骨髓内皮祖细胞治疗糖尿病大鼠后肢缺血的实验性研究
|
摘要
研究背景:众所周知,糖尿病(DM)是目前威胁人类生命健康的常见病、多发病。肢体动脉闭塞导致的下肢缺血是DM的四大血管并发症之一,临床治疗困难,截肢率高,占非创伤性截肢的50%。随着干细胞移植技术的不断发展,人们已开始将这一技术应用于糖尿病下肢缺血的研究。内皮祖细胞(endothelial progenitor cells,EPCs)最早于1997年在外周血中被发现,并被证明其在出生后的血管新生(angiogenesis)中发挥着重要作用。本研究试图通过EPCs移植治疗糖尿病下肢缺血的动物实验,为其进一步合理地临床应用奠定初步理论基础。
第一部分内皮祖细胞在骨髓单个核细胞移植治疗糖尿病下肢缺血中的作用
目的:通过将糖尿病大鼠骨髓密度梯度离心后所得单个核细胞(mononuclear cells,MNCs)直接向糖尿病大鼠下肢缺血部位进行局部注射与从相同数量的MNCs中培养所得的EPCs注射后的促血管新生效果进行比较,以及对等量骨髓MNCs和EPCs的促血管新生作用进行比较,了解骨髓干细胞移植及EPCs在其中的作用。
方法:取健康Wistar大鼠98只,将10g/L的链脲佐菌素(STZ)溶液按65mg/kg体重腹腔注射达到糖尿病标准后,将其中56只分别结扎左下肢股动脉及主要分支制作糖尿病下肢缺血模型,随机分为A、B、C、D四组(每组14只)。MNCs应用密度离心法从抽取的骨髓液中获得,EPCs由MNCs接种于含10%FBS及促生长细胞因子的α-MEM培养基中培养7获得,得率约为10%。移植方法为取细胞PBS悬液分10个位点均匀注射于实验大鼠的缺血下肢。A组移植细胞为4×106个MNCs;B组移植细胞为从与A组细胞数量相等的MNCs经体外培养获得的EPCs(数量约为4×105个);C组移植细胞为与B组EPCs细胞数量相等的MNCs; D组注射液体总量相同的PBS液。注射后7天,各组取6只大鼠,分别将手术部位肌肉取材称重匀浆后,ELISA方法测VEGF含量。注射后第28天,各组大鼠取材免疫组化计数血管密度。
结果:A组、B组、C组均较D组在局部VEGF含量和血管密度两项指标上明显增高,有统计学意义;但A组和B组两组间无论VEGF含量还是血管密度均没有显著性差异;B组与C组间比较,B组的VEGF含量和微血管密度两项指标均高于C组,结果有统计学差异。
结论:
1.采用局部注射骨髓干细胞移植方式可以促进糖尿病大鼠缺血下肢的血管新生。
2.局部注射EPCs量与MNCs量相等情况下,EPCs局部注射后缺血下肢的血管新生优于MNCs的局部注射。
3.将骨髓MNCs直接注射入糖尿病下肢缺血局部与先将此数量的MNCs培养纯化获得内皮祖细胞后再局部注射,在促进血管新生方面二者无明显差别,说明内皮祖细胞在骨髓单个核细胞移植后的血管新生中发挥主导作用。
第二部分糖尿病对骨髓来源内皮祖细胞移植在下肢缺血中促血管新生作用的影响
目的:比较受体相同(均为糖尿病下肢缺血大鼠或均为非糖尿病的下肢缺血大鼠)情况下,糖尿病大鼠和正常大鼠骨髓EPCs局部注射后的血管新生作用有无不同。比较相同移植物(均为糖尿病大鼠骨髓EPCs或均为正常大鼠骨髓EPCs)注射情况下,糖尿病下肢缺血大鼠和非糖尿病下肢缺血大鼠血管新生作用有无不同。
方法:将24只糖尿病大鼠与相同数量的正常大鼠,同时取骨髓MNCs并分别进行EPCs培养,另各取36只大鼠制作糖尿病下肢缺血模型和非糖尿病下肢缺血模型,待造模成功并稳定1周后,将各模型组随机分为糖尿病下肢缺血组:A1、B1、C1,每组均12只;非糖尿病下肢缺血组:A2、B2、C2,每组12只。将上述培养7天后的糖尿病大鼠EPCs注射A1和A2组各大鼠左下肢缺血局部,取同样培养天数的正常大鼠EPCs以相同数量、方法注入B1和B2组各大鼠左下肢缺血局部,C1和C2组左下肢缺血局部以相同方式注入等量PBS溶液。注射7天后各组取6只大鼠取材测VEGF含量。其余28天后取材,免疫组化染色计数各组大鼠左下肢微血管密度。
结果:受体相同,各移植物组间比较:糖尿病下肢缺血各组VEGF含量和微血管密度比较,P值均<0.05,结果有显著性差异,其中A1组与B1组局部VEGF含量和血管密度均较C1组增高有显著性差异,但A1组与B1组两组间无显著性差异。非糖尿病下肢缺血各组VEGF含量和微血管密度比较,P值均<0.05,结果有显著性差异,其中A2组与B2组局部VEGF含量和血管密度均较C2组增高,差异有显著性,但A2组与B2组两组间无显著性差异。
移植物相同,各受者组间比较:移植相同骨髓EPCs或PBS溶液后,A2、B2组与C2组(非糖尿病下肢缺血组)无论是局部VEGF含量和血管密度都较相应的A1、B1与C1组(糖尿病下肢缺血组)增高,P值均<0.05,结果有统计学显著性差异。
结论:
1局部注射糖尿病大鼠骨髓内皮祖细胞与注射正常大鼠内皮祖细胞均有明显促进血管新生作用,但两者间差异没有显著性,说明糖尿病对移植的“种子”—骨髓来源内皮祖细胞无明显影响。
2注射相同骨髓来源内皮祖细胞,非糖尿病大鼠下肢缺血的血管新生作用较糖尿病大鼠明显,说明糖尿病对移植的“土壤”—下肢缺血微环境有明显影响。
Background: Diabetes mellitus (DM) is one of the most common and frequently-occurring diseases and it threats the human health seriously. The lower limb ischemia indused by extremity arterial occlusion is one of the four major vascular complications of DM, which gives more difficulties to clinical treatment accounting high amputation rate by 50% of non-traumatic amputation. With continuous development of the stem cell transplantation, many doctors have started to apply this technology to treat diabetic lower limb ischemia. The endothelial progenitor cells (EPCs) was found in peripheral blood as early as 1997, which was proved to play a important role in angiogenesis after birth. In this study, we attempted to use the EPCs transplantation as a treatment for the diabetic lower limb ischemia in rat DM model, in order to establish a reasonable theoretical basis for more clinical applications.
Part 1 Effect of Endothelial Progenitor Cells in the Treatment of the Diabetic Lower Limb Ischemia by Bone Marrow Mononuclear Cells Transplantation
Objective: To compare the transplantation angiogenesis-promoting effect of bone marrow mononuclear cells (MNCs), which are obtained from the bone marrow, with post-cultivated EPCs derived from the same amount of MNCs by injecting directly to the site of lower limb ischemia in diabetic rats, and compare the angiogenesis effect of the same number of EPCs and MNCs in the same way in ordes to research their different effect in angiogenesis-promoting.
