组织激肽释放酶在糖尿病视网膜病变患者血清中的变化及其调节血管新生机制

Changes of tissue kallikrein in serum of patients with diabetic retinopathy and its role in retinal angiogensis

目的检测组织激肽释放酶(tissue kallikrein,TKLK)、血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)和可溶性细胞间黏附分子-1(soluble intracellular adhension molecul-1,s ICAM-1)在糖尿病视网膜病变(diabetic retinopathy,DR)患者血清中的变化,观察TKLK在低氧条件下对人视网膜微血管内皮细胞(human retinal microvascular endothelial cells,HRMECs)中VEGF和ICAM-1表达的影响。方法收集2型糖尿病患者60例,按照DR分期标准将患者分为糖尿病无DR组(DM组)、非增生性DR组(NPDR组)和增生性DR组(PDR组),收集同期在本院体检的志愿者作为对照组,每组20例。ELISA法检测血清中TKLK、VEGF和s ICAM-1的水平。体外HRMECs分别进行常氧和低氧培养,不同浓度重组TKLK处理后,检测细胞增殖、凋亡以及VEGF和ICAM-1的表达。结果 DM、NPDR和PDR组患者血清中TKLK、VEGF和s ICAM-1水平明显高于对照组,4组总体差异有统计学意义(F=28.805,P=0.002;F=32.041,P=0.002;F=26.169,P=0.001);PDR患者血清中TKLK、VEGF和s ICAM-1水平显著高于DM和NPDR组(均为P<0.001)。TKLK与VEGF(r=0.623,P<0.01)和s ICAM-1水平(r=0.598,P<0.01)均呈正相关。10μg·m L^(-1)rh TKLK可显著抑制低氧诱导的HRMECs增殖以及VEGF和ICAM-1的表达,并可促进细胞凋亡(P<0.05)。结论 TKLK通过与VEGF和ICAM-1相互作用而影响DR的进展。

Objective To determine the levels of serum tissue kallikrein （TKLK）, vascular endothelial growth factor （VEGF） and soluble intracellular adhension molecul-1 （ sICAM-1 ） in diabetic retinopathy （DR） patients,and evaluate the effects of TKLK on the expression of VEGF and ICAM-1 in human retinal vascular endothelial cells （HRMECs） under hypoxia. Methods Sixty DM patients were enrolled and grouped according to the staging criteria of DR into： DM patients without DR group （DM group）, non-proliferative DR group （NPDR group） and proliferative DR group （PDR group ）. Simultaneously ,20 healthy volunteers were collected as the controls. ELISA was applied to measure the levels of serum TKLK, VEGF and sICAM- 1 in all the eligible subjects,and the correlations between them were analyzed. HRMECs were incubated in vitro with various concentrations of rhTKLK, and then were grown in normoxia and hypoixa conditions. Cell viability and apoptosis as well as the expression of VEGF and ICAM-1 were examined. Results The serum levels of TKLK,VEGF and sICAM-1 in DM, NPDR and PDR groups were higher than that in control group, there were significant differences among four groups （F = 28. 805 ,P = 0. 002 ;F = 32.041 ,P = 0. 002, F = 26. 159,P=0. 001 ）. Meanwhile,the serum levels of TKLK,VEGF and sICAM-1 in PDR group were higher than those in DM and NPDR groups （ all P 〈 0.001 ）. There were significant positive correlations between the levels of TKLK and VEGF （ r = 0.623 ,P 〈 0.01 ） as well as TKLK and sICAM-1 （ r = 0.598 ,P 〈 0.01 ）. 10 μg · mL^-1 rhTKLK dramatically inhibited the hypoxia-induced proliferation of HRMECs and expