高糖诱导氧化应激对大鼠内皮祖细胞VEGFR表达的影响
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摘要
目的：糖尿病(Diabetes Mellitus, DM)已经成为继肿瘤、心血管疾病之后第三大严重威胁人类健康的慢性疾病。DM合并血管病变是DM的主要并发症之一,也是患者致死、致残的主要原因。目前的研究认为DM大血管病变后侧枝形成能力受损是引起DM患者血管病变最重要原因的之一。关于DM合并血管病变的发病机制一直是研究的热点,既往提出四种机制,即多元醇通路的激活、晚期糖基化终末产物(Advanced glycation end products, AGEs)的形成、PKC途径及氨基已糖途径的激活,这些均不能够完全解释DM血管并发症的发病机制。最近的研究表明,氧化应激参与了心血管疾病发生、发展过程,Banting奖获得者Brownlee M提出“糖尿病并发症的共同机制”学说,指出线粒体电子传递链过氧化物产生过量是高血糖导致血管损伤的共同机制。该学说合理解释了氧化应激与糖尿病血管损伤的内在联系,被认为是糖尿病并发症研究领域的一个突破性进展,获得了广泛关注。氧化应激增强所致的血管病变与DM患者血管病变临床后果及其病理改变相似,都涉及到内皮祖细胞(endothelial progenitor cells, EPCs)增殖能力和新生血管的形成能力受损。血管内皮生长因子(vascular endothelial growth factor, VEGF)是血管新生最重要的因子之一,有人研究认为VEGF分泌减少导致了血管新生功能障碍,但在DM大鼠后肢缺血模型中发现VEGF的分泌并未减少,研究还发现VEGF治疗DM合并的缺血性疾病效果更差。提示DM患者肢体缺血后血管快速血运重建受阻与VEGF无关。目前,高糖对EPCs自身氧化应激水平的影响以及这种影响对EPCs表面血管内皮细胞生长因子受体(vascular endothelial growth factor Receptor VEGFR)表达产生的影响国内外还尚无相关研究。我们利用大鼠淋巴细胞分离液分离大鼠骨髓冲洗液中的单个核细胞,培养3天后用Dil-标记的乙酰化低密度脂蛋白(Dil-labeled acetylated LDL, DiI-acLDL)和FITC标记荆豆凝集素-1 (fluorescein isothiocyanate-labeled Ulex europaeus agglutinin-1, FITC-UEA-1)双染法进行的鉴定,显示双荧光阳性的细胞被认为是内皮祖细胞(Endothelial progenitor cells, EPCs)。完成鉴定后的大鼠EPCs给予不同浓度(培养液终末葡萄糖浓度分别15mmol/L、30mmol/L、60mmol/L)的高糖干预,分别在24h、48h、96h进行EPCs增殖能力检测和自身氧化应激标志物的检测,评估高糖对EPCs增殖和自身氧化应激水平的影响。最后,观察高糖对大鼠EPCs表面VEGFR表达的影响,并通过抗氧化剂干预治疗研究,初步探讨高糖诱导的氧化应激对EPCs表面VEGFR表达的影响,为糖尿病患者肢体缺血后快速血运重建受阻提供理论支持。
方法：将12只SD大鼠脱颈处死,用75%酒精浸泡消毒15 min。置于超净台中,在无菌条件下分离其股骨和胫骨,剪去长骨两端,暴露骨髓腔,用5ml注射器抽取含有1%肝素的PBS液冲洗骨髓腔,直至骨髓腔发白。利用大鼠淋巴细胞分离液分离大鼠骨髓冲洗液中的单个核细胞,培养3天后用DiI-acLDL和FITC-UEA-1双染法进行的鉴定,显示双荧光阳性的细胞被认为是内皮祖细胞(Endothelial progenitor cells, EPCs)。5天后进行细胞传代和试验分组,根据培养液中终末葡萄糖浓度的不同分成两组：正常对照组(培养液终末葡萄糖浓度为5.5mmol/L)和高糖干预组。高糖干预组又分为三个亚组：高糖1组(培养液终末葡萄糖浓度为15 mmol/L葡萄糖)；高糖2组(培养液终末葡萄糖浓度为30mmol/L);高糖3组(培养液终末葡萄糖浓度为60mmol/L)。分别在24h、48h、96h用MTT法进行EPCs增殖检测和检测试剂盒进行氧化应激标志物抗02-、丙二醛(Maleic Dialdehyde, MDA)、谷胱甘肽(glutathione, GSH)检测,最后在有和无抗氧化剂的治疗干预下用免疫组化进行EPCs VEGFR表达检测。
结果：1.光镜下大鼠EPCs的形态观察发现,刚分离的单个核细胞呈圆形,体型较小,在培养2天后出现贴壁,3d后有明显集落形成,可见梭形细胞和细胞簇出现,其结构与血岛相似,梭形细胞既有单个细胞也有线带样结构。7d后梭形细胞线样排列,随培养时间增加,细胞形态逐渐变大,呈现出典型铺路石样改变。2.培养3d后的细胞呈现典型的Dil-acLDL和FITC-UEA-1双阳性染色,证实为EPCs。3. EPCs增殖能力检测。高糖作用24h后与正常对照组(5.5 mmol/L)比较,高糖3组(60 mmol/L)的EPCs增殖能力显著增强(P<0.05),高糖1组(15mmol/L)和高糖2组(30 mmol/L)无明显差异；高糖作用48h后,高糖2组和高糖3组EPCs增殖能力显著减弱(P<0.05)；作用96h后,高糖2组和高糖3组EPCs增殖能力进一步减弱(P<0.05),正常对照组与高糖1组无明显差异。各实验组不同时间点与24h比较,正常对照组和高糖1组在48h和96h EPCs增殖能力分别显著增强(P<0.05)；高糖2组EPCs增殖能力无明显差异；高糖3组EPCs增殖能力在96h显著减弱(P<0.05)。4. EPCs抗O2-浓度检测。高糖作用24h后与正常对照组比较,高糖1组的EPCs抗O2-浓度明显增加(P<0.05)；作用48h后与正常对照组比较,高糖干预组EPCs抗O2-浓度明显减少(P<0.05)；作用96h后与正常对照组比较高糖干预组EPCs抗O2-浓度进一步减少(P<0.05)。各实验组不同时间点与24h相比,正常对照组在48h后抗O2-浓度明显增加(P<0.05)；高糖1组无明显差异；高糖2组和高糖3组在48h和96h抗O2-浓度均明显减少(P<0.05)。5.EPCs MDA浓度检测。高糖作用24h后与正常对照组比较,高糖干预组EPCs MDA浓度均无明显差异；作用48h后,高糖2组和高糖3组EPCs MDA浓度显著增加(P<0.05)；作用96h后,高糖干预组EPCs MDA浓度均显著增加(P<0.05)。