Methods: 98 healthy Wistar rats were used to make the diabetes mellitus model by intraperitoneal injecting with 65mg/kg streptozotocin (STZ) solution, the left femoral artery and its main branch of 56 rats of them were ligated respectively to product diabetic lower limb ischemia. Then they were randomly divided into A, B, C, D group (n = 14). MNCs were acquired from bone marrow by density gradient centrifugation. EPCs were acquired from MNCs cultured with 10% FBS and growth-promoting cytokines inα-MEM medium for 7 days. Transplantation was operated by sub-10-point injecting in left hind limb of each mouse. In group A,injected cells were 4×106 MNCs ; In group B,injected cells were EPCs acquired from 4×106 MNCs ; In group C,injected cells were MNCs as same as the Cell Number of group B; The group D was injected with the same amount of PBS solution. After injecting 7 days, 6 rats in each group were killed to take the muscles from the surgical site, and weighted, homogenized, and measured the VEGF levels with ELISA. In the 28th day, vascular density was counted by immunohistochemistry from the rats remained in each group.
Results: The group A, group B and group C were all more increased than the group D in the local VEGF levels and microvessel density, and the difference was significant. But there was no significant difference between group A and group B. Comparing the group B and group C, it was concluded that the group B was more increased than the groupC in the local VEGF levels and microvessel density , and the difference was significant.
Conclusion:
1. Stem Cell Transplantation by local injection of bone marrow stem cells in diabetic rats could promote angiogenesis in their ischemic lower extremities.
2. In the condition of transplanting with the same amount of cells, the angiogenesis role of injection with bone marrow EPCs was stronger than the role of bone marrow MNCs.
3. In the condition of the same amount of original MNCs, the role local injection of EPCs and the same way of MNCs in the promoting of angiogenesis in diabetic lower limb ischemia was same .The research showed that EPCs played important role in angiogenesis after stem cell transplantion.
Part 2 The effection of Diabetes on Bone Marrow EPCs Transplantation in the Angiogenesis of Hind Limb Ischemia in Diabetic Rats
Objective: To research the effection of Diabetes on bone marrow EPCs and microenvironment in transplantation in the angiogenesis of hind limb ischemia in diabetic rats.
Methods: 24 diabetic rats and 24 normal rats were killed, their bone marrow MNCs were insolation and cultured to aquire enough amounts of EPCs. 36 diabetic hind limb ischemia rats were randomly divided into A1, B1, C1 groups, each group included 12 rats. 36 non-diabetic hind limb ischemia rats were randomly divided into A2, B2, C2 groups, each group included 12 rats. After cultured for 7 days, EPCs from diabetic rats were injected into the left hind ischemic limb of rats of A1 and A2. With the same methods and the same number, EPCs derived from the normal rats were injected into the rats of group of B1 and B2.C1 and C2 rats were injected with the the equivalent PBS. 7 days after injection, 6 rats in each group were used to measure the VEGF levels. After 28 days, the rest were to be drawn to account of microvessel density in the local muscle of left hind limb through immunohistochemical staining.
Results: When comparing among groups of same acceptor, there were significant difference among the A1, B1, and C1 in the contents of VEGF and microvessel density. The Group A1 and group B1 were all higher in local expressing levels of VEGF and microvessel density than the group C1, but the distinction between the group A1 and B1 were not significant. When comparing the groups of non-diabetic lower limb ischemia, there were significant difference among A2, B2, and C2 in the contents of VEGF and vascular density. The group A2 and group B2 were also separately higher than the group C2 on both local VEGF levels and microvessel density, but the distinction between the group A2 and B2 were not significant.When comparing among groups of same transplants, the VEGF and microvessel density of group A2, B2, C2 were higher than group A1, B1, C1, P<0.05.
Conclusion:
1. Local injection of EPCs from the bone marrow of diabetic rats or from the normal rat was significantly in promoting angiogenesis, but there was no significant difference between the two ways. The result showed that diabetes had not efection on EPCs cultured from bone marrow.
2. Injecting the same EPCs, the angiogenesis of normal rats was better than the diabetic ones. The result showed that diabetes had efection on microenvironment of transplantion.
第一部分内皮祖细胞在骨髓单个核细胞移植治疗糖尿病下肢缺血中的作用
目的:通过将糖尿病大鼠骨髓密度梯度离心后所得单个核细胞(mononuclear cells,MNCs)直接向糖尿病大鼠下肢缺血部位进行局部注射与从相同数量的MNCs中培养所得的EPCs注射后的促血管新生效果进行比较,以及对等量骨髓MNCs和EPCs的促血管新生作用进行比较,了解骨髓干细胞移植及EPCs在其中的作用。
方法:取健康Wistar大鼠98只,将10g/L的链脲佐菌素(STZ)溶液按65mg/kg体重腹腔注射达到糖尿病标准后,将其中56只分别结扎左下肢股动脉及主要分支制作糖尿病下肢缺血模型,随机分为A、B、C、D四组(每组14只)。MNCs应用密度离心法从抽取的骨髓液中获得,EPCs由MNCs接种于含10%FBS及促生长细胞因子的α-MEM培养基中培养7获得,得率约为10%。移植方法为取细胞PBS悬液分10个位点均匀注射于实验大鼠的缺血下肢。A组移植细胞为4×106个MNCs;B组移植细胞为从与A组细胞数量相等的MNCs经体外培养获得的EPCs(数量约为4×105个);C组移植细胞为与B组EPCs细胞数量相等的MNCs; D组注射液体总量相同的PBS液。注射后7天,各组取6只大鼠,分别将手术部位肌肉取材称重匀浆后,ELISA方法测VEGF含量。注射后第28天,各组大鼠取材免疫组化计数血管密度。
结果:A组、B组、C组均较D组在局部VEGF含量和血管密度两项指标上明显增高,有统计学意义;但A组和B组两组间无论VEGF含量还是血管密度均没有显著性差异;B组与C组间比较,B组的VEGF含量和微血管密度两项指标均高于C组,结果有统计学差异。
结论:
1.采用局部注射骨髓干细胞移植方式可以促进糖尿病大鼠缺血下肢的血管新生。
2.局部注射EPCs量与MNCs量相等情况下,EPCs局部注射后缺血下肢的血管新生优于MNCs的局部注射。
3.将骨髓MNCs直接注射入糖尿病下肢缺血局部与先将此数量的MNCs培养纯化获得内皮祖细胞后再局部注射,在促进血管新生方面二者无明显差别,说明内皮祖细胞在骨髓单个核细胞移植后的血管新生中发挥主导作用。
第二部分糖尿病对骨髓来源内皮祖细胞移植在下肢缺血中促血管新生作用的影响
目的:比较受体相同(均为糖尿病下肢缺血大鼠或均为非糖尿病的下肢缺血大鼠)情况下,糖尿病大鼠和正常大鼠骨髓EPCs局部注射后的血管新生作用有无不同。比较相同移植物(均为糖尿病大鼠骨髓EPCs或均为正常大鼠骨髓EPCs)注射情况下,糖尿病下肢缺血大鼠和非糖尿病下肢缺血大鼠血管新生作用有无不同。
方法:将24只糖尿病大鼠与相同数量的正常大鼠,同时取骨髓MNCs并分别进行EPCs培养,另各取36只大鼠制作糖尿病下肢缺血模型和非糖尿病下肢缺血模型,待造模成功并稳定1周后,将各模型组随机分为糖尿病下肢缺血组:A1、B1、C1,每组均12只;非糖尿病下肢缺血组:A2、B2、C2,每组12只。将上述培养7天后的糖尿病大鼠EPCs注射A1和A2组各大鼠左下肢缺血局部,取同样培养天数的正常大鼠EPCs以相同数量、方法注入B1和B2组各大鼠左下肢缺血局部,C1和C2组左下肢缺血局部以相同方式注入等量PBS溶液。注射7天后各组取6只大鼠取材测VEGF含量。其余28天后取材,免疫组化染色计数各组大鼠左下肢微血管密度。
结果:受体相同,各移植物组间比较:糖尿病下肢缺血各组VEGF含量和微血管密度比较,P值均<0.05,结果有显著性差异,其中A1组与B1组局部VEGF含量和血管密度均较C1组增高有显著性差异,但A1组与B1组两组间无显著性差异。非糖尿病下肢缺血各组VEGF含量和微血管密度比较,P值均<0.05,结果有显著性差异,其中A2组与B2组局部VEGF含量和血管密度均较C2组增高,差异有显著性,但A2组与B2组两组间无显著性差异。
移植物相同,各受者组间比较:移植相同骨髓EPCs或PBS溶液后,A2、B2组与C2组(非糖尿病下肢缺血组)无论是局部VEGF含量和血管密度都较相应的A1、B1与C1组(糖尿病下肢缺血组)增高,P值均<0.05,结果有统计学显著性差异。
结论:
1局部注射糖尿病大鼠骨髓内皮祖细胞与注射正常大鼠内皮祖细胞均有明显促进血管新生作用,但两者间差异没有显著性,说明糖尿病对移植的“种子”—骨髓来源内皮祖细胞无明显影响。
2注射相同骨髓来源内皮祖细胞,非糖尿病大鼠下肢缺血的血管新生作用较糖尿病大鼠明显,说明糖尿病对移植的“土壤”—下肢缺血微环境有明显影响。
Background: Diabetes mellitus (DM) is one of the most common and frequently-occurring diseases and it threats the human health seriously. The lower limb ischemia indused by extremity arterial occlusion is one of the four major vascular complications of DM, which gives more difficulties to clinical treatment accounting high amputation rate by 50% of non-traumatic amputation. With continuous development of the stem cell transplantation, many doctors have started to apply this technology to treat diabetic lower limb ischemia. The endothelial progenitor cells (EPCs) was found in peripheral blood as early as 1997, which was proved to play a important role in angiogenesis after birth. In this study, we attempted to use the EPCs transplantation as a treatment for the diabetic lower limb ischemia in rat DM model, in order to establish a reasonable theoretical basis for more clinical applications.
