葡聚糖/聚乳酸-乙醇酸微球联合3种因子促进缺血下肢的血管再生
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摘要
背景:葡聚糖/聚乳酸-乙醇酸微球药物缓释系统可使药物在作用部位维持有效药物浓度。目的:探讨携载粒细胞集落刺激因子、促红细胞生成素或血管内皮生长因子葡聚糖/聚乳酸-乙醇酸微球对缺血下肢血管再生的影响。方法:制备分别携载粒细胞集落刺激因子、促红细胞生成素与血管内皮生长因子的葡聚糖/聚乳酸-乙醇酸微球,通过扫描电镜观察其表面形态,利用ELISA法检测其包封率和累积释放率。将3种携载生长因子的微球与纤维蛋白胶混合,制备携载生长因子葡聚糖/聚乳酸纤维蛋白胶复合物。取24只雄性SD大鼠(江西省中医学院实验动物中心提供),制作右下肢缺血模型,随机分成2组干预,每组12只:观察组大腿肌肉内侧分5点注射携载3种生长因子葡聚糖/聚乳酸-乙醇酸微球的纤维蛋白胶复合物,对照组大腿肌肉内侧注射葡聚糖/聚乳酸-乙醇酸微球纤维蛋白凝胶复合物。术后第1周取注射部位肌肉组织,免疫组织化学染色观察增殖细胞核抗原、Bcl-2、基质细胞衍生因子1和趋化因子基质细胞衍生因子4的表达;术后第4周取注射部位肌肉组织,碱性磷酸酶与免疫组织化学染色观察毛细血管密度。实验方案经南昌大学医学院动物实验伦理委员会批准。结果与结论:(1)葡聚糖/聚乳酸-乙醇酸微球呈球形,表面光滑,直径40-120μm;搭载粒细胞集落刺激因子、促红细胞生成素与血管内皮生长因子葡聚糖/聚乳酸-乙醇酸微球的包封率分别为84%,85%,82%,4周的累积释放率分别为89.5%,90.3%,91.2%;(2)观察组增殖细胞核抗原、Bcl-2、基质细胞衍生因子1和趋化因子基质细胞衍生因子4阳性表达高于对照组;(3)观察组毛细血管密度与α-平滑肌肌动蛋白阳性血管密度高于对照组(P <0.05);(4)结果表明利用葡聚糖/聚乳酸-乙醇酸微球联合多种血管再生因子治疗下肢缺血,是一种有前途的血管再生方法。
BACKGROUND: The dextran/poly(lactic-co-glycolic acid)(PLGA) microspheres as a sustained release drug delivery system enables the drug to maintain an effective concentration at the site of action.OBJECTIVE: To investigate the effects of dextran/PLGA microspheres carrying granulocyte colony-stimulating factor, erythropoietin and vascular endothelial growth factor on the neovascularization of ischemic lower limb of rats.METHODS: Dextran/PLGA microspheres carrying granulocyte colony-stimulating factor, erythropoietin, and vascular endothelial growth factor were prepared and their surface morphology was observed by scanning electron microscopy. The encapsulation efficiency and cumulative release rate were detected by ELISA method. Dextran/PLGA microspheres carrying three growth factors were mixed with fibrin glue to prepare a growth factor dextran/PLGA/fibrin glue complex. Right lower limb ischemia models were produced in 24 male Sprague-Dawley rats(Laboratory Animal Center, Jiangsu University of Traditional Chinese Medicine, China). These animal models were randomly divided into two groups(n = 12/group). In the experimental group, dextran/PLGA/fibrin glue complex carrying three growth factors was injected into the inner thigh muscle at 5 points. In the control group, dextran/PLGA/fibrin glue complex without growth factors was identically injected. At 1 week after surgery, the muscle tissue around the injection site was harvested for immunohistochemistry to detect the expression of proliferating cell nuclear antigen, Bcl-2, stromal cell-derived factor 1, and C-X-C chemokine receptor type 4. At 4 weeks after surgery, the muscle tissue around the injection site was harvested for histological and immunohistochemical examination to measure capillary density. This study was approved by Animal Ethics Committee of Nanchang University School of Medicine, China.RESULTS AND CONCLUSION: The dextran/PLGA microspheres were spherical and had a smooth surface with a diameter of 40-120 μm.The encapsulation efficiency of dextran/PLGA microspheres carrying granulocyte colony-stimulating factor, erythropoietin, and vascular endothelial growth factor was 84%, 85%, and 82%, respectively. The 4-week cumulative release rate was 89.5%, 90.3%, 91.2%, respectively.The expression of proliferating cell nuclear antigen, Bcl~(-2), stromal cell-derived factor 1, and C-X-C chemokine receptor type 4 in the experimental group was significantly higher than that in the control group. Capillary density and α-smooth muscle actin-positive vascular density in the experimental group were significantly higher than those in the control group(P < 0.05). These results suggest that dextran/PLGA microspheres carrying multiple growth factors for treatment of lower limb ischemia is a promising therapeutic method.