各实验组不同时间点与24h比较,正常对照组在48h和96hEPCs MDA浓度均无明显变化；高糖1组在96h后EPCs MDA浓度显著增加(P<0.05)；高糖2组和高糖3组在48h和96h后EPCs MDA浓度均显著增加(P<0.05)。6.EPCs的GSH含量检测。高糖作用24h后与正常对照组比较,高糖干预组EPCs的GSH含量无明显变化；作用48h和96h后高糖2组和高糖3组EPCs的GSH含量均显著降低(P<0.05)；高糖1组无明显变化。各实验组不同时间点与24h相比,正常对照组在48h和96h后EPCs的GSH含量均显著增加(P<0.05)；高糖1组EPCs的GSH含量无明显差异；高糖2组和高糖3组在48h和96h后EPCs的GSH含量均显著减弱(P<0.05)。7.EPCs VEGFR表达检测。(1).高糖分别作用24h和48h后与正常对照组比较,高糖2组和高糖3组EPCs VEGFR表达均显著减少(P<0.05)；作用96h后,与正常对照组比较高糖干预组EPCs VEGFR表达均明显减少(P<0.05)。各实验组不同时间点与24h相比,正常对照组无明显变化；高糖1组和高糖3组在96h后EPCs VEGFR表达明显减少(P<0.05)；高糖2组EPCs VEGFR表达在48h和96h后均明显减少(P<0.05)。(2).在有抗氧化剂存在条件下,各实验组EPCs VEGFR表达随时间,浓度变化没有明显的差异。(3).与无抗氧化剂比较,抗氧化剂提高了高糖干预组EPCs VEGFR表达,在一定的程度上挽救了高糖干预组EPCs VEGFR表达的严重缺陷。
结论：1.高糖明显的抑制了大鼠EPCs增殖,且随着葡萄糖浓度的增加和作用时间的延长抑制作用愈来愈强。2.高糖诱导大鼠EPCs自身氧化应激增强,抗氧化应激明显减弱。且随葡萄糖浓度的增加,时间的延长作用愈来愈强。3.高糖明显抑制大鼠EPCs VEGFR的表达,但在抗氧化剂的治疗干预下,明显提高了高糖干预组EPCs VEGFR的表达,并在一定程度上挽救了高糖干预组EPCs VEGFR表达的严重缺陷。提示大鼠EPCs自身的氧化应激增强可能是抑制大鼠EPCs VEGFR表达的重要原因之一。
bjective:Diabetes has become the third largest serious chronic diseases threated to human health, after the cancer and cardiovascular disease. Vascular disease is one of the major complications in diabetes, as well as the main cause for disability and death. The current studies have suggested that collateral formation impaired is one of the most important reasons of diabetic vascular disease. Many pathogenesis have proposed on diabetic vascular disease, including four the mechanism—activation of polyol pathway、PKC pathway、and amino sugar pathway and formation of AGEs, for its own shortcomings, all of them can not fully explain diabetic vascular complications. Recent studies have shown that oxidative stress involved in the occurrence and development of cardiovascular disease, and Banting Award winner Brownlee.M has proposed the theory of "common mechanism of diabetic vascular complications", which points out that an excessive peroxidase in mitochondrial electron transport chain is common mechanism of vascular injury caused by hyperglycemia. the theory can reasonably explain the internal relations of oxidative stress and diabetic vascular injury in diabetic complications, as has been considered a breakthrough in the field and has caused wide public concern. Vascular disease caused by enhancing oxidative stress and vascular disease in diabetic patients have the same pathological changes and the clinical consequences, all relating to the EPC proliferation impaired and formation of new blood vessels impaired. VEGF is one of the most important angiogenesis factor, some studies once suggested that angiogenesis dysfunction is caused by VEGF reduced, but in the diabetic ischemic hindlimb model, many studies found VEGF secretion did not decrease. Many studies also found that treatment of diabetic ischemic disease with VEGF are less