Part 1 Effect of Endothelial Progenitor Cells in the Treatment of the Diabetic Lower Limb Ischemia by Bone Marrow Mononuclear Cells Transplantation
Objective: To compare the transplantation angiogenesis-promoting effect of bone marrow mononuclear cells (MNCs), which are obtained from the bone marrow, with post-cultivated EPCs derived from the same amount of MNCs by injecting directly to the site of lower limb ischemia in diabetic rats, and compare the angiogenesis effect of the same number of EPCs and MNCs in the same way in ordes to research their different effect in angiogenesis-promoting.
Methods: 98 healthy Wistar rats were used to make the diabetes mellitus model by intraperitoneal injecting with 65mg/kg streptozotocin (STZ) solution, the left femoral artery and its main branch of 56 rats of them were ligated respectively to product diabetic lower limb ischemia. Then they were randomly divided into A, B, C, D group (n = 14). MNCs were acquired from bone marrow by density gradient centrifugation. EPCs were acquired from MNCs cultured with 10% FBS and growth-promoting cytokines inα-MEM medium for 7 days. Transplantation was operated by sub-10-point injecting in left hind limb of each mouse. In group A,injected cells were 4×106 MNCs ; In group B,injected cells were EPCs acquired from 4×106 MNCs ; In group C,injected cells were MNCs as same as the Cell Number of group B; The group D was injected with the same amount of PBS solution. After injecting 7 days, 6 rats in each group were killed to take the muscles from the surgical site, and weighted, homogenized, and measured the VEGF levels with ELISA. In the 28th day, vascular density was counted by immunohistochemistry from the rats remained in each group.
Results: The group A, group B and group C were all more increased than the group D in the local VEGF levels and microvessel density, and the difference was significant. But there was no significant difference between group A and group B. Comparing the group B and group C, it was concluded that the group B was more increased than the groupC in the local VEGF levels and microvessel density , and the difference was significant.
Conclusion:
1. Stem Cell Transplantation by local injection of bone marrow stem cells in diabetic rats could promote angiogenesis in their ischemic lower extremities.
2. In the condition of transplanting with the same amount of cells, the angiogenesis role of injection with bone marrow EPCs was stronger than the role of bone marrow MNCs.
3. In the condition of the same amount of original MNCs, the role local injection of EPCs and the same way of MNCs in the promoting of angiogenesis in diabetic lower limb ischemia was same .The research showed that EPCs played important role in angiogenesis after stem cell transplantion.
Part 2 The effection of Diabetes on Bone Marrow EPCs Transplantation in the Angiogenesis of Hind Limb Ischemia in Diabetic Rats
Objective: To research the effection of Diabetes on bone marrow EPCs and microenvironment in transplantation in the angiogenesis of hind limb ischemia in diabetic rats.
Methods: 24 diabetic rats and 24 normal rats were killed, their bone marrow MNCs were insolation and cultured to aquire enough amounts of EPCs. 36 diabetic hind limb ischemia rats were randomly divided into A1, B1, C1 groups, each group included 12 rats. 36 non-diabetic hind limb ischemia rats were randomly divided into A2, B2, C2 groups, each group included 12 rats. After cultured for 7 days, EPCs from diabetic rats were injected into the left hind ischemic limb of rats of A1 and A2. With the same methods and the same number, EPCs derived from the normal rats were injected into the rats of group of B1 and B2.C1 and C2 rats were injected with the the equivalent PBS. 7 days after injection, 6 rats in each group were used to measure the VEGF levels. After 28 days, the rest were to be drawn to account of microvessel density in the local muscle of left hind limb through immunohistochemical staining.
Results: When comparing among groups of same acceptor, there were significant difference among the A1, B1, and C1 in the contents of VEGF and microvessel density. The Group A1 and group B1 were all higher in local expressing levels of VEGF and microvessel density than the group C1, but the distinction between the group A1 and B1 were not significant. When comparing the groups of non-diabetic lower limb ischemia, there were significant difference among A2, B2, and C2 in the contents of VEGF and vascular density. The group A2 and group B2 were also separately higher than the group C2 on both local VEGF levels and microvessel density, but the distinction between the group A2 and B2 were not significant.When comparing among groups of same transplants, the VEGF and microvessel density of group A2, B2, C2 were higher than group A1, B1, C1, P<0.05.
Conclusion:
1. Local injection of EPCs from the bone marrow of diabetic rats or from the normal rat was significantly in promoting angiogenesis, but there was no significant difference between the two ways. The result showed that diabetes had not efection on EPCs cultured from bone marrow.
2. Injecting the same EPCs, the angiogenesis of normal rats was better than the diabetic ones. The result showed that diabetes had efection on microenvironment of transplantion.
