| 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.
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