effective. It is suggested the rapid revascularization blocked after limb ischemia in diabetic patients has nothing to do with the VEGF. Currently, the level of oxidative stress in EPCs and the expression of VEGFR in EPCs affected by the high glucose have no studies at home and abroad, So this experiment were designed. The rat bone marrow mononuclear cells isolated by using rat lymphocyte separation medium fluid, and the cell isolated and cultured were identified applaying with DiI-acLDL and FITC-UEA-1 double staining afrer 3 days. dual fluorescence-positive cells were considered to be endothelial progenitor cells (EPCs). EPCs identified were treated with various concentrations of high glucose (terminal glucose concentration in medium 15 mmol/L,30 mmol/L,60 mmol/L respectively), to detect the proliferation in EPCs and the markers of oxidative stress in EPCs at 24h、48h、96h respectively to assess the level of oxidative stress and the proliferation in EPCs affected by high glucose. Finally, the expression of VEGFR in EPCs affected by high glucose were observed, and through comparing with antioxidant treatment, Preliminary discussing high glucose induced oxidative stress on expression of VEGFR in EPCs, to provide theoretical support for diabetic ischemic limb revascularization blocked.
Methods:12 SD rats were harrested, immersed in 75% alcohol for 15 minites. Place rats on clean workbench, isolate the femur and tibia under sterile conditions, cut off the ends of long bones, expose bone marrow cavity, extract PBS containing 1% heparin with 5ml Syringe to wash the marrow cavity, use rat lymphocyte separation medium fluid to isolate the rat bone marrow mononuclear cells. the cell were identified applaying with DiI-acLDL and FITC-UEA-1 double staining afrer 3 days. dual fluorescence-positive cells were considered to be endothelial progenitor cells (EPCs). Subcultured and divided groups after 5 days, according to the various glucose concentrations,the cell were divided into two groups:the control group (terminal glucose concentration in medium was 5.5 mmol/L) and the high glucose intervention group. The high glucose intervention group were divided into three subgroups:high glucose group 1 (terminal glucose concentration in medium was 15 mmol/L glucose); high glucose group 2 (terminal glucose concentration in medium was was 30 mmol/L); high glucose group 3 (terminal glucose concentration in medium was was 60 mmol/L). The proliferation in EPCs were detected by MTT method and the markers of oxidative stress as anti-02-、MDA、GSH in EPCs were detected by the colorimetric method at 24h、48h、96h respectively. Finally, the expression of VEGFR in EPCs were detected by immunohistochemical method with and without the intervention of antioxidant therapy.
