去铁胺促进BMSCs靶向归巢和血管新生的实验研究
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摘要
目的应用低氧模拟剂去铁胺(desferrioxamine,DFO)模拟组织缺氧环境,观察其是否能促进BMSCs在大鼠随意皮瓣中的归巢和血管新生。方法分离、培养荧光素酶转基因Lewis大鼠的BMSCs和成纤维细胞(fibroblast,FB)。选用4周龄Lewis雄性大鼠40只,在其背部形成10 cm×3 cm大小矩形皮瓣,然后随机分成4组,每组10只:A组于大鼠球后静脉丛注射200μL PBS;B、C组同上法分别注射浓度为1×10~6个/mL的FB和BMSCs 200μL;D组同C组方法注射BMSCs后,腹腔注射DFO[100 mg/(kg·d)],连续7 d。术后7 d,观察各组大鼠皮瓣成活情况并计算皮瓣成活率,采用激光散斑血流成像仪检测皮瓣血流情况;术后30 min及1、4、7、14 d采用生物发光成像检测移植细胞在大鼠体内分布情况;术后7 d行CD31免疫荧光染色计算毛细血管密度,免疫荧光检测基质细胞衍生因子1(stromal cell derived factor 1,SDF-1)、EGF、FGF及Ki67的表达情况;利用荧光素酶抗体标记移植的BMSCs,免疫荧光染色观察其是否参与损伤组织修复。结果术后7 d各组缺血皮瓣坏死边界已明确,C、D组皮瓣成活率明显高于A、B组,D组高于C组(P<0.05)。激光散斑血流成像仪检测示,C、D组皮瓣血流值显著高于A、B组,D组高于C组(P<0.05)。生物发光成像示BMSCs随时间变化逐渐向缺血缺氧区迁移,最终分布到缺血组织中;术后14 d D组的光子信号明显强于其他组(P<0.05)。CD31免疫荧光染色示,C、D组毛细血管密度显著高于A、B组,D组高于C组(P<0.05)。C、D组SDF-1、EGF、FGF及Ki67的表达明显强于A、B组,D组强于C组。术后7 d移植的荧光素酶标记BMSCs表达于组织的动脉弹力层、毛细血管处和毛囊处。结论 DFO可以加速BMSCs向随意皮瓣缺氧区域的迁移归巢,加速BMSCs在缺血组织中的分化,同时促进缺血组织血管新生。
Objective To investigate whether desferrioxamine(DFO) can enhance the homing of bone marrow mesenchymal stem cells(BMSCs) and improve neovascularization in random flaps of rats. Methods BMSCs and fibroblasts(FB) of luciferase transgenic Lewis rats were isolated and cultured. Forty 4-week-old Lewis male rats were used to form a 10 cm×3 cm rectangular flap on their back. The experimental animals were randomly divided into 4 groups with 10 rats in each group: in group A, 200 μL PBS were injected through retrobulbar venous plexus; in group B, 200 μL FB with a concentration of 1×10~6 cells/mL were injected; in group C, 200 μL BMSCs with a concentration of 1×10~6 cells/mL were injected; in group D, cells transplantation was the same as that in group C, after cells transplantation, DFO[100 mg/(kg·d)] were injected intraperitoneally for 7 days. On the 7 th day after operation, the survival rate of flaps in each group was observed and calculated; the blood perfusion was observed by laser speckle imaging. Bioluminescence imaging was used to detect the distribution of transplanted cells in rats at 30 minutes and 1, 4, 7, and 14 days after operation.Immunofluorescence staining was performed at 7 days after operation to observe CD31 staining and count capillary density under 200-fold visual field and to detect the expressions of stromal cell derived factor 1(SDF-1), epidermal growth factor(EGF), fibroblast growth factor(FGF), and Ki67. Transplanted BMSCs were labeled with luciferase antibody and observed by immunofluorescence staining whether they participated in the repair of injured tissues. Results The necrosis boundary of ischemic flaps in each group was clear at 7 days after operation. The survival rate of flaps in groups C and D was significantly higher than that in groups A and B, and in group D than in group C(P<0.05). Laser speckle imaging showed that the blood perfusion units of flaps in groups C and D was significantly higher than that in groups A and B, and in group D than in group C(P<0.05). Bioluminescence imaging showed that BMSCs gradually migrated to the ischemia and hypoxia area and eventually distributed to the ischemic tissues. The photon signal of group D was significantly stronger than that of other groups at 14 days after operation(P<0.05). CD31 immunofluorescence staining showed that capillary density in groups C and D was significantly higher than that in groups A and B, and in group D than in group C(P<0.05). The expressions of SDF-1, EGF, FGF, and Ki67 in groups C and D were significantly stronger than those in groups A and B, and in group D than in group C. Luciferase-labeled BMSCs were expressed in the elastic layer of arteries,capillaries, and hair follicles at 7 days after transplantation. Conclusion DFO can enhance the migration and homing of BMSCs to the hypoxic area of random flap, accelerate the differentiation of BMSCs in ischemic tissue, and improve the neovascularization of ischemic tissue.
