【作者】 周晨光；

【导师】 孟建波；

【作者基本信息】 河北医科大学 ， 内科学， 2010， 硕士

【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。更多还原

【Abstract】 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 TransplantationObjective: 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 RatsObjective: 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.更多还原