Results:1. Cell morphology of EPCs was observed with light microscope, mononuclear cells just isolated were round, a little. Cultural mononuclear cells started to appear adherent growth after 2 days and started to appear colony formation after 3 days, and spindle cells and cell clusters can be seen clearly,and its structure was similar to blood island, and spindle cells were both wired -like structure and single cell. The line-like arrangement of spindle cells, and with time increasing, the cells become bigger, appearing a typical cobblestone-like changes after 7 days.2. The cultural cell appeared typical DiI-acLDL and FITC-UEA-1 double positive staining after 3 days.3. The high glucose intervention group compared with the control group at 24h point, the proliferation in EPCs were significantly enhanced in high glucose groups 3 (P<0.05); the high glucose intervention group compared with the control group at 48h point, the proliferation in EPCs were significantly reduced in high glucose group 2 and group 3 (P<0.05); the high glucose intervention group compared with the control group at 96h point, the proliferation in EPCs was significantly reduced further in high glucose group 2 and group 3 (P <0.05), and control group and high glucose group 1 have no significant difference. Different time points in each experimental group compared with the 24h point, the proliferation in EPCs was significantly enhanced (P<0.05) at the 48h and 96h point in high glucose group 1; the proliferation in EPCs has no significant difference in high glucose group 2; the proliferation in EPCs were significantly decreased at the 96h point in high glucose group 3 (P<0.05).4. The high glucose intervention group compared with the control group at 24h point, the concentration of anti-O2-in EPCs significantly increased in high glucose group 1 (P<0.05); the high glucose intervention group compared with the control group at 48h point, the concentration of anti-O2- in EPCs significantly reduced in high glucose intervention group(P<0.05); the high glucose intervention group compared with the control group at 96h point, the concentration of anti-O2- in EPCs was significantly reduced in high glucose intervention group (P<0.05). Different time points in each experimental group compared with the 24h point, the concentration of anti-O2- in EPCs significantly increased at the 48h point in the control group (P<0.05); the concentration of anti-O2- in EPCs had no significant difference in high glucose group 1; the concentration of anti-O2- in EPCs reduced in high glucose group 2 and group 3 at the 48h and 96h point (P<0.05).5. the high glucose intervention group compared with the control group at 24h point, the concentrations of MDA in EPCs had no significant difference in high glucose intervention group; the high glucose intervention group compared with the control group at 48h point, the concentrations of MDA in EPCs increased significantly in high glucose group 2 and group 3 (P<0.05); the high glucose intervention group compared with the control group at 96h point, the concentrations of MDA in EPCs significantly increased in high glucose intervention group (P<0.05). Different time points in each experimental group compared with the 24h point, the concentrations of MDA in EPCs had no significant difference in control group at the 48h and 96h point; the concentrations of MDA in EPCs increased significantly in high glucose group 1 at the 96h point (P<0.05); the concentrations of MDA in EPCs increased significantly in high glucose group 2 and group 3 at the 48h and 96h point (P<0.05).6. the high glucose intervention group compared with the control group at 24h point, the concentration of GSH in EPCs have had no significant difference in high glucose intervention group; the concentration of GSH in EPCs were significantly decreased in the high glucose group 2 and group 3 at 48h and 96h point (P<0.05); the concentration of GSH in EPCs have had no significant difference in high glucose group 1. Different time points in each experimental group compared with the 24h point, the concentration of GSH in EPCs significantly increased in the control group at the 48h and 96h point (P<0.05); the concentration of GSH in EPCs had no significant difference in high glucose group 1; the concentration of GSH in EPCs significantly reduced in high glucose group 2 and group 3 at the 48h and 96h point (P<0.05).7. (1). High glucose intervention group compared with the control group at 24h and 48h point respectively, the expression of VEGFR in EPCs significantly reduced (P<0.05) in the high glucose group 2 and group 3; high glucose intervention group compared with the control group at 96h point, the expression of VEGFR in EPCs significantly decreased in the high glucose intervention group (P<0.05). Different time points in each experimental group compared with the 24h point, the expression of VEGFR in EPCs had no significant difference in the control group; the expression of VEGFR in EPCs significantly reduced in high glucose group 1 and group 3 at the 96h point (P<0.05); the expression of VEGFR in EPCs significantly decreased in high glucose group 2 at the 48h and 96h point (P<0.05). (2). Under antioxidants therapy, the expression of VEGFR in EPCs had no significant difference in each experimental group with the time and concentration increasing. (3). Antioxidants therapy compared with non-antioxidants therapy, the expression of VEGFR in EPCs significantly increased in high glucose intervention group, in a certain extent, the serious defects of expression of VEGFR in EPCs saved by antioxidants therapy in the intervention group.
