Lv F,Liu T.A pro-angiogenic degradable Mg-poly(lacticco-glycolic acid)implant combined with rhbFGF in a rat limb ischemia model[J].Acta Biomaterialia,2017,64:279-289.
[2]Layman H,Sacasa M,Murphy AE,et al.Co-delivery of FGF-2 and G-CSF from gelatin-based hydrogels as angiogenic therapy in a murine critical limb ischemic model[J].Acta Biomater,2009,5(1):230-239.
[3]Li L,Okada H,Takemura G,et al.Sustained release of erythropoietin using biodegradable gelatin hydrogel microspheres persistently improves lower leg ischemia[J].J Am Coll Cardiol,2009,53(25):2378-2388.
[4]Kumari A,Yadav SK,Yadav SC.Biodegradable polymeric nanoparticles based drug delivery systems[J].Colloids Surf B Biointerfaces,2010,75(1):1-18.
[5]Ribeiro MP,Morgado PI,Miguel SP,et al.Dextran-based hydrogel containing chitosan microparticles loaded with growth factors to be used in wound healing[J].Mater Sci Eng C Mater Biol Appl,2013,33(5):2958-2966.
[6]Raza K,Kumar N,Misra C,et al.Dextran-PLGA-loaded docetaxel micelles with enhanced cytotoxicity and better pharmacokinetic profile[J].Int J Biol Macromol,2016,88:206-212.
[7]Zhu G,Mallery SR,Schwendeman SP.Stabilization of proteins encapsulated in injectable poly(lactide-co-glycolide)[J].Nat Biotechnol,2000,18(1):52-57.
[8]Maas C,Hermeling S,Bouma B,et al.A role for protein misfolding in immunogenicity of biopharmaceuticals[J].J Biol Chem,2007,282(4):2229-2236.
[9]Kang J,Wu F,Cai Y,et al.Development of recombinant human growth hormone(rhGH)sustained-release microspheres by a low temperature aqueous phase/aqueous phase emulsion method[J].Eur J Pharm Sci,2014,62:141-147.
[10]Ouma GO,Zafrir B,Mohler ER,et al.Therapeutic angiogenesis in critical limb ischemia[J].Angiology,2013,64(6):466-480.
[11]Giacca M,Zacchigna S.VEGF gene therapy:therapeutic angiogenesis in the clinic and beyond[J].Gene Ther,2012,19(6):622-629.
[12]Dor Y,Djonov V,Abramovitch R,et al.Conditional switching of VEGF provides new insights into adult neovascularization and proangiogenic therapy[J].EMBO J,2002,21(8):1939-1947.
[13]Ozawa CR,Banfi A,Glazer NL,et al.Microenvironmental VEGFconcentration,not total dose,determines a threshold between normal and aberrant angiogenesis[J].J Clin Invest,2004,4(113):516-527.
[14]Springer ML,Ozawa CR,Banfi A,et al.Localized arteriole formation directly adjacent to the site of VEGF-induced angiogenesis in muscle[J].Mol Ther,2003,7(4):441-449.
[15]Heil M,Eitenmuller I,Schmitz-Rixen T,et al.Arteriogenesis versus angiogenesis:similarities and differences[J].J Cell Mol Med,2006,10(1):45-55.
[16]张航,王照飞,张百灵,等.祖细胞和干细胞与缺血性疾病的治疗[J].中国动脉硬化杂志,2017,25(5):536-540.
[17]Avraham-Davidi I,Yona S,Grunewald M,et al.On-site education of VEGF-recruited monocytes improves their performance as angiogenic and arteriogenic accessory cells[J].J Exp Med,2013,210(12):2611-2625.
[18]Grunewald M,Avraham I,Dor Y,et al.VEGF-induced adult neovascularization:recruitment,retention,and role of accessory cells[J].Cell,2006,124(1):175-189.
[19]杨向红.生长因子的协同作用与新血管形成的研究进展[J].中国动脉硬化杂志,2011,19(1):1-5.
[2]Layman H,Sacasa M,Murphy AE,et al.Co-delivery of FGF-2 and G-CSF from gelatin-based hydrogels as angiogenic therapy in a murine critical limb ischemic model[J].Acta Biomater,2009,5(1):230-239.
[3]Li L,Okada H,Takemura G,et al.Sustained release of erythropoietin using biodegradable gelatin hydrogel microspheres persistently improves lower leg ischemia[J].J Am Coll Cardiol,2009,53(25):2378-2388.
[4]Kumari A,Yadav SK,Yadav SC.Biodegradable polymeric nanoparticles based drug delivery systems[J].Colloids Surf B Biointerfaces,2010,75(1):1-18.
[5]Ribeiro MP,Morgado PI,Miguel SP,et al.Dextran-based hydrogel containing chitosan microparticles loaded with growth factors to be used in wound healing[J].Mater Sci Eng C Mater Biol Appl,2013,33(5):2958-2966.
[6]Raza K,Kumar N,Misra C,et al.Dextran-PLGA-loaded docetaxel micelles with enhanced cytotoxicity and better pharmacokinetic profile[J].Int J Biol Macromol,2016,88:206-212.
[7]Zhu G,Mallery SR,Schwendeman SP.Stabilization of proteins encapsulated in injectable poly(lactide-co-glycolide)[J].Nat Biotechnol,2000,18(1):52-57.
[8]Maas C,Hermeling S,Bouma B,et al.A role for protein misfolding in immunogenicity of biopharmaceuticals[J].J Biol Chem,2007,282(4):2229-2236.
[9]Kang J,Wu F,Cai Y,et al.Development of recombinant human growth hormone(rhGH)sustained-release microspheres by a low temperature aqueous phase/aqueous phase emulsion method[J].Eur J Pharm Sci,2014,62:141-147.
[10]Ouma GO,Zafrir B,Mohler ER,et al.Therapeutic angiogenesis in critical limb ischemia[J].Angiology,2013,64(6):466-480.
[11]Giacca M,Zacchigna S.VEGF gene therapy:therapeutic angiogenesis in the clinic and beyond[J].Gene Ther,2012,19(6):622-629.
[12]Dor Y,Djonov V,Abramovitch R,et al.Conditional switching of VEGF provides new insights into adult neovascularization and proangiogenic therapy[J].EMBO J,2002,21(8):1939-1947.
[13]Ozawa CR,Banfi A,Glazer NL,et al.Microenvironmental VEGFconcentration,not total dose,determines a threshold between normal and aberrant angiogenesis[J].J Clin Invest,2004,4(113):516-527.
[14]Springer ML,Ozawa CR,Banfi A,et al.Localized arteriole formation directly adjacent to the site of VEGF-induced angiogenesis in muscle[J].Mol Ther,2003,7(4):441-449.
[15]Heil M,Eitenmuller I,Schmitz-Rixen T,et al.Arteriogenesis versus angiogenesis:similarities and differences[J].J Cell Mol Med,2006,10(1):45-55.
[16]张航,王照飞,张百灵,等.祖细胞和干细胞与缺血性疾病的治疗[J].中国动脉硬化杂志,2017,25(5):536-540.
[17]Avraham-Davidi I,Yona S,Grunewald M,et al.On-site education of VEGF-recruited monocytes improves their performance as angiogenic and arteriogenic accessory cells[J].J Exp Med,2013,210(12):2611-2625.
[18]Grunewald M,Avraham I,Dor Y,et al.VEGF-induced adult neovascularization:recruitment,retention,and role of accessory cells[J].Cell,2006,124(1):175-189.
[19]杨向红.生长因子的协同作用与新血管形成的研究进展[J].中国动脉硬化杂志,2011,19(1):1-5.