Objective To investigate whether desferrioxamine(DFO) can enhance the homing of bone marrow mesenchymal stem cells(BMSCs) and improve neovascularization in random flaps of rats. Methods BMSCs and fibroblasts(FB) of luciferase transgenic Lewis rats were isolated and cultured. Forty 4-week-old Lewis male rats were used to form a 10 cm×3 cm rectangular flap on their back. The experimental animals were randomly divided into 4 groups with 10 rats in each group: in group A, 200 μL PBS were injected through retrobulbar venous plexus; in group B, 200 μL FB with a concentration of 1×10~6 cells/mL were injected; in group C, 200 μL BMSCs with a concentration of 1×10~6 cells/mL were injected; in group D, cells transplantation was the same as that in group C, after cells transplantation, DFO[100 mg/(kg·d)] were injected intraperitoneally for 7 days. On the 7 th day after operation, the survival rate of flaps in each group was observed and calculated; the blood perfusion was observed by laser speckle imaging. Bioluminescence imaging was used to detect the distribution of transplanted cells in rats at 30 minutes and 1, 4, 7, and 14 days after operation.Immunofluorescence staining was performed at 7 days after operation to observe CD31 staining and count capillary density under 200-fold visual field and to detect the expressions of stromal cell derived factor 1(SDF-1), epidermal growth factor(EGF), fibroblast growth factor(FGF), and Ki67. Transplanted BMSCs were labeled with luciferase antibody and observed by immunofluorescence staining whether they participated in the repair of injured tissues. Results The necrosis boundary of ischemic flaps in each group was clear at 7 days after operation. The survival rate of flaps in groups C and D was significantly higher than that in groups A and B, and in group D than in group C(P<0.05). Laser speckle imaging showed that the blood perfusion units of flaps in groups C and D was significantly higher than that in groups A and B, and in group D than in group C(P<0.05). Bioluminescence imaging showed that BMSCs gradually migrated to the ischemia and hypoxia area and eventually distributed to the ischemic tissues. The photon signal of group D was significantly stronger than that of other groups at 14 days after operation(P<0.05). CD31 immunofluorescence staining showed that capillary density in groups C and D was significantly higher than that in groups A and B, and in group D than in group C(P<0.05). The expressions of SDF-1, EGF, FGF, and Ki67 in groups C and D were significantly stronger than those in groups A and B, and in group D than in group C. Luciferase-labeled BMSCs were expressed in the elastic layer of arteries,capillaries, and hair follicles at 7 days after transplantation. Conclusion DFO can enhance the migration and homing of BMSCs to the hypoxic area of random flap, accelerate the differentiation of BMSCs in ischemic tissue, and improve the neovascularization of ischemic tissue.
引文
1Granero-MoltóF, Weis JA, Miga MI, et al. Regenerative effects of transplanted mesenchymal stem cells in fracture healing. Stem Cells, 2009, 27(8):1887-1898.