BACKGROUND: The dextran/poly(lactic-co-glycolic acid)(PLGA) microspheres as a sustained release drug delivery system enables the drug to maintain an effective concentration at the site of action.OBJECTIVE: To investigate the effects of dextran/PLGA microspheres carrying granulocyte colony-stimulating factor, erythropoietin and vascular endothelial growth factor on the neovascularization of ischemic lower limb of rats.METHODS: Dextran/PLGA microspheres carrying granulocyte colony-stimulating factor, erythropoietin, and vascular endothelial growth factor were prepared and their surface morphology was observed by scanning electron microscopy. The encapsulation efficiency and cumulative release rate were detected by ELISA method. Dextran/PLGA microspheres carrying three growth factors were mixed with fibrin glue to prepare a growth factor dextran/PLGA/fibrin glue complex. Right lower limb ischemia models were produced in 24 male Sprague-Dawley rats(Laboratory Animal Center, Jiangsu University of Traditional Chinese Medicine, China). These animal models were randomly divided into two groups(n = 12/group). In the experimental group, dextran/PLGA/fibrin glue complex carrying three growth factors was injected into the inner thigh muscle at 5 points. In the control group, dextran/PLGA/fibrin glue complex without growth factors was identically injected. At 1 week after surgery, the muscle tissue around the injection site was harvested for immunohistochemistry to detect the expression of proliferating cell nuclear antigen, Bcl-2, stromal cell-derived factor 1, and C-X-C chemokine receptor type 4. At 4 weeks after surgery, the muscle tissue around the injection site was harvested for histological and immunohistochemical examination to measure capillary density. This study was approved by Animal Ethics Committee of Nanchang University School of Medicine, China.RESULTS AND CONCLUSION: The dextran/PLGA microspheres were spherical and had a smooth surface with a diameter of 40-120 μm.The encapsulation efficiency of dextran/PLGA microspheres carrying granulocyte colony-stimulating factor, erythropoietin, and vascular endothelial growth factor was 84%, 85%, and 82%, respectively. The 4-week cumulative release rate was 89.5%, 90.3%, 91.2%, respectively.The expression of proliferating cell nuclear antigen, Bcl~(-2), stromal cell-derived factor 1, and C-X-C chemokine receptor type 4 in the experimental group was significantly higher than that in the control group. Capillary density and α-smooth muscle actin-positive vascular density in the experimental group were significantly higher than those in the control group(P < 0.05). These results suggest that dextran/PLGA microspheres carrying multiple growth factors for treatment of lower limb ischemia is a promising therapeutic method.
引文
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[24]Kawachi K,Iso Y,Sato T,et al.Effects of erythropoietin on angiogenesis after myocardial infarction in porcine.Heart Vessels.2012;27(1):79-88.
[25]Lin JS,Chen YS,Chiang HS,et al.Hypoxic preconditioning protects rat hearts against ischaemia-reperfusion injury:role of erythropoietin on progenitor cell mobilization.J Physiol.2008;586(23):5757-5769.
[26]Kato S,Amano H,Ito Y,et al.Effect of erythropoietin on angiogenesis with the increased adhesion of platelets to the microvessels in the hind-limb ischemia model in mice.J Pharmacol Sci.2010;112(2):167-175.
[27]Jin DK,Shido K,Kopp HG,et al.Cytokine-mediated deployment of SDF-1 induces revascularization through recruitment of CXCR4+hemangiocytes.Nat Med.2006;12(5):557-567.
[28]Petit I,Jin D,Rafii S.The SDF-1-CXCR4 signaling pathway:a molecular hub modulating neo-angiogenesis.Trends Immunol.2007;28(7):299-307.
