2型糖尿病患者CD14^+CD16^+单核细胞的水平及其对LPS和IL-15刺激的反应

Proportion of CD14^+CD16^+ monocytes in peripheral blood from patients with type 2 diabetic mellitus and effect of LPS and IL-15 on expression of STAT5 in monocytes

目的:研究2型糖尿病(T2DM)患者外周血CD14+CD16+单核细胞的比例及脂多糖(LPS)联合白细胞介素15(IL-15)对其的影响,以了解炎症性免疫反应在T2DM中的可能作用机制。方法:对28例T2DM患者和20例健康志愿者外周血用流式细胞术检测CD14+CD16+单核细胞的比例,并分离其外周血单个核细胞(PB-MC),用LPS和IL-15干预4 h,收集PBMC和培养上清。分别采用Western blotting检测PBMC内STAT5和p-STAT5蛋白表达。免疫荧光检测p-STAT5蛋白表达,ELISA法检测外周血25-羟维生素D3[25(OH)D3]和IL-6浓度,免疫比浊法检测其外周血C-反应蛋白(CRP)水平,ELISA法检测LPS和IL-15干预后细胞培养上清IL-6和单核细胞趋化蛋白-1(MCP-1)的浓度。结果:T2DM组外周血CD14+CD16+单核细胞数量明显高于正常对照组(P<0.01),并与血清CRP和IL-6水平呈正相关(r=0.394,P<0.05和r=0.741,P<0.01),与25(OH)D3浓度呈负相关(r=0.409,P<0.01),且25(OH)D3水平与CRP和IL-6水平均呈负相关(r=-0.479和r=-0.774,均P<0.01),LPS联合IL-15刺激后PBMC的p-STAT5蛋白表达水平和PBMC培养上清IL-6、MCP-1浓度明显高于正常对照组(P<0.01),且T2DM患者p-STAT5的表达存在激活现象。结论:T2DM患者体内存在单核细胞功能紊乱,这种功能紊乱可能与活性维生素D3不足有关,二者可能参与了T2DM患者体内免疫炎症反应并与微炎症互为因果,从而导致了T2DM及其并发症的发生发展;其作用机制可能与STAT5信号通路的激活有关。

AIM： To characterize the proportion of CD14＋CD16＋ monocytes in peripheral blood from type 2 diabetes（T2DM） patients and to observe the response of CD14＋CD16＋ monocytes to lipopolysaccharide（LPS） and interleukin-15（IL-15） for further exploring the potential mechanism of inflammatory immune response in the pathogenesis of T2DM.METHODS： Twenty-eight patients with T2DM and 20 healthy volunteers were enrolled in the study.The peripheral blood was collected for determining the percentage of CD14＋CD16＋ monocytes by flow cytometry.The peripheral blood mononuclear cells（PBMC） were isolated and subject to stimulation with LPS and IL-15 for 4 h.The protein expression of STAT5 was detected by Western blotting and the phosphorylated（p）-STAT5 was determined by Western blotting and immunofluorescence.Serum levels of 25-hydroxyvitamin D3 [25（OH） D3]and IL-6,and the concentrations of IL-6 and monocyte chemoattractant protein-1（MCP-1） in the culture supernatants were assessed by ELISA.Serum level of C-reactive protein（CRP） was measured by immunoturbidimetry.RESULTS： There were positive correlations between the quantity of CD14＋CD16＋ monocytes and serum levels of CRP and IL-6（r=0.394,P0.05 and r=0.741,P0.01）,while serum 25（OH） D3 was negatively correlated with the quantity of CD14＋CD16＋ monocytes（r=-0.409,P0.01）,serum CRP（r=-0.479,P0.01） and serum IL-6（r=-0.774,P 0.01）.After stimulated with LPS and IL-15,PBMC showed significant up-regulation of p-STAT5 protein expression,and significant increases in the supernatant levels of IL-6 and MCP-1 were observed（P0.05）.The expression of p-STAT5 existed in the nucleus.CONCLUSION： These findings suggest that the functional disturbance in monocytes occurs in T2DM,which may be related to insufficiency of vitamin D3.The aberrant activation of STAT5 signaling pathway underlies the functional abnormalities of the monocytes in T2DM.