2 Falanga V. Wound healing and its impairment in the diabetic foot.Lancet, 2005, 366(9498):1736-1743.
3Misao Y, Takemura G, Arai M, et al. Bone marrow-derived myocyte-like cells and regulation of repair-related cytokines after bone marrow cell transplantation. Cardiovasc Res, 2006, 69(2):476-490.
4Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999, 284(5411):143-147.
5Javazon EH, Keswani SG, Badillo AT, et al. Enhanced epithelial gap closure and increased angiogenesis in wounds of diabetic mice treated with adult murine bone marrow stromal progenitor cells.Wound Repair Regen, 2007, 15(3):350-359.
6McFarlane RM, Deyoung G, Henry RA. The design of a pedicle flap in the rat to study necrosis and its prevention. Plast Reconstr Surg, 1965, 35:177-182.
7Giordano A, Galderisi U, Marino IR. From the laboratory bench to the patient’s bedside:an update on clinical trials with mesenchymal stem cells. J Cell Physiol, 2007, 211(1):27-35.
8邓蓉蓉,谢伊旻,谢林.间充质干细胞归巢的研究与进展.中国组织工程研究, 2016, 20(19):2879-2888.
9Liu N, Tian J, Cheng J, et al. Migration of CXCR4 gene-modified bone marrow-derived mesenchymal stem cells to the acute injured kidney. J Cell Biochem, 2013, 114(12):2677-2689.
10Chiang KH, Cheng WL, Shih CM, et al. Statins, HMG-CoA reductase inhibitors, improve neovascularization by increasing the expression density of CXCR4 in endothelial progenitor cells. PLoS One, 2015, 10(8):e0136405.
11Ceradini DJ, Kulkarni AR, Callaghan MJ, et al. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1induction of SDF-1. Nat Med, 2004, 10(8):858-864.
12Oladipupo S, Hu S, Kovalski J, et al. VEGF is essential for hypoxiainducible factor-mediated neovascularization but dispensable for endothelial sprouting. Proc Natl Acad Sci U S A, 2011, 108(32):13264-13269.
13 Hou Z, Nie C, Si Z, et al. Deferoxamine enhances neovascularization and accelerates wound healing in diabetic rats via the accumulation of hypoxia-inducible factor-1α. Diabetes Res Clin Pract, 2013, 101(1):62-71.
14Mehrabani M, Najafi M, Kamarul T, et al. Deferoxamine preconditioning to restore impaired HIF-1α-mediated angiogenic mechanisms in adipose-derived stem cells from STZ-induced type 1 diabetic rats. Cell Prolif, 2015, 48(5):532-549.
15Ikeda Y, Tajima S, Yoshida S, et al. Deferoxamine promotes angiogenesis via the activation of vascular endothelial cell function.Atherosclerosis, 2011, 215(2):339-347.
16Du Z, Zan T, Huang X, et al. DFO enhances the targeting of CD34-positive cells and improves neovascularization. Cell Transplant,2015, 24(11):2353-2366.
17Ichioka S, Kudo S, Shibata M, et al. Bone marrow cell implantation improves flap viability after ischemia-reperfusion injury. Ann Plast Surg, 2004, 52(4):414-418.
18Zheng Y, Yi C, Xia W, et al. Mesenchymal stem cells transduced by vascular endothelial growth factor gene for ischemic random skin flaps. Plast Reconstr Surg, 2008, 121(1):59-69.
19Wu Y, Chen L, Scott PG, et al. Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells, 2007, 25(10):2648-2659.
20Chen L, Tredget EE, Wu PY, et al. Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing. PLoS One, 2008, 3(4):e1886.
21Gnecchi M, He H, Noiseux N, et al. Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement. FASEB J, 2006,20(6):661-669.
22Kinnaird T, Stabile E, Burnett MS, et al. Local delivery of marrowderived stromal cells augments collateral perfusion through paracrine mechanisms. Circulation, 2004, 109(12):1543-1549.
23 Kinnaird T, Stabile E, Burnett MS, et al. Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ Res, 2004, 94(5):678-685.