引文
1 Asahara T, Murohara T, Sullivan A, et a1. Isolation of putative progenitor endothelial cells for angiogenesis [J]. Science, 1997, 275(5302): 964-967
2孙敬方.动物实验方法学.北京:人民卫生出版社, 2001
3司徒镇强,吴军正.细胞培养.西安:世界图书出版西安公司, 2004, 45
4赵春华.干细胞原理、技术与临床.北京:化学工业出版社, 2006
5谷涌泉, Royal JP. DSA检查对预测血管性截肢平面的初步研究[J].外科理论与实践, 2001, 6(5): 298-300
6侯剑峰,法宪恩,张蕾, et al.同种异体骨髓单个核细胞移植对急性心肌梗死后大鼠心肌细胞凋亡及相关基因表达的影响[J].郑州大学学报(医学版), 2007, 42(1): 78-81
7李凤奎,王纯耀.实验动物学.郑州:郑州大学出版社, 2001. 121
8 Cross ML, C1aesson-Welsh L. FGF and VEGF function in angiogenesis: signaling pathways, biological response and therapeutic inhibition [J]. Trends Pharma Sci, 2001, 22: 201-207
9 Tilton RG, Chang KC, Leieune WS, et al. Role of nitric oxide in the hyperpermeability and hemodynamic changes induced by intravenous VEGE [J]. Invest Ophthalmol Vis Sci. 1999, 40: 689-696
10 Canneliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med, 2000, 6(10): 389-395
11 Isner J, Asahara T. Angiogenesis and vasculogenesis as therapeuticstrategies for postnatal neovascularization [J]. J Clin Invest, 1999, 103 (9): 1231-1236
12 Murayama T, Teppor OM, Silver M, et a1. Determination of bone marrow-derived endothelial progenitor cell significance in angiogenic growth factor-inducde neovascularizlation in vivo [J]. Exp Hematol, 2002, 30(8): 967-972
13 Abdulaziz AK, Hilal A, Jacques G, et a1. Therapeutic angio genesis using autologous bone marrow stromla cells: improved blood flow in a chronic limb ischemia model [J]. Ann Thorac Surg, 2003, 75(1): 204-209
14 Kalka C, Macuda H, Takahashi T, et a1. Transplantation of expanded endothelial progenitor cells for therapeutic neovascularization [J]. Proc Natl Acad Sci USA, 2000, 97(7): 3422-3427
15 Tateishi-Yuyama E, Matsubara H, Murohara T, et al. Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomized controlled trial [J]. Lancet, 2002, 360 (9331): 427-435
16谷涌泉,郭连瑞,张建,等.自体骨髓于细胞移植治疗严重下肢缺血1例.中国实用外科杂志, 2003, 23: 670
17 Werner N, Kosiol S, Schiegl T, et a1. Circulating endothelial progenitor cells and cardiovascular out comes [J]. N Engl J Med, 2005, 353(10): 999
18 Huang PP, Yang XF, Li SZ, et al.VRandomised comparison of G-CSF-mobilized peripheral blood mononuclear cells versus bone marrow-mononuclear cells for the treatment of patients with lower limb arteriosclerosis obliterans [J]. Thromb Haemost, 2007, 98(6): 1335-1342
19 Iwaguro H, Yamaguchi J, Kalka C, et a1. Endothelial progenitor cell vascular endothelial growth factor gene transfer for vascular regeneration [J]. Circulation, 2002, 105(6): 732-738
20 Jakosson L, Kreuger J, Holmborn K, et al. Heparan sulfate in transpotentiates VEGFR—mediated angiogenesis [J]. Dev Cell, 2006, 1 (5): 625-634
21 Schmidtl C, Rossig L, Fichtlscherer S, et a1. Reduced number ofcirculating endothelial progenitor ceils predicts future cardiovascular events: proof of concept for the clinical importance of endogenous vascular repair [J]. Circulation, 2005, 111(22): 2981-2987.
22 Isner JM, Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization [J]. Clin Invest, 1999, 103(9): 1231
23 Ferrara N. Vascular endothelial growth factor and the regulation of angiogenesis [J]. Recent Prog Horm Res, 2000, 55: 15
24 Suzukj T, NishdaM, Frtami S, et a1. Neoendothelializalion after peripheral blood stem cell transplantation in humans: a case report of a Tokaimura nuclear accident victim [J]. Cardiovasc Res, 2003, 58(2): 487-492
25 Gehling UM, Ergün S, Schumacher U, et a1. In vitro differentiation of endothelial cells form AC133-positive progenitor cells [J]. Blood, 2000, 95(10): 3106-311
26 Sagrario O, Michael I, Stephen H, et a1. Neuronal defects and delayed wound healing in the mice lacking fibroblast growth factor2 [J]. Proc Natl Acad Sci USA, 1998, 95: 5672-5677
27 Schmidtl C, Rossig L, Fichtlscherer S, et a1. Reduced number of circulating endothelial progenitor ceils predicts future cardiovascular events:proof of concept for the clinical importance of endogenous vascular repair [J]. Circulation, 2005, 111(22): 2981-2987
28 McClung JA, Nasser N, Saleem M, et al. Circulating endothelial cells are elevated in patients with type 2 diabetes mellitus independently of HbA1c [J]. Diabetologia, 2005, 48(2): 345-350
29 Loomans CJ, de Koning EJ, Staal FJ, et al. Endothelial progenitor cell dysfunction: a novel concept in the pathogenesis of vascular complications of type 1 diabetes [J]. Diabetes, 2004, 53(1): 195-199
30 Kuki S, Imanishi T, Kobayashi K. Hyperglycemia accelerated endothelial progenitor cell senescence via the activation of p38 mitogen activated protein kinase [J]. Circ J, 2006, 70(8): 1076-1081
31 Ceradini DJ, Yao D, Grogan RH, et al. Decreasing intracellular superoxidecorrects defective ischemia-induced new vessel formation in diabeticmice [J]. Biol Chem, 2008, 283(16): 10930-10938
32 Landmesser U, Bahlmann F, Mueller M, et al. Simvastatin versus ezetimibe: pleiotropic and lipid-lowering effects on endothelial function in humans [J]. Circulation, 2005, 111: 2356-2363
33 W emer N, Priller J, Laufs U, et al. Bone m arrow-derived progenitor cells modulate vascular reendothelialization and neointimal formation:effect of 3-hydroxy-3-m ethylglutaryl coenzyme A reductase inhibition [J]. Arterioscler Thromb Vasc Biol, 2002, 22: 1567-1572
34 Assmus B, Urbich C, Aicher A, et al. HM G-CoA reductase inhibitors reduce senescence and increase proliferation of endothelial progenitor cells via regulation of cell cycle regulatory genes [J]. Circ Res, 2003, 92:1049-1055
35 Hristov M, Fach C, Becker C, et al. Reduced numbers of circulating endothelial progenitor cells in patients with coronary artery disease associated with longterm statin treatment [J]. Atherosclerosis, 2007, 192(2): 413-420
1 Badorff C, Brandes RP, Popp R, et a1. Transdifferentiation of blood—derived human adult endothelial progenitor cells into functionally active cardiomyocytes [J]. Circulation, 2003, 107(7): 1024-1032
2 Thum T, Bauersachs J. Endothelial progenitor cells as potential drug targets [J]. Curr Durg Targets Cardiovasc Haematol Disord, 2005, 5(4): 277-286
3 Werner N, Kosiol S, Schiegl T, et al. Circulating endothelial progenitor cells and cardiovascular outcomes [J]. N Engl J Med, 2005, 353(10): 999
4 Boyer M, Townsend LE, Vogel LM, et al. Isolation of endothelial cells and their progenitor cells from human peripheral blood [J]. Vasc Surg 2000, 31(1Pt1): 181-189
5 Falon P, Arentson E, Kazarov A, et a1. bFGF positively regulates hematopoitic development. Development, 2000, 127(9): 1931-194l
