| Objective:To establish a simple and cheap method to culture EPCs and fulfill the needs of EPCs based on cell therapy in patients with diabetes; to investigate different subpopulations of endothelial progenitor cells, such as CD34+, CD133+, CD133+KDR+cells in patients with type2diabetes mellitus (T2DM), with or without diabetic complications; to contrast the express of TLR4and eNOS in diabetic rats’ and normal rats’ EPCs, and observe the impact of the LPS stimulation to TLR4/NF-kappa B in EPCs; to compare number and function of EPCs after co-culture with SDF-1alpha, and the curative effect of transplantation using EPCs co-cultured with SDF-1alpha. Methods: Separate mononuclear cells from human peripheral blood mononuclear cells and umbilical cord blood mononuclear cells by modified density-gradient centrifugation and hydroxyethyl starch combination with density-gradient centrifugation, and they were cultured in endothelial cell (EC) basal medium-2(EBM-2, Clonetics) for7days.Different EPC subpopulations were enumerated by flow cytometry using triple staining (CD34+, CD133+, KDR+).150patients with T2DM and32controls were enrolled in a case-control study. Another part of the test is to observe rat bone marrow mononuclear cells derived endothelial progenitor cells:1) The proliferation and adhesion of EPCs from diabetic rats and normal rats were measured. The expression of TLR4in these two derived EPCs were analyzed by flow cytometry, the concentration of TNF alpha and IL-6 in the cell culture medium were detected by ELISA, the amount of eNOS produced by EPCs were measured by spectrophotometric method. In vitro, EPCs, macrophages and a mixture of this two kinds of cells were co-cultured with different concentration of LPS in the culture medium, TLR4, NF-kappa B, IL-6and TNF alpha were detected;2) The proliferation and adhesion ability of EPCs were detected after co-cultured with SDF-1alpha (10ng/mL) for48hours. Diabetes model were induced by STZ injection, The rats were anesthetized with sodium pentobarbital.Lower limb ischemia was induced by ligating the right femoral artery. Immediately, the EPCs, co-cultured with SDF-1alpha, were intramuscular injection, after28days, limb vascular were observed by angiography. Results:1) Characterization of peripheral blood derived EPCs and umbilical cord blood derived EPCs. We observed that different derived EPCs changed morphology and size over time within the cultures, peripheral blood derived EPCs have cell colony formation and umbilical cord blood derived EPCs form spindle cells, which arranged forming typical line structure.②The survival rate of peripheral blood and umbilical cord blood EPCs were97.6%and99.3%, respectively.③The number of EPCs Source of cord blood cells higher than that of peripheral blood.④The proliferation rate of umbilical cord blood derived endothelial progenitor cells is significantly higher than that of peripheral blood EPCs.⑤By flow cytometry detection, two types of cells express CD133, CD34and VEGR-2. CD34+cells in Umbilical cord blood EPC is higher.⑥Immunofluorescence detection FITC-UEA1and Dil-acLDL shows double-positive.2) The number of EPCs were lower in patients with diabetes than control. The concentration of LDL-c and urine microalbumin, the number of BMI and waist were negative correlation with number of EPCs in patients with diabetes. And in patients with PAD, circulating CD34+, CD133+, CD133+KDR+EPCs showed a significant decline with advancing stages of nephropathy. But the number of CD34+cells decreased slowly with advancing stages of nephropathy, CD133+and CD133+KDR+EPCs just declined in patients with massive albuminuria.3) Characterization of bone marrow mononuclear cell derived EPCs in diabetes rat and control rat.. The number of EPCs in diabetic rats was decreased significantly than normal control rats (29.21+/-1.56vs.49.08+5.83)/(view x200)(P<0.05), the proliferation and adhesion ability of EPCs in diabetes rat was lower than that in normal control group (0.51+0.05vs.0.69+/-0.02)(28.03+/-3.51vs.46.24+2.51)/(view x200)(P<0.05). eNOS activity of EPCs in diabetes rat was significantly lower than EPC in normal control rat (1.47+/-0.46vs.3.23+/-0.74U/ml)(P<0.05). TLR4expression of EPCs in diabetes rats is significantly higher than control (3.8%vs 1.6%), concentration of IL-6and TNF alpha were higher than control, in which, IL-6expression between the two groups was statistically difference, IL-6(pg/ml),1039±28vs675±32, P<0.05; TNF-α(pg/ml),216±31vs198±29. After co-culture with LPS, TLR4expression in EPCs is higher than macrophages, but with concentration of LPS increase, TLR4expression rate increased slightly. So, we found that lower concentration of LPS (10ng/ml) can have effective stimulus on EPCs; there followed higher concentration of TNF alpha and IL-6, especially IL-6.4) SDF-1alpha (10ng/mL) can significantly increase the proliferation and adhesion ability of EPCs. Cell transplantation for the treatment of lower limb ischemia in diabetic rats, the results showed that treatment effect using EPCs co-cultured with SDF-1is better than that of using EPCs or saline.Total antioxidant capacity, eNOS and nitric oxide of EPCs co-cultured with SDF-1were higher than using EPCs or saline. Conclusion:1) Cord blood derived EPC has higher proliferation and expansion ability, which has better potential for transplantation.2) The number of different subpopulations of EPCs in T2DM patients with different diabetic complications were not declined simply.3) The number, the adhesion and proliferation ability of EPCs were impaired in patients with2diabetes. In diabetes, TLR4express higher in EPCs, with reduced secretion of eNOS, which makes the cells in a low-grade inflammatory state. LPS stimulation can produce more TLR4expression in EPCs, and there is proinflammatory synergy effect when EPCs co-cultured with macrophage.4) SDF-1alpha can significantly enhance the adhesion and proliferation ability of EPCs, this ability have similar effect on diabetic rats.5) SDF-1alpha and EPCs transplantation after cocultivation, improved the curative effect of lower limb ischemia in diabetic rats., the SDF-1alpha improved mobilization and homing of EPCs with diabetic state. |
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