[29]Kucia M,Ratajczak J,Ratajczak MZ.Bone marrow as a source of circulating CXCR4+tissue-committed stem cells.Biol Cell.2005;97(2):133-146.
[30]Askari AT,Unzek S,Popovic ZB,et al.Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy.Lancet.2003;362(9385):697-703.
[31]Abbott JD,Huang Y,Liu D,et al.Stromal cell-derived factor-1alpha plays a critical role in stem cell recruitment to the heart after myocardial infarction but is not sufficient to induce homing in the absence of injury.Circulation.2004;110(21):3300-3305.
[32]Ceradini DJ,Kulkarni AR,Callaghan MJ,et al.Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1.Nat Med.2004;10(8):858-864.
[33]De Falco E,Porcelli D,Torella AR,et al.SDF-1 involvement in endothelial phenotype and ischemia-induced recruitment of bone marrow progenitor cells.Blood.2004;104(12):3472-3482.
[34]Lamagna C,Bergers G.The bone marrow constitutes a reservoir of pericyte progenitors.J Leukoc Biol.2006;80(4):677-681.
[35]Seeger FH,Rasper T,Koyanagi M,et al.CXCR4 expression determines functional activity of bone marrow-derived mononuclear cells for therapeutic neovascularization in acute ischemia.Arterioscler Thromb Vasc Biol.2009;29(11):1802-1809.
[2]Grunewald M,Avraham I,Dor Y,et al.VEGF-induced adult neovascularization:recruitment,retention,and role of accessory cells.Cell.2006;124(1):175-189.
[3]Klopsch C,Skorska A,Ludwig M,et al.Intramyocardial angiogenetic stem cells and epicardial erythropoietin save the acute ischemic heart.Dis Model Mech.2018;11(6).pii:dmm033282.doi:10.1242/dmm.033282.
[4]KimákováP,Solár P,SolárováZ,et al.Erythropoietin and Its Angiogenic Activity.Int J Mol Sci 2017;18(7).pii:E1519.doi:10.3390/ijms18071519.
[5]Spadaccio C,Rainer A,Trombetta M,et al.A G-CSF functionalized scaffold for stem cells seeding:a differentiating device for cardiac purposes.J Cell Mol Med.2011;15(5):1096-108.
[6]Cleland JL,Duenas ET,Park A,et al.Development of poly-(D,L-lactide--coglycolide)microsphere formulations containing recombinant human vascular endothelial growth factor to promote local angiogenesis.J Control Release.2001;72(1-3):13-24.
[7]Iwanaga K,Takano H,Ohtsuka M,et al.Effects of G-CSF on cardiac remodeling after acute myocardial infarction in swine.Biochem Biophys Res Commun.2004;325(4):1353-1359.
[8]K?rbling M,Reuben JM,Gao H,et al.Recombinant human granulocyte-colony-stimulating factor-mobilized and apheresiscollected endothelial progenitor cells:a novel blood cell component for therapeutic vasculogenesis.Transfusion.2006;46(10):1795-1802.
[9]Misao Y,Takemura G,Arai M,et al.Importance of recruitment of bone marrow-derived CXCR4+cells in post-infarct cardiac repair mediated by G-CSF.Cardiovasc Res.2006;71(3):455-465.
[10]Deindl E,Zaruba MM,Brunner S,et al.G-CSF administration after myocardial infarction in mice attenuates late ischemic cardiomyopathy by enhanced arteriogenesis.FASEB J.2006;20(7):956-958.
[11]Ohki Y,Heissig B,Sato Y,et al.Granulocyte colony-stimulating factor promotes neovascularization by releasing vascular endothelial growth factor from neutrophils.FASEB J.2005;19(14):2005-2007.
[12]Ohtsuka M,Takano H,Zou Y,et al.Cytokine therapy prevents left ventricular remodeling and dysfunction after myocardial infarction through neovascularization.FASEB J.2004;18(7):851-853.
[13]Janmaat ML,Heerkens JL,de Bruin AM,et al.Erythropoietin accelerates smooth muscle cell-rich vascular lesion formation in mice through endothelial cell activation involving enhanced PDGF-BB release.Blood.2010;115(7):1453-1460.