6 Asahara T, Murohara T, Sullivan A, et a1. Isolation of putative progenitorendothelial cells for angiogenesis [J]. Science, 1997, 275(5302): 964-967
7 Matsumoto T, Mifune Y, Kawamoto A, et a1. Fracture induced mobilization and incorporation of bone marrow-derived endothelial progenitor cells for bone healing [J]. Cell Physiol, 2008, 215 (1): 234-242
8 Au P, Daheron LM, Duda DG, et al. Differentia1in vivo potentia1 of endothelial progenitor cells from human umbilical cord blood and adult peripheral blood to form functional long-lasting vessels [J]. Blood, 2008, l11 (3): 1302-1305
9 Cherqui S, Kurian SM, Schussler O, et a1. Isolation and angiogenesis by endothelial progenitors in the fetal liver [J]. Stem Cells, 2006, 24(1): 44-54
10 Yamashita J, Itoh H, Hirashima M, et al. Flkl-positive cells derived from embryonic stem cells serve as vascular progenitors [J]. Nature, 2000, 408(6808): 92-96
11 Martinez-Estrada OM, Munoz-Santos Y, Julve J, et a1. Human adipose tissue as a source of Flk-1+ cells: new method of differentiation and expansion [J]. Cardiovasc Res, 2005, 65(2): 328-333
12 Peichev M, Naiyer AJ, Pereira D, et a1. Expression of VEGFR-2 and AC133 by circulating human CD34-cells identifies a population of functional endothelial precursors [J]. Blood, 2000, 95(3): 952-958
13 Suzukj T, NishdaM, Frtami S, et a1. Neoendothelializalion after peripheral blood stem cell transplantation in humans:a case report of a Tokaimura nuclear accident victim [J]. Cardiovasc Res, 2003, 58(2): 487-492
14 Aicher A, Rentsch M, Sasaki K, et al. Nonbone marrow-derived circulating progenitor cells contribute to postnatal neovascularization following tissue ischemia [J]. Circ Res, 2007, 100(4): 581-589
15 Domenico R, Angelo V, Beatrice N, et a1. Postnatal vasculogenesis. Mechanism of Development, 2001, 100(1): 157- 163
16 Gehling UM, Ergun S, Schumacher U, et a1. In vitro diferentiaiton of endothelial cell from AC133+ progenitor cel1 [J]. Blood, 2000, 95 (10): 3106-3112
17 Harraz M, Jiao C, Hanlon HD, et a1. CD34 - blood-derived humanendothelial cell progenitors [J]. Stem Cells, 2001, 19(4): 304-312
18 Gill M, Dias S, Hattori K, et al. Vascular trauma induces rapid but transient mobilization of VEGFR2+AC133+ endothelial precursor cells. Circ Res, 2001, 88(22): 167-174
19 Deschaseaux F, Selmani Z, Falcoz PE, et al. Two types of circulating endothelial progenitor cells in patients receiving long term therapy by HMG-CoA reductase inhibitors [J]. Eur J Pharmacol 2007; 562(1-2): 111-118
20 Kim S, Park S, Kim J, et al. Differentiation of endothelial cells from human umbilical cord blood AC133- CD14+ cells. Ann Hematol 2005; 84(7): 417-422
21 Krenning G, Dankers PY, Jovanovic D, et al. Efficient differentiation of CD14+ monocytic cells into endothelial cells on degradable biomaterials [J]. Biomaterials 2007; 28(8): 1470-1479
22 Li TS, Hamano K, Nishida M, et al. CD117+stem cells play a key role in therapeutic angiogenesis induced by bone marrow cell implantation. Am J Physiol Heart Circ Physiol 2003; 285(3): H931-937
23 Zhang W, Zhang G, Jin H, et al. Characteristics of bone marrow-derived endothelial progenitor cells in aged mice [J]. Biochem Biophys Res Commun 2006; 348(3): 1018-1023
24赵春华.干细胞原理、技术与临床.北京:化学工业出版社, 2006, 178
25 Risau W, Flamme I, et al. Vasculogenesis [J]. Annu Rev Cell Biol, 1995, 11: 73-91
26 Isner J, Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization [J]. Clin Invest, 1999, 103(9): 1231-1236
27 Tepper OM, Galiano RD, Kalka C, et al. Endothelial progenitor cells: the promise of vascular stem cells for plastic surgery [J]. Plast Reconstr Surg, 2003, 111(2): 846-854
28 Hofer T, Desbaillets I, Hopfl G, et al. Dissecting hypoxia-dependent and hypoxia-independent steps in the HIF-1αactivation cascade: implicationsfor HIF-1αgene therapy [J]. Faseb J, 200l, 15(14): 2715-2717
29 Heissig B, Hattori K, Dias S, et al. Recruitment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kit-ligand [J]. Cell, 2002, l09(5): 625-637
30 Levesque JP, Takamatsu Y, Nilsson SK, et a1. Vascular cell adhesion molecule-1(CD106)is cleaved by neutrophil proteases in the bone marrow following hematopoietic progenitor cell mobilization by granulocyte colony-stimulating factor [J]. Blood, 200l, 98(5): 1289-1297
31 Ceradini DJ, Kulkarni AR, Callaghan MJ, et a1. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1induction of SDF-1 [J]. Nat Med, 2004, l0(8): 858-864
32 Hiasa K, Ishibashi M, Ohtani K, et a1. Gene transfer of stromla cell-derived factor--1enhances ischemic vasculogenesis and angiogenesis via vascular endothelial growth factor / endothelial nitrie oxide synthase-related pathway [J]. Circulation, 2004, 109(20): 2454-2461
33 Mlhle R, Bsutz F, Rsfii S, et a1. The chemokine receptor CXCR-4 is expressed on CD34+ hematopoietic progenitors and leukemic cells and mediates transendothelial migration induced by stromal cell-derived factor [J]. Blood, 1998, 91(12): 4523-4530
34 Quesenberry PJ, Becker PS. Stem cell homi ng: Rolling, crawling, and nesting. Proc Natl Acad Sci USA, 1998, 95 (26): 15155-151557
35 Walter DH, Rittig K, Bahlmann FH, et a1. Statin therapy accelerates reendothelialization: a novel effect involving mobilization and incorporation of bone marrow derived endothelial progenitor cells [J]. Circulation, 2002, 105(25): 3017-3024
36 Grunewald M, Avraham I, Dor Y, et a1. VEGF-induced adult neovascularizaiton:recruitment,retention,and role of accessory cells [J]. Cell, 2006, l24(1): 175-89
37 Butler JM, Guthrie SM, Koc M, et a1. SDF-1 is both necessary and sufficient to promote proliferative retinopathy [J]. Clin Invest, 2005, ll5(1): 86-93
38 Cao Y, Hong A, Schulten H, et al. Update on therapeutic neovascularization [J]. Cardiovasc Res, 2005: 65(3): 639-648
39 Rsfii S, Meeus S, Dias S, et a1. Contribution of marrow-derived progenitors to vascular and cardiac regeneration [J]. Semin Cel1 Dev Bio1, 2002, 13(1): 61-67
40 Wang JF, Park IW, Groopman JE. Stromal cell-derived factor-1alpha stimulates tyrosine phosphory1ation of multiple focal adhesion proteins and induces migration of hematopoietic progenitor cells: roles of phosphoinositide-3 kinase and protein kinase C [J]. Blood, 2000, 95(8): 2505-2513
41 Mace KA, Yu DH, Paydar KZ, et al. Sustained expression of Hif-1alpha in the diabetic environment promotes angiogenesis and cutaneous wound repair [J]. Wound Repair Regen, 2007, 15(5): 636-645
