雌激素对糖尿病患者内皮祖细胞数量和功能的影响

Effect of estrogen on the number and function of endothelial progenitor cells of diabetic patients

目的:观察雌激素对2型糖尿病患者外周血内皮祖细胞数量和功能的改变,并检测趋化因子受体CXCR4的表达。方法:实验于2005-02/09在华中科技大学同济医学院附属协和医院完成。选取武汉市中心医院收治的20例2型糖尿病患者,均根据1999年中国糖尿病学会新的诊断标准确诊。①抽取2型糖尿病患者外周静脉血,常规密度梯度离心法获取单个核细胞,进行内皮祖细胞的分离培养。加入雌二醇培养3d,按其浓度分为雌激素0nmol/L组、雌激素50nmol/L组、雌激素100nmol/L组。②检测内皮祖细胞的生长增殖和凋亡情况,测定其黏附能力。分别以反转录-聚合酶链法及流式细胞术对内皮祖细胞表达CXCR4的水平进行检测。结果:按意向处理分析,实验选取20例2型糖尿病患者血样全部进入结果分析。①内皮祖细胞的培养、鉴定及计数情况:培养4d后可见贴壁的单个核细胞,7d后细胞变为梭形,并慢慢变长。流式细胞仪鉴定细胞表型,可见内皮祖细胞大多表达CD34,且60%～80%表达CD133。对贴壁细胞行荧光染色后,>80%的细胞双染色阳性,可认为是正在分化的内皮祖细胞。计数结果显示,随着加入雌激素浓度的增加,各组内皮祖细胞数量差异具有显著性意义(P<0.05)。②内皮祖细胞增殖及凋亡的检测结果:各组内皮祖细胞增殖率基本相似(P>0.05)。随着雌激素浓度的增加,雌激素0nmol/L组、雌激素50nmol/L组、雌激素100nmol/L组的凋亡率呈下降趋势,差异有显著性意义(P<0.05)。③内皮祖细胞黏附能力检测结果:各组内皮祖细胞黏附能力基本相似(P>0.05)。④CXCR4在内皮祖细胞上的表达情况:流式细胞术检测各组内皮祖细胞上CXCR4的表达阳性率,可见随着雌激素浓度增加,各组CXCR4表达率也随之增加,差异具有显著性意义(P<0.05)。反转录-聚合酶链反应法检测CXCR4的表达,亦得到相似结果。结论:雌激素对2型糖尿病患者内皮祖细胞的数量及功能均产生影响,可明显抑制内皮祖细胞凋亡及上调CXCR4的表达。提示雌激素可能在防止糖尿病周围血管病变上发挥一定作用。

AIM： To observe the effect of estrogen on the number and function of endothelial progenitor cells of patients with type Ⅱ diabetes mellitus and detect the expression of chemotactic factor receptor CXCR4. METHODS： This experiment was conducted at the Union Hospital, Tongji Medical College, Huazhong University of Science and Technology from February to September 2005. Twenty patients with type Ⅱ diabetes mellitus who were diagnosed according to new diagnosis standard of China Diabetes Association in 1999 and received treatment in the Wuhan Central Hospital were enrolled. （1）Peripheral venous blood was extracted from type Ⅱ diabetes mellitus. Monocytes were isolated and induced to endothelial progenitor cells （EPCs） with routine density gradient centrifugation. Estradiol was added for culture for 3 days. Estrogen 0 nmol/L group, estrogen 50 nmol/L group, estrogen 100 nmol/L group were divided according to concentration. （2） Proliferation and apeptosis of EPCs were detected and adhesion ability was measared. The level of CXCR4 expressed by EPCS was detected with reverse transcription-pelymerase chain reaction and flow cytometer separately. RESULTS： According to intention-to-treat analysis, blood sample of 20 patients with type Ⅱ diabetes mellitus all entered the stage of result analysis. （1）4 days after culture, adhered monocytes were found. Monocytes was shuttle-shape and extended slowly 7 days later. We detected cellular phenotype with flow cytometer and found that EPCs mostly expressed CD34 and 60%-80% expressed CD133. The adhered cells were given fluorescence staining. 〉 80% cells were double-staining positive, which were considered to be differentiating EPCs . The results of counting showed that with the increase of concentration of cestrogen, there was significant difference of the number of EPCs in each group （P 〈 0.05）. （2） The proliferative rate of EPCs was basically the same in each group （P 〉 0.05）. With the increase of concentration of cestrogen, the apoptosis rate of oestrngen 0 nmo1/L group, cestrogen 50 nmol/L group and oestrogen 100 nmol/L group was in descending manner, with significant difference （P 〈 0.05）. （3） Adhesion ability of EPCs was basically the same in each group（P 〉 0.05）. （4）CXCR4 expression positive rate of EPCs was detected with flow cytometer . Results showed that with the increase of concentration of cestrogen, CXCR4 expression rate was increased in each group, with significant difference （P 〈 0.05 ）. Similar results were obtained through detecting the expression of CXCR4 with reverse transcriptionpolymerase chain reaction. CONCLUSION： Estrogen affects the number and function of EPCs of patients with type Ⅱ diabetes mellitus. It can obviously inhibit the apeptosis of EPCs and up-regulate the expression of CXCR4, suggesting that estrogen exerts some effect in preventing diabetic perivascular disesease.