Conclusion:1. High glucose significantly inhibite the proliferation in EPCs, and with the glucose concentrations and time increasing,the role of inhibition become stronger and stronger.2. Oxidative stress induced by high glucose in EPCs significantly increase, and anti-oxidation significantly decreased, with the concentration and time increasing, the role of oxidative stress become stronger and stronger.3. High glucose significantly inhibite the expression of VEGFR in EPCs, But under antioxidants therapy, expression of VEGFR in EPCs in high glucose significantly increase, in a certain extent, the serious defects of expression of VEGFR in EPCs saved by antioxidants therapy in the intervention group. Suggesting that increasing own oxidative stress in EPCs is one of the important reasons which inhibited the expression of VEGFR in EPCs.
方法：将12只SD大鼠脱颈处死,用75%酒精浸泡消毒15 min。置于超净台中,在无菌条件下分离其股骨和胫骨,剪去长骨两端,暴露骨髓腔,用5ml注射器抽取含有1%肝素的PBS液冲洗骨髓腔,直至骨髓腔发白。利用大鼠淋巴细胞分离液分离大鼠骨髓冲洗液中的单个核细胞,培养3天后用DiI-acLDL和FITC-UEA-1双染法进行的鉴定,显示双荧光阳性的细胞被认为是内皮祖细胞(Endothelial progenitor cells, EPCs)。5天后进行细胞传代和试验分组,根据培养液中终末葡萄糖浓度的不同分成两组：正常对照组(培养液终末葡萄糖浓度为5.5mmol/L)和高糖干预组。高糖干预组又分为三个亚组：高糖1组(培养液终末葡萄糖浓度为15 mmol/L葡萄糖)；高糖2组(培养液终末葡萄糖浓度为30mmol/L);高糖3组(培养液终末葡萄糖浓度为60mmol/L)。分别在24h、48h、96h用MTT法进行EPCs增殖检测和检测试剂盒进行氧化应激标志物抗02-、丙二醛(Maleic Dialdehyde, MDA)、谷胱甘肽(glutathione, GSH)检测,最后在有和无抗氧化剂的治疗干预下用免疫组化进行EPCs VEGFR表达检测。
结果：1.光镜下大鼠EPCs的形态观察发现,刚分离的单个核细胞呈圆形,体型较小,在培养2天后出现贴壁,3d后有明显集落形成,可见梭形细胞和细胞簇出现,其结构与血岛相似,梭形细胞既有单个细胞也有线带样结构。7d后梭形细胞线样排列,随培养时间增加,细胞形态逐渐变大,呈现出典型铺路石样改变。2.培养3d后的细胞呈现典型的Dil-acLDL和FITC-UEA-1双阳性染色,证实为EPCs。3. EPCs增殖能力检测。高糖作用24h后与正常对照组(5.5 mmol/L)比较,高糖3组(60 mmol/L)的EPCs增殖能力显著增强(P<0.05),高糖1组(15mmol/L)和高糖2组(30 mmol/L)无明显差异；高糖作用48h后,高糖2组和高糖3组EPCs增殖能力显著减弱(P<0.05)；作用96h后,高糖2组和高糖3组EPCs增殖能力进一步减弱(P<0.05),正常对照组与高糖1组无明显差异。各实验组不同时间点与24h比较,正常对照组和高糖1组在48h和96h EPCs增殖能力分别显著增强(P<0.05)；高糖2组EPCs增殖能力无明显差异；高糖3组EPCs增殖能力在96h显著减弱(P<0.05)。4. EPCs抗O2-浓度检测。高糖作用24h后与正常对照组比较,高糖1组的EPCs抗O2-浓度明显增加(P<0.05)；作用48h后与正常对照组比较,高糖干预组EPCs抗O2-浓度明显减少(P<0.05)；作用96h后与正常对照组比较高糖干预组EPCs抗O2-浓度进一步减少(P<0.05)。各实验组不同时间点与24h相比,正常对照组在48h后抗O2-浓度明显增加(P<0.05)；高糖1组无明显差异；高糖2组和高糖3组在48h和96h抗O2-浓度均明显减少(P<0.05)。5.EPCs MDA浓度检测。高糖作用24h后与正常对照组比较,高糖干预组EPCs MDA浓度均无明显差异；作用48h后,高糖2组和高糖3组EPCs MDA浓度显著增加(P<0.05)；作用96h后,高糖干预组EPCs MDA浓度均显著增加(P<0.05)。各实验组不同时间点与24h比较,正常对照组在48h和96hEPCs MDA浓度均无明显变化；高糖1组在96h后EPCs MDA浓度显著增加(P<0.05)；高糖2组和高糖3组在48h和96h后EPCs MDA浓度均显著增加(P<0.05)。6.EPCs的GSH含量检测。高糖作用24h后与正常对照组比较,高糖干预组EPCs的GSH含量无明显变化；作用48h和96h后高糖2组和高糖3组EPCs的GSH含量均显著降低(P<0.05)；高糖1组无明显变化。各实验组不同时间点与24h相比,正常对照组在48h和96h后EPCs的GSH含量均显著增加(P<0.05)；高糖1组EPCs的GSH含量无明显差异；高糖2组和高糖3组在48h和96h后EPCs的GSH含量均显著减弱(P<0.05)。7.EPCs VEGFR表达检测。(1).高糖分别作用24h和48h后与正常对照组比较,高糖2组和高糖3组EPCs VEGFR表达均显著减少(P<0.05)；作用96h后,与正常对照组比较高糖干预组EPCs VEGFR表达均明显减少(P<0.05)。各实验组不同时间点与24h相比,正常对照组无明显变化；高糖1组和高糖3组在96h后EPCs VEGFR表达明显减少(P<0.05)；高糖2组EPCs VEGFR表达在48h和96h后均明显减少(P<0.05)。(2).在有抗氧化剂存在条件下,各实验组EPCs VEGFR表达随时间,浓度变化没有明显的差异。(3).与无抗氧化剂比较,抗氧化剂提高了高糖干预组EPCs VEGFR表达,在一定的程度上挽救了高糖干预组EPCs VEGFR表达的严重缺陷。
结论：1.高糖明显的抑制了大鼠EPCs增殖,且随着葡萄糖浓度的增加和作用时间的延长抑制作用愈来愈强。2.高糖诱导大鼠EPCs自身氧化应激增强,抗氧化应激明显减弱。且随葡萄糖浓度的增加,时间的延长作用愈来愈强。3.高糖明显抑制大鼠EPCs VEGFR的表达,但在抗氧化剂的治疗干预下,明显提高了高糖干预组EPCs VEGFR的表达,并在一定程度上挽救了高糖干预组EPCs VEGFR表达的严重缺陷。提示大鼠EPCs自身的氧化应激增强可能是抑制大鼠EPCs VEGFR表达的重要原因之一。