42 Phillpott NJ, Prue RI, Mash JC, et a1. G-CSF-mobilized CD34 peripheral blood stem cells are significantly less apoptotic than unstimulated peripheral blood CD34 cells: role of G-CSF as survival factor [J]. Br J Haematol, 1997, 97(1): 146-152
43 Heeschen C, Aicher A, Lehmann R. Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization [J]. Blood, 2003, 102: 1340-1346
44 Thomas T, Sarah H, Sabrina F, et al. Age-dependent impairment of endothelial progenitor cells is corrected by growth hormone mediated increase of insulin-like growth factor-1 [J]. Circ Res, 2007, 100(3): 434-443
45 Vantle M, Huntgeburth M, Caglayen E, et a1. PI3-kinase/Akt-dependent antiapoptotic signaling by the PDGF alpha receptor is negatively regulated by src family kinases [J]. FEBS Lett, 2006, 580(30): 6769-6776
46 McClung JA, Nasser N, Saleem M, et al. Circulating endothelial cells are elevated in patients with type 2 diabetes mellitus independently of HbA1c [J] . Diabetologia, 2005, 48(2): 345-350
47 Capla JM, Grogan RH. Diabetes impairs endothelial progenitor cellsmediated blood vessel formation in response to hypoxia [J] . Plast Reconstr Surg, 2007, 119(1): 59-70
48 Kuki S, Imanishi T, Kobayashi K. Hyperglycemia accelerated endothelial progenitor cell senescence via the activation of p38 mitogen activated protein kinase [J]. Circ J, 2006, 70(8): 1076-1081
49 Ceradini DJ, Yao D, Grogan RH, et al. Decreasing intracellular superoxide corrects defective ischemia-induced new vessel formation in diabeticmice [J] . J Biol Chem, 2008, 283(16): 10930-10938
50 Schmidtl C, Rossig L, Fichtlscherer S, et a1. Reduced number of circulating endothelial progenitor ceils predicts future cardiovascular events: proof of concept for the clinical importance of endogenous vascular repair [J]. Circulation, 2005, 111(22): 2981-2987
51 Vasa M, Fichtlscherer S, Aicher, et a1. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronaryartery disease [J]. Circ Res, 2001, 89(1): E1-E7
52 Damas JK, WahreT, Yndestad A, et a1. Stromal cell-derived factor-1 alpha in unstable angina: potential antiinflammatory and matrix-stabilizing effects [J]. Circulation, 2002, 106 (1): 36-42
53 Wang Y, Dong Y, Li R, et a1. Transmyocardial revascularization and vascular endothelial growth factor administration enhance effect of cell transplantation for myocardial infarct repair in rats [J]. Coron Artery Dis, 2006, 17(3): 275-281
54 Ma FX, Zhou B, Chen z, et a1. Oxidized low density lipoprotein impairs endothelial progenitor cells by regulation of endothelial nitric oxide synthase [J]. J Lipid Res, 2006, 47(6): 1227-1237
55 Kerstin S, Nikos W, Jan B, et a1. Estrogen increases bone marrow-derived endothelial progenitor cell production and diminish neointima formation [J]. Circulation, 2003, 107(24): 3059-3065
56 Fujii H, Li SH, Szmitko PE, et a1. C-reactive protein alters antioxidant defenses and promotes apoptosis in endothelial progenitor cells. Arterioscler Thromb Vasc Biol, 2006, 26 (11): 2476-82
57 Kahrstedt S, Weber H, Holtz J, et a1. Depression of progenitor cell function by advanced glycation endproducts (AGEs): Potential relevance for impaired angiogenesis in advanced age and diabetes[J]. Exp Gerontol, 2006, 41 (5): 540-548
58 Kobayashi K, Imanishi T, Akasaka T. Endothelial progenitor cell differentiation and senescence in an angiotensin II-infusion rat model [J]. Hypertens Res, 2006, 29 (6): 49-455
59 Wang CH, Ting MK, Verma S. Pioglitazone increases the numbers and improves the functional capacity of endothelial progenitor cells in patients with diabetes mellitus[ J] . Am Heart J, 2006, 152 (6): 1051
60 Madeddu P, Kraenkel N, Barcelos LS, et al. Phosphoinositide 3-kinase gamma gene knockout impairs postischemic neovascularization and endothelial progenitor cell functions [J]. Arterioscler ThrombVasc Biol 2008, 28(1): 68-76
2孙敬方.动物实验方法学.北京:人民卫生出版社, 2001
3司徒镇强,吴军正.细胞培养.西安:世界图书出版西安公司, 2004, 45
4赵春华.干细胞原理、技术与临床.北京:化学工业出版社, 2006
5谷涌泉, Royal JP. DSA检查对预测血管性截肢平面的初步研究[J].外科理论与实践, 2001, 6(5): 298-300
6侯剑峰,法宪恩,张蕾, et al.同种异体骨髓单个核细胞移植对急性心肌梗死后大鼠心肌细胞凋亡及相关基因表达的影响[J].郑州大学学报(医学版), 2007, 42(1): 78-81
7李凤奎,王纯耀.实验动物学.郑州:郑州大学出版社, 2001. 121
8 Cross ML, C1aesson-Welsh L. FGF and VEGF function in angiogenesis: signaling pathways, biological response and therapeutic inhibition [J]. Trends Pharma Sci, 2001, 22: 201-207
9 Tilton RG, Chang KC, Leieune WS, et al. Role of nitric oxide in the hyperpermeability and hemodynamic changes induced by intravenous VEGE [J]. Invest Ophthalmol Vis Sci. 1999, 40: 689-696
10 Canneliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med, 2000, 6(10): 389-395
11 Isner J, Asahara T. Angiogenesis and vasculogenesis as therapeuticstrategies for postnatal neovascularization [J]. J Clin Invest, 1999, 103 (9): 1231-1236
12 Murayama T, Teppor OM, Silver M, et a1. Determination of bone marrow-derived endothelial progenitor cell significance in angiogenic growth factor-inducde neovascularizlation in vivo [J]. Exp Hematol, 2002, 30(8): 967-972
13 Abdulaziz AK, Hilal A, Jacques G, et a1. Therapeutic angio genesis using autologous bone marrow stromla cells: improved blood flow in a chronic limb ischemia model [J]. Ann Thorac Surg, 2003, 75(1): 204-209
14 Kalka C, Macuda H, Takahashi T, et a1. Transplantation of expanded endothelial progenitor cells for therapeutic neovascularization [J]. Proc Natl Acad Sci USA, 2000, 97(7): 3422-3427
15 Tateishi-Yuyama E, Matsubara H, Murohara T, et al. Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomized controlled trial [J]. Lancet, 2002, 360 (9331): 427-435
16谷涌泉,郭连瑞,张建,等.自体骨髓于细胞移植治疗严重下肢缺血1例.中国实用外科杂志, 2003, 23: 670
17 Werner N, Kosiol S, Schiegl T, et a1. Circulating endothelial progenitor cells and cardiovascular out comes [J]. N Engl J Med, 2005, 353(10): 999
18 Huang PP, Yang XF, Li SZ, et al.VRandomised comparison of G-CSF-mobilized peripheral blood mononuclear cells versus bone marrow-mononuclear cells for the treatment of patients with lower limb arteriosclerosis obliterans [J]. Thromb Haemost, 2007, 98(6): 1335-1342
19 Iwaguro H, Yamaguchi J, Kalka C, et a1. Endothelial progenitor cell vascular endothelial growth factor gene transfer for vascular regeneration [J]. Circulation, 2002, 105(6): 732-738
20 Jakosson L, Kreuger J, Holmborn K, et al. Heparan sulfate in transpotentiates VEGFR—mediated angiogenesis [J]. Dev Cell, 2006, 1 (5): 625-634
21 Schmidtl C, Rossig L, Fichtlscherer S, et a1. Reduced number ofcirculating endothelial progenitor ceils predicts future cardiovascular events: proof of concept for the clinical importance of endogenous vascular repair [J]. Circulation, 2005, 111(22): 2981-2987.
