微小RNA-200b-GATA2-血管内皮生长因子受体2通路在糖尿病创面愈合中的作用及其机制

Mechanism of action of the microRNA-200b-GATA2-vascular endothelial growth factor receptor 2 signal axis on wound healing of diabetic wounds

目的探讨糖尿病创面不愈合是否与微小RNA(miR)-200b-GATA2-血管内皮生长因子受体(VEGFR)2通路表达异常引起的血管再生障碍有关。方法将12只C57/BL6小鼠作为对照组,24只db/db糖尿病小鼠采用随机数字表法分为模型组和miR-200b敲低组,每组12只,在每只小鼠背面制作直径10 mm圆形创面;分别于造模后第1、7和14天记录各组小鼠创面愈合率;造模后第14天取材,采用苏木精-伊红(HE)染色观察小鼠创面组织形态,免疫组织化学检测CD34阳性表达,实时定量反转录聚合酶链反应(RT-qPCR)检测miR-200b表达,蛋白质印迹法(Western blot)检测GATA2、VEGFR2蛋白相对表达量。结果造模后第7天和第14天,模型组的创面愈合率低于对照组[(22.46±2.33)%比(34.63±1.42)%,(49.53±2.81)%比(72.48±2.54)%,F=9.37、8.42,P均<0.01];造模后第7天miR-200b敲低组的创面愈合率增加高于模型组[(27.52±1.64)%比(22.46±2.33)%,F=9.37、8.42,P<0.05],造模后第14天miR-200b敲低组的创面愈合率明显高于模型组[(61.93±2.55)%比(49.53±2.81)%,F=9.37、8.42,P<0.01]。对照组创面皮肤细胞排列整齐,无炎性细胞浸润;模型组创面表皮坏死,原纤维结构消失;miR-200b敲低组创面有肉芽组织形成,修复明显。模型组的CD34阳性表达量低于对照组(0.21±0.08比0.63±0.06,F=12.32,P<0.05);而miR-200b敲低组CD34阳性表达量高于模型组(0.48±0.05比0.21±0.08,F=12.32,P<0.01)。模型组miR-200b相对表达量高于对照组(1.43±0.18比0.73±0.12,F=6.44,P<0.01);miR-200b敲低组miR-200b相对表达量低于模型组(0.92±0.15比1.43±0.18,F=6.44,P<0.01)。模型组GATA2蛋白和VEGFR2蛋白相对表达量低于对照组(0.36±0.07比0.78±0.09,0.43±1.41比0.86±1.32,F=4.85、3.79,P均<0.01);miR-200b敲低组GATA2和VEGFR2蛋白相对表达量高于模型组(0.64±0.07比0.36±0.07,0.68±1.57比0.43±1.41,F=4.85、3.79,P均<0.01),差异均有统计学意义。结论 miR-200b-GATA2-VEGFR2信号轴对糖尿病创面愈合有重要的调控作用,调控该信号轴可以改善糖尿病足创面愈合过程,缩短创面愈合时间。

Objective To investigate whether diabetic wound nonunion is related to vascular regeneration dysfunction caused by abnormal expression of microRNA(miR)-200b-GATA2-vascular endothelial growth factor receptor(VEGFR)2 pathway.Methods Twelve C57/BL6 mice were used as the control group,and 24 db/db diabetic mice were divided into a model group and a miR-200b knockdown group by random number table,with 12 mice in each group,made on the back of each mouse Round wounds with a diameter of 10mm;wound healing rates of mice in each group were recorded on the 1,7 and 14 days after modeling;samples were taken on the 14th day after modeling,and hematoxylin and eosin(HE)staining was used to observe the morphology of the wound tissue of the mice and immunize them.The positive expression of CD34 was detected by histochemistry,the expression of miR-200b was detected by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR),and the relative expression of GATA2 and VEGFR2 protein was detected by Western blotting.Results On the 7th and 14th day after modeling,the wound healing rate of the model group was lower than that of the control group[(22.46±2.33)%vs.(34.63±1.42)%,(49.53±2.81)%vs.(72.48±2.54)%,F=9.37,8.42,P<0.01];The wound healing rate of the miR-200b knockdown group increased higher than that of the model group on the 7th day after modeling[(27.52±1.64)%vs.(22.46±2.33)%,F=9.37,8.42,P<0.05],and the wound healing rate of the miR-200b knockdown group was increased on the 14th day after modeling.It was significantly higher than the model group[(61.93±2.55)%vs.(49.53±2.81)%,F=9.37,8.42,P<0.01].The skin cells of the wound in the control group are arranged neatly without inflammatory cell infiltration;the wound in the model group is necrotic,the collagen of the dermis is loose,the cell level is blurred,the arrangement is irregular,and the fibril structure disappears;the wound in the miR-200b knockdown group has the granulation tissue is formed,and there is scar formation,and the repair is evident.The positive expression of CD34 in the model group was lower than that in the control group(0.21±0.08 vs.0.63±0.06,F=12.32,P<0.05);while the positive expression of CD34 in the miR-200b knockdown group was higher than that in the model group(0.48±0.05 vs.0.21±0.08,F=12.32,P<0.01).The relative expression of miR-200b in the model group was higher than that in the control group(1.43±0.18 vs.0.73±0.12,F=6.44,P<0.01);the relative expression of miR-200b in the miR-200b knockdown group was lower than that in the model group(0.92±0.15 vs.1.43±0.18,F=6.44,P<0.01).The relative expression levels of GATA2 protein and VEGFR2 protein in the model group were lower than those in the control group(0.36±0.07 vs.0.78±0.09,0.43±1.41 vs.0.86±1.32,F=4.85,3.79,P<0.01);In the miR-200b knockdown group,the relative expression level of GATA2 and VEGFR2 proteins were higher than that of the model group(0.64±0.07 vs.0.36±0.07,0.68±1.57 vs.0.43±1.41,F=4.85,3.79,P<0.01).Conclusion miR-200b-GATA2-VEGFR2 signal axis plays an essential role in regulating the wound healing of diabetic foot,which can improve the wound healing process and shorten the wound healing time.