2 Falanga V. Wound healing and its impairment in the diabetic foot.Lancet, 2005, 366(9498):1736-1743.
3Misao Y, Takemura G, Arai M, et al. Bone marrow-derived myocyte-like cells and regulation of repair-related cytokines after bone marrow cell transplantation. Cardiovasc Res, 2006, 69(2):476-490.
4Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999, 284(5411):143-147.
5Javazon EH, Keswani SG, Badillo AT, et al. Enhanced epithelial gap closure and increased angiogenesis in wounds of diabetic mice treated with adult murine bone marrow stromal progenitor cells.Wound Repair Regen, 2007, 15(3):350-359.
6McFarlane RM, Deyoung G, Henry RA. The design of a pedicle flap in the rat to study necrosis and its prevention. Plast Reconstr Surg, 1965, 35:177-182.
7Giordano A, Galderisi U, Marino IR. From the laboratory bench to the patient’s bedside:an update on clinical trials with mesenchymal stem cells. J Cell Physiol, 2007, 211(1):27-35.
8邓蓉蓉,谢伊旻,谢林.间充质干细胞归巢的研究与进展.中国组织工程研究, 2016, 20(19):2879-2888.
9Liu N, Tian J, Cheng J, et al. Migration of CXCR4 gene-modified bone marrow-derived mesenchymal stem cells to the acute injured kidney. J Cell Biochem, 2013, 114(12):2677-2689.
10Chiang KH, Cheng WL, Shih CM, et al. Statins, HMG-CoA reductase inhibitors, improve neovascularization by increasing the expression density of CXCR4 in endothelial progenitor cells. PLoS One, 2015, 10(8):e0136405.
11Ceradini DJ, Kulkarni AR, Callaghan MJ, et al. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1induction of SDF-1. Nat Med, 2004, 10(8):858-864.
12Oladipupo S, Hu S, Kovalski J, et al. VEGF is essential for hypoxiainducible factor-mediated neovascularization but dispensable for endothelial sprouting. Proc Natl Acad Sci U S A, 2011, 108(32):13264-13269.
13 Hou Z, Nie C, Si Z, et al. Deferoxamine enhances neovascularization and accelerates wound healing in diabetic rats via the accumulation of hypoxia-inducible factor-1α. Diabetes Res Clin Pract, 2013, 101(1):62-71.
14Mehrabani M, Najafi M, Kamarul T, et al. Deferoxamine preconditioning to restore impaired HIF-1α-mediated angiogenic mechanisms in adipose-derived stem cells from STZ-induced type 1 diabetic rats. Cell Prolif, 2015, 48(5):532-549.
15Ikeda Y, Tajima S, Yoshida S, et al. Deferoxamine promotes angiogenesis via the activation of vascular endothelial cell function.Atherosclerosis, 2011, 215(2):339-347.
16Du Z, Zan T, Huang X, et al. DFO enhances the targeting of CD34-positive cells and improves neovascularization. Cell Transplant,2015, 24(11):2353-2366.
17Ichioka S, Kudo S, Shibata M, et al. Bone marrow cell implantation improves flap viability after ischemia-reperfusion injury. Ann Plast Surg, 2004, 52(4):414-418.
18Zheng Y, Yi C, Xia W, et al. Mesenchymal stem cells transduced by vascular endothelial growth factor gene for ischemic random skin flaps. Plast Reconstr Surg, 2008, 121(1):59-69.
19Wu Y, Chen L, Scott PG, et al. Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells, 2007, 25(10):2648-2659.
20Chen L, Tredget EE, Wu PY, et al. Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing. PLoS One, 2008, 3(4):e1886.
21Gnecchi M, He H, Noiseux N, et al. Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement. FASEB J, 2006,20(6):661-669.
22Kinnaird T, Stabile E, Burnett MS, et al. Local delivery of marrowderived stromal cells augments collateral perfusion through paracrine mechanisms. Circulation, 2004, 109(12):1543-1549.
23 Kinnaird T, Stabile E, Burnett MS, et al. Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ Res, 2004, 94(5):678-685.