22 Isner JM, Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization [J]. Clin Invest, 1999, 103(9): 1231
23 Ferrara N. Vascular endothelial growth factor and the regulation of angiogenesis [J]. Recent Prog Horm Res, 2000, 55: 15
24 Suzukj T, NishdaM, Frtami S, et a1. Neoendothelializalion after peripheral blood stem cell transplantation in humans: a case report of a Tokaimura nuclear accident victim [J]. Cardiovasc Res, 2003, 58(2): 487-492
25 Gehling UM, Ergün S, Schumacher U, et a1. In vitro differentiation of endothelial cells form AC133-positive progenitor cells [J]. Blood, 2000, 95(10): 3106-311
26 Sagrario O, Michael I, Stephen H, et a1. Neuronal defects and delayed wound healing in the mice lacking fibroblast growth factor2 [J]. Proc Natl Acad Sci USA, 1998, 95: 5672-5677
27 Schmidtl C, Rossig L, Fichtlscherer S, et a1. Reduced number of circulating endothelial progenitor ceils predicts future cardiovascular events:proof of concept for the clinical importance of endogenous vascular repair [J]. Circulation, 2005, 111(22): 2981-2987
28 McClung JA, Nasser N, Saleem M, et al. Circulating endothelial cells are elevated in patients with type 2 diabetes mellitus independently of HbA1c [J]. Diabetologia, 2005, 48(2): 345-350
29 Loomans CJ, de Koning EJ, Staal FJ, et al. Endothelial progenitor cell dysfunction: a novel concept in the pathogenesis of vascular complications of type 1 diabetes [J]. Diabetes, 2004, 53(1): 195-199
30 Kuki S, Imanishi T, Kobayashi K. Hyperglycemia accelerated endothelial progenitor cell senescence via the activation of p38 mitogen activated protein kinase [J]. Circ J, 2006, 70(8): 1076-1081
31 Ceradini DJ, Yao D, Grogan RH, et al. Decreasing intracellular superoxidecorrects defective ischemia-induced new vessel formation in diabeticmice [J]. Biol Chem, 2008, 283(16): 10930-10938
32 Landmesser U, Bahlmann F, Mueller M, et al. Simvastatin versus ezetimibe: pleiotropic and lipid-lowering effects on endothelial function in humans [J]. Circulation, 2005, 111: 2356-2363
33 W emer N, Priller J, Laufs U, et al. Bone m arrow-derived progenitor cells modulate vascular reendothelialization and neointimal formation:effect of 3-hydroxy-3-m ethylglutaryl coenzyme A reductase inhibition [J]. Arterioscler Thromb Vasc Biol, 2002, 22: 1567-1572
34 Assmus B, Urbich C, Aicher A, et al. HM G-CoA reductase inhibitors reduce senescence and increase proliferation of endothelial progenitor cells via regulation of cell cycle regulatory genes [J]. Circ Res, 2003, 92:1049-1055
35 Hristov M, Fach C, Becker C, et al. Reduced numbers of circulating endothelial progenitor cells in patients with coronary artery disease associated with longterm statin treatment [J]. Atherosclerosis, 2007, 192(2): 413-420
1 Badorff C, Brandes RP, Popp R, et a1. Transdifferentiation of blood—derived human adult endothelial progenitor cells into functionally active cardiomyocytes [J]. Circulation, 2003, 107(7): 1024-1032
2 Thum T, Bauersachs J. Endothelial progenitor cells as potential drug targets [J]. Curr Durg Targets Cardiovasc Haematol Disord, 2005, 5(4): 277-286
3 Werner N, Kosiol S, Schiegl T, et al. Circulating endothelial progenitor cells and cardiovascular outcomes [J]. N Engl J Med, 2005, 353(10): 999
4 Boyer M, Townsend LE, Vogel LM, et al. Isolation of endothelial cells and their progenitor cells from human peripheral blood [J]. Vasc Surg 2000, 31(1Pt1): 181-189
5 Falon P, Arentson E, Kazarov A, et a1. bFGF positively regulates hematopoitic development. Development, 2000, 127(9): 1931-194l
6 Asahara T, Murohara T, Sullivan A, et a1. Isolation of putative progenitorendothelial cells for angiogenesis [J]. Science, 1997, 275(5302): 964-967
7 Matsumoto T, Mifune Y, Kawamoto A, et a1. Fracture induced mobilization and incorporation of bone marrow-derived endothelial progenitor cells for bone healing [J]. Cell Physiol, 2008, 215 (1): 234-242
8 Au P, Daheron LM, Duda DG, et al. Differentia1in vivo potentia1 of endothelial progenitor cells from human umbilical cord blood and adult peripheral blood to form functional long-lasting vessels [J]. Blood, 2008, l11 (3): 1302-1305
9 Cherqui S, Kurian SM, Schussler O, et a1. Isolation and angiogenesis by endothelial progenitors in the fetal liver [J]. Stem Cells, 2006, 24(1): 44-54
10 Yamashita J, Itoh H, Hirashima M, et al. Flkl-positive cells derived from embryonic stem cells serve as vascular progenitors [J]. Nature, 2000, 408(6808): 92-96
11 Martinez-Estrada OM, Munoz-Santos Y, Julve J, et a1. Human adipose tissue as a source of Flk-1+ cells: new method of differentiation and expansion [J]. Cardiovasc Res, 2005, 65(2): 328-333
12 Peichev M, Naiyer AJ, Pereira D, et a1. Expression of VEGFR-2 and AC133 by circulating human CD34-cells identifies a population of functional endothelial precursors [J]. Blood, 2000, 95(3): 952-958
13 Suzukj T, NishdaM, Frtami S, et a1. Neoendothelializalion after peripheral blood stem cell transplantation in humans:a case report of a Tokaimura nuclear accident victim [J]. Cardiovasc Res, 2003, 58(2): 487-492
14 Aicher A, Rentsch M, Sasaki K, et al. Nonbone marrow-derived circulating progenitor cells contribute to postnatal neovascularization following tissue ischemia [J]. Circ Res, 2007, 100(4): 581-589
15 Domenico R, Angelo V, Beatrice N, et a1. Postnatal vasculogenesis. Mechanism of Development, 2001, 100(1): 157- 163
16 Gehling UM, Ergun S, Schumacher U, et a1. In vitro diferentiaiton of endothelial cell from AC133+ progenitor cel1 [J]. Blood, 2000, 95 (10): 3106-3112
17 Harraz M, Jiao C, Hanlon HD, et a1. CD34 - blood-derived humanendothelial cell progenitors [J]. Stem Cells, 2001, 19(4): 304-312
18 Gill M, Dias S, Hattori K, et al. Vascular trauma induces rapid but transient mobilization of VEGFR2+AC133+ endothelial precursor cells. Circ Res, 2001, 88(22): 167-174
19 Deschaseaux F, Selmani Z, Falcoz PE, et al. Two types of circulating endothelial progenitor cells in patients receiving long term therapy by HMG-CoA reductase inhibitors [J]. Eur J Pharmacol 2007; 562(1-2): 111-118
20 Kim S, Park S, Kim J, et al. Differentiation of endothelial cells from human umbilical cord blood AC133- CD14+ cells. Ann Hematol 2005; 84(7): 417-422
21 Krenning G, Dankers PY, Jovanovic D, et al. Efficient differentiation of CD14+ monocytic cells into endothelial cells on degradable biomaterials [J]. Biomaterials 2007; 28(8): 1470-1479
22 Li TS, Hamano K, Nishida M, et al. CD117+stem cells play a key role in therapeutic angiogenesis induced by bone marrow cell implantation. Am J Physiol Heart Circ Physiol 2003; 285(3): H931-937
23 Zhang W, Zhang G, Jin H, et al. Characteristics of bone marrow-derived endothelial progenitor cells in aged mice [J]. Biochem Biophys Res Commun 2006; 348(3): 1018-1023
24赵春华.干细胞原理、技术与临床.北京:化学工业出版社, 2006, 178
25 Risau W, Flamme I, et al. Vasculogenesis [J]. Annu Rev Cell Biol, 1995, 11: 73-91