[14]Darnell JE,Kerr IM,Stark GR.Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins.Science.1994;264(5164):1415-1421.
[15]Watowich SS,Hilton DJ,Lodish HF.Activation and inhibition of erythropoietin receptor function:role of receptor dimerization.Mol Cell Biol.1994;14(6):3535-3549.
[16]Haller H,Christel C,Dannenberg L,et al.Signal transduction of erythropoietin in endothelial cells.Kidney Int.1996;50(2):481-488.
[17]Pelekanou V,Kampa M,Kafousi M,et al.Erythropoietin and its receptor in breast cancer:correlation with steroid receptors and outcome.Cancer Epidemiol Biomarkers Prev.2007;16(10):2016-2023.
[18]Heeschen C,Aicher A,Lehmann R,et al.Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization.Blood.2003;102(4):1340-1346.
[19]Brines M,Cerami A.Discovering erythropoietin's extra-hematopoietic functions:biology and clinical promise.Kidney Int.2006;70(2):246-250.
[20]Westenbrink BD,Lipsic E,van der Meer P,et al.Erythropoietin improves cardiac function through endothelial progenitor cell and vascular endothelial growth factor mediated neovascularization.Eur Heart J.2007;28(16):2018-2027.
[21]Kobayashi H,Minatoguchi S,Yasuda S,et al.Post-infarct treatment with an erythropoietin-gelatin hydrogel drug delivery system for cardiac repair.Cardiovasc Res.2008;79(4):611-620.
[22]Noguchi CT,Wang L,Rogers HM,et al.Survival and proliferative roles of erythropoietin beyond the erythroid lineage.Expert Rev Mol Med.2008;10:e36.
[23]Santhanam AV,d'Uscio LV,Peterson TE,et al.Activation of endothelial nitric oxide synthase is critical for erythropoietin-induced mobilization of progenitor cells.Peptides.2008;29(8):1451-1455.
[24]Kawachi K,Iso Y,Sato T,et al.Effects of erythropoietin on angiogenesis after myocardial infarction in porcine.Heart Vessels.2012;27(1):79-88.
[25]Lin JS,Chen YS,Chiang HS,et al.Hypoxic preconditioning protects rat hearts against ischaemia-reperfusion injury:role of erythropoietin on progenitor cell mobilization.J Physiol.2008;586(23):5757-5769.
[26]Kato S,Amano H,Ito Y,et al.Effect of erythropoietin on angiogenesis with the increased adhesion of platelets to the microvessels in the hind-limb ischemia model in mice.J Pharmacol Sci.2010;112(2):167-175.
[27]Jin DK,Shido K,Kopp HG,et al.Cytokine-mediated deployment of SDF-1 induces revascularization through recruitment of CXCR4+hemangiocytes.Nat Med.2006;12(5):557-567.
[28]Petit I,Jin D,Rafii S.The SDF-1-CXCR4 signaling pathway:a molecular hub modulating neo-angiogenesis.Trends Immunol.2007;28(7):299-307.
[29]Kucia M,Ratajczak J,Ratajczak MZ.Bone marrow as a source of circulating CXCR4+tissue-committed stem cells.Biol Cell.2005;97(2):133-146.
[30]Askari AT,Unzek S,Popovic ZB,et al.Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy.Lancet.2003;362(9385):697-703.
[31]Abbott JD,Huang Y,Liu D,et al.Stromal cell-derived factor-1alpha plays a critical role in stem cell recruitment to the heart after myocardial infarction but is not sufficient to induce homing in the absence of injury.Circulation.2004;110(21):3300-3305.
[32]Ceradini DJ,Kulkarni AR,Callaghan MJ,et al.Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1.Nat Med.2004;10(8):858-864.
[33]De Falco E,Porcelli D,Torella AR,et al.SDF-1 involvement in endothelial phenotype and ischemia-induced recruitment of bone marrow progenitor cells.Blood.2004;104(12):3472-3482.
[34]Lamagna C,Bergers G.The bone marrow constitutes a reservoir of pericyte progenitors.J Leukoc Biol.2006;80(4):677-681.
[35]Seeger FH,Rasper T,Koyanagi M,et al.CXCR4 expression determines functional activity of bone marrow-derived mononuclear cells for therapeutic neovascularization in acute ischemia.Arterioscler Thromb Vasc Biol.2009;29(11):1802-1809.