bjective:Diabetes has become the third largest serious chronic diseases threated to human health, after the cancer and cardiovascular disease. Vascular disease is one of the major complications in diabetes, as well as the main cause for disability and death. The current studies have suggested that collateral formation impaired is one of the most important reasons of diabetic vascular disease. Many pathogenesis have proposed on diabetic vascular disease, including four the mechanism—activation of polyol pathway、PKC pathway、and amino sugar pathway and formation of AGEs, for its own shortcomings, all of them can not fully explain diabetic vascular complications. Recent studies have shown that oxidative stress involved in the occurrence and development of cardiovascular disease, and Banting Award winner Brownlee.M has proposed the theory of "common mechanism of diabetic vascular complications", which points out that an excessive peroxidase in mitochondrial electron transport chain is common mechanism of vascular injury caused by hyperglycemia. the theory can reasonably explain the internal relations of oxidative stress and diabetic vascular injury in diabetic complications, as has been considered a breakthrough in the field and has caused wide public concern. Vascular disease caused by enhancing oxidative stress and vascular disease in diabetic patients have the same pathological changes and the clinical consequences, all relating to the EPC proliferation impaired and formation of new blood vessels impaired. VEGF is one of the most important angiogenesis factor, some studies once suggested that angiogenesis dysfunction is caused by VEGF reduced, but in the diabetic ischemic hindlimb model, many studies found VEGF secretion did not decrease. Many studies also found that treatment of diabetic ischemic disease with VEGF are less effective. It is suggested the rapid revascularization blocked after limb ischemia in diabetic patients has nothing to do with the VEGF. Currently, the level of oxidative stress in EPCs and the expression of VEGFR in EPCs affected by the high glucose have no studies at home and abroad, So this experiment were designed. The rat bone marrow mononuclear cells isolated by using rat lymphocyte separation medium fluid, and the cell isolated and cultured were identified applaying with DiI-acLDL and FITC-UEA-1 double staining afrer 3 days. dual fluorescence-positive cells were considered to be endothelial progenitor cells (EPCs). EPCs identified were treated with various concentrations of high glucose (terminal glucose concentration in medium 15 mmol/L,30 mmol/L,60 mmol/L respectively), to detect the proliferation in EPCs and the markers of oxidative stress in EPCs at 24h、48h、96h respectively to assess the level of oxidative stress and the proliferation in EPCs affected by high glucose. Finally, the expression of VEGFR in EPCs affected by high glucose were observed, and through comparing with antioxidant treatment, Preliminary discussing high glucose induced oxidative stress on expression of VEGFR in EPCs, to provide theoretical support for diabetic ischemic limb revascularization blocked.