26 Isner J, Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization [J]. Clin Invest, 1999, 103(9): 1231-1236
27 Tepper OM, Galiano RD, Kalka C, et al. Endothelial progenitor cells: the promise of vascular stem cells for plastic surgery [J]. Plast Reconstr Surg, 2003, 111(2): 846-854
28 Hofer T, Desbaillets I, Hopfl G, et al. Dissecting hypoxia-dependent and hypoxia-independent steps in the HIF-1αactivation cascade: implicationsfor HIF-1αgene therapy [J]. Faseb J, 200l, 15(14): 2715-2717
29 Heissig B, Hattori K, Dias S, et al. Recruitment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kit-ligand [J]. Cell, 2002, l09(5): 625-637
30 Levesque JP, Takamatsu Y, Nilsson SK, et a1. Vascular cell adhesion molecule-1(CD106)is cleaved by neutrophil proteases in the bone marrow following hematopoietic progenitor cell mobilization by granulocyte colony-stimulating factor [J]. Blood, 200l, 98(5): 1289-1297
31 Ceradini DJ, Kulkarni AR, Callaghan MJ, et a1. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1induction of SDF-1 [J]. Nat Med, 2004, l0(8): 858-864
32 Hiasa K, Ishibashi M, Ohtani K, et a1. Gene transfer of stromla cell-derived factor--1enhances ischemic vasculogenesis and angiogenesis via vascular endothelial growth factor / endothelial nitrie oxide synthase-related pathway [J]. Circulation, 2004, 109(20): 2454-2461
33 Mlhle R, Bsutz F, Rsfii S, et a1. The chemokine receptor CXCR-4 is expressed on CD34+ hematopoietic progenitors and leukemic cells and mediates transendothelial migration induced by stromal cell-derived factor [J]. Blood, 1998, 91(12): 4523-4530
34 Quesenberry PJ, Becker PS. Stem cell homi ng: Rolling, crawling, and nesting. Proc Natl Acad Sci USA, 1998, 95 (26): 15155-151557
35 Walter DH, Rittig K, Bahlmann FH, et a1. Statin therapy accelerates reendothelialization: a novel effect involving mobilization and incorporation of bone marrow derived endothelial progenitor cells [J]. Circulation, 2002, 105(25): 3017-3024
36 Grunewald M, Avraham I, Dor Y, et a1. VEGF-induced adult neovascularizaiton:recruitment,retention,and role of accessory cells [J]. Cell, 2006, l24(1): 175-89
37 Butler JM, Guthrie SM, Koc M, et a1. SDF-1 is both necessary and sufficient to promote proliferative retinopathy [J]. Clin Invest, 2005, ll5(1): 86-93
38 Cao Y, Hong A, Schulten H, et al. Update on therapeutic neovascularization [J]. Cardiovasc Res, 2005: 65(3): 639-648
39 Rsfii S, Meeus S, Dias S, et a1. Contribution of marrow-derived progenitors to vascular and cardiac regeneration [J]. Semin Cel1 Dev Bio1, 2002, 13(1): 61-67
40 Wang JF, Park IW, Groopman JE. Stromal cell-derived factor-1alpha stimulates tyrosine phosphory1ation of multiple focal adhesion proteins and induces migration of hematopoietic progenitor cells: roles of phosphoinositide-3 kinase and protein kinase C [J]. Blood, 2000, 95(8): 2505-2513
41 Mace KA, Yu DH, Paydar KZ, et al. Sustained expression of Hif-1alpha in the diabetic environment promotes angiogenesis and cutaneous wound repair [J]. Wound Repair Regen, 2007, 15(5): 636-645
42 Phillpott NJ, Prue RI, Mash JC, et a1. G-CSF-mobilized CD34 peripheral blood stem cells are significantly less apoptotic than unstimulated peripheral blood CD34 cells: role of G-CSF as survival factor [J]. Br J Haematol, 1997, 97(1): 146-152
43 Heeschen C, Aicher A, Lehmann R. Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization [J]. Blood, 2003, 102: 1340-1346
44 Thomas T, Sarah H, Sabrina F, et al. Age-dependent impairment of endothelial progenitor cells is corrected by growth hormone mediated increase of insulin-like growth factor-1 [J]. Circ Res, 2007, 100(3): 434-443
45 Vantle M, Huntgeburth M, Caglayen E, et a1. PI3-kinase/Akt-dependent antiapoptotic signaling by the PDGF alpha receptor is negatively regulated by src family kinases [J]. FEBS Lett, 2006, 580(30): 6769-6776
46 McClung JA, Nasser N, Saleem M, et al. Circulating endothelial cells are elevated in patients with type 2 diabetes mellitus independently of HbA1c [J] . Diabetologia, 2005, 48(2): 345-350
47 Capla JM, Grogan RH. Diabetes impairs endothelial progenitor cellsmediated blood vessel formation in response to hypoxia [J] . Plast Reconstr Surg, 2007, 119(1): 59-70
48 Kuki S, Imanishi T, Kobayashi K. Hyperglycemia accelerated endothelial progenitor cell senescence via the activation of p38 mitogen activated protein kinase [J]. Circ J, 2006, 70(8): 1076-1081
49 Ceradini DJ, Yao D, Grogan RH, et al. Decreasing intracellular superoxide corrects defective ischemia-induced new vessel formation in diabeticmice [J] . J Biol Chem, 2008, 283(16): 10930-10938
50 Schmidtl C, Rossig L, Fichtlscherer S, et a1. Reduced number of circulating endothelial progenitor ceils predicts future cardiovascular events: proof of concept for the clinical importance of endogenous vascular repair [J]. Circulation, 2005, 111(22): 2981-2987
51 Vasa M, Fichtlscherer S, Aicher, et a1. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronaryartery disease [J]. Circ Res, 2001, 89(1): E1-E7
52 Damas JK, WahreT, Yndestad A, et a1. Stromal cell-derived factor-1 alpha in unstable angina: potential antiinflammatory and matrix-stabilizing effects [J]. Circulation, 2002, 106 (1): 36-42
53 Wang Y, Dong Y, Li R, et a1. Transmyocardial revascularization and vascular endothelial growth factor administration enhance effect of cell transplantation for myocardial infarct repair in rats [J]. Coron Artery Dis, 2006, 17(3): 275-281
54 Ma FX, Zhou B, Chen z, et a1. Oxidized low density lipoprotein impairs endothelial progenitor cells by regulation of endothelial nitric oxide synthase [J]. J Lipid Res, 2006, 47(6): 1227-1237
55 Kerstin S, Nikos W, Jan B, et a1. Estrogen increases bone marrow-derived endothelial progenitor cell production and diminish neointima formation [J]. Circulation, 2003, 107(24): 3059-3065
56 Fujii H, Li SH, Szmitko PE, et a1. C-reactive protein alters antioxidant defenses and promotes apoptosis in endothelial progenitor cells. Arterioscler Thromb Vasc Biol, 2006, 26 (11): 2476-82
57 Kahrstedt S, Weber H, Holtz J, et a1. Depression of progenitor cell function by advanced glycation endproducts (AGEs): Potential relevance for impaired angiogenesis in advanced age and diabetes[J]. Exp Gerontol, 2006, 41 (5): 540-548
58 Kobayashi K, Imanishi T, Akasaka T. Endothelial progenitor cell differentiation and senescence in an angiotensin II-infusion rat model [J]. Hypertens Res, 2006, 29 (6): 49-455
59 Wang CH, Ting MK, Verma S. Pioglitazone increases the numbers and improves the functional capacity of endothelial progenitor cells in patients with diabetes mellitus[ J] . Am Heart J, 2006, 152 (6): 1051
60 Madeddu P, Kraenkel N, Barcelos LS, et al. Phosphoinositide 3-kinase gamma gene knockout impairs postischemic neovascularization and endothelial progenitor cell functions [J]. Arterioscler ThrombVasc Biol 2008, 28(1): 68-76