Methods:12 SD rats were harrested, immersed in 75% alcohol for 15 minites. Place rats on clean workbench, isolate the femur and tibia under sterile conditions, cut off the ends of long bones, expose bone marrow cavity, extract PBS containing 1% heparin with 5ml Syringe to wash the marrow cavity, use rat lymphocyte separation medium fluid to isolate the rat bone marrow mononuclear cells. the cell were identified applaying with DiI-acLDL and FITC-UEA-1 double staining afrer 3 days. dual fluorescence-positive cells were considered to be endothelial progenitor cells (EPCs). Subcultured and divided groups after 5 days, according to the various glucose concentrations,the cell were divided into two groups:the control group (terminal glucose concentration in medium was 5.5 mmol/L) and the high glucose intervention group. The high glucose intervention group were divided into three subgroups:high glucose group 1 (terminal glucose concentration in medium was 15 mmol/L glucose); high glucose group 2 (terminal glucose concentration in medium was was 30 mmol/L); high glucose group 3 (terminal glucose concentration in medium was was 60 mmol/L). The proliferation in EPCs were detected by MTT method and the markers of oxidative stress as anti-02-、MDA、GSH in EPCs were detected by the colorimetric method at 24h、48h、96h respectively. Finally, the expression of VEGFR in EPCs were detected by immunohistochemical method with and without the intervention of antioxidant therapy.
Results:1. Cell morphology of EPCs was observed with light microscope, mononuclear cells just isolated were round, a little. Cultural mononuclear cells started to appear adherent growth after 2 days and started to appear colony formation after 3 days, and spindle cells and cell clusters can be seen clearly,and its structure was similar to blood island, and spindle cells were both wired -like structure and single cell. The line-like arrangement of spindle cells, and with time increasing, the cells become bigger, appearing a typical cobblestone-like changes after 7 days.2. The cultural cell appeared typical DiI-acLDL and FITC-UEA-1 double positive staining after 3 days.3. The high glucose intervention group compared with the control group at 24h point, the proliferation in EPCs were significantly enhanced in high glucose groups 3 (P<0.05); the high glucose intervention group compared with the control group at 48h point, the proliferation in EPCs were significantly reduced in high glucose group 2 and group 3 (P<0.05); the high glucose intervention group compared with the control group at 96h point, the proliferation in EPCs was significantly reduced further in high glucose group 2 and group 3 (P <0.05), and control group and high glucose group 1 have no significant difference. Different time points in each experimental group compared with the 24h point, the proliferation in EPCs was significantly enhanced (P<0.05) at the 48h and 96h point in high glucose group 1; the proliferation in EPCs has no significant difference in high glucose group 2; the proliferation in EPCs were significantly decreased at the 96h point in high glucose group 3 (P<0.05).4. The high glucose intervention group compared with the control group at 24h point, the concentration of anti-O2-in EPCs significantly increased in high glucose group 1 (P<0.05); the high glucose intervention group compared with the control group at 48h point, the concentration of anti-O2- in EPCs significantly reduced in high glucose intervention group(P<0.05); the high glucose intervention group compared with the control group at 96h point, the concentration of anti-O2- in EPCs was significantly reduced in high glucose intervention group (P<0.05). Different time points in each experimental group compared with the 24h point, the concentration of anti-O2- in EPCs significantly increased at the 48h point in the control group (P<0.05); the concentration of anti-O2- in EPCs had no significant difference in high glucose group 1; the concentration of anti-O2- in EPCs reduced in high glucose group 2 and group 3 at the 48h and 96h point (P<0.05).5. the high glucose intervention group compared with the control group at 24h point, the concentrations of MDA in EPCs had no significant difference in high glucose intervention group; the high glucose intervention group compared with the control group at 48h point, the concentrations of MDA in EPCs increased significantly in high glucose group 2 and group 3 (P<0.05); the high glucose intervention group compared with the control group at 96h point, the concentrations of MDA in EPCs significantly increased in high glucose intervention group (P<0.05). Different time points in each experimental group compared with the 24h point, the concentrations of MDA in EPCs had no significant difference in control group at the 48h and 96h point; the concentrations of MDA in EPCs increased significantly in high glucose group 1 at the 96h point (P<0.05); the concentrations of MDA in EPCs increased significantly in high glucose group 2 and group 3 at the 48h and 96h point (P<0.05).6. the high glucose intervention group compared with the control group at 24h point, the concentration of GSH in EPCs have had no significant difference in high glucose intervention group; the concentration of GSH in EPCs were significantly decreased in the high glucose group 2 and group 3 at 48h and 96h point (P<0.05); the concentration of GSH in EPCs have had no significant difference in high glucose group 1. Different time points in each experimental group compared with the 24h point, the concentration of GSH in EPCs significantly increased in the control group at the 48h and 96h point (P<0.05); the concentration of GSH in EPCs had no significant difference in high glucose group 1; the concentration of GSH in EPCs significantly reduced in high glucose group 2 and group 3 at the 48h and 96h point (P<0.05).7. (1). High glucose intervention group compared with the control group at 24h and 48h point respectively, the expression of VEGFR in EPCs significantly reduced (P<0.05) in the high glucose group 2 and group 3; high glucose intervention group compared with the control group at 96h point, the expression of VEGFR in EPCs significantly decreased in the high glucose intervention group (P<0.05). Different time points in each experimental group compared with the 24h point, the expression of VEGFR in EPCs had no significant difference in the control group; the expression of VEGFR in EPCs significantly reduced in high glucose group 1 and group 3 at the 96h point (P<0.05); the expression of VEGFR in EPCs significantly decreased in high glucose group 2 at the 48h and 96h point (P<0.05). (2). Under antioxidants therapy, the expression of VEGFR in EPCs had no significant difference in each experimental group with the time and concentration increasing. (3). Antioxidants therapy compared with non-antioxidants therapy, the expression of VEGFR in EPCs significantly increased in high glucose intervention group, in a certain extent, the serious defects of expression of VEGFR in EPCs saved by antioxidants therapy in the intervention group.
Conclusion:1. High glucose significantly inhibite the proliferation in EPCs, and with the glucose concentrations and time increasing,the role of inhibition become stronger and stronger.2. Oxidative stress induced by high glucose in EPCs significantly increase, and anti-oxidation significantly decreased, with the concentration and time increasing, the role of oxidative stress become stronger and stronger.3. High glucose significantly inhibite the expression of VEGFR in EPCs, But under antioxidants therapy, expression of VEGFR in EPCs in high glucose significantly increase, in a certain extent, the serious defects of expression of VEGFR in EPCs saved by antioxidants therapy in the intervention group. Suggesting that increasing own oxidative stress in EPCs is one of the important reasons which inhibited the expression of VEGFR in EPCs.
引文
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25. Polakis P.The mang ways of Wnt in cancer[J].Curr Opin Genet Dev,2007,17(1):45-51.
26. Venkiteswaran k,xiao K,Summers S et al.Regulation of endothelial barrier function and growth by VE.cadherin plakoglobin,and beta. catenin[J].Am J Physiol Cell Physiol 2002,283(8):811-815.
27. Chen CH, Dixon RA, Ke LY, et al.Vascular Progenitor Cells in Diabetes Mellitus roles of Wnt Signaling and Negatively charged Low-Density lipoprotein[J]. Am J Physiol Cell Physiol 2009,104(9):1038-1040.
28.28,Kim KI,Cho HJ,Hahn JY,et al.β-catenin overexpression augments angiogenesis and skeletai muscle regeneration through dual mechanism of vascular endothelial grouth factor-mediated endothelial cell prolifetion and progenitor cell mobilization[J]. Arterioscler Thromb Vasc Biol,2006,26(3):91-98.
29. Silvestre JS,Levy BL. Molecular basis of angiopathy in diabetes mellitus[J]. Circ Res,2006,98(1):4-6.
30. Roguin A,Nitecki S, Rubinstein I,et al.Vascular endothelial growth factor (VEGF) fail to improve blood flow and to promote collateralization in a diabetic mouse ischemic hindlimb model[J]. Card Dia,2003,2(18):1-6.
31. Tuo QH,Zeng H,Stinnett A,et al. Critical role of angiopoietins/Tie-2 in hyperglycemic exacerbation of myocardial infarction and impaired angiogenesis[J]. Am J Physiol Heart Circ Physiol,2008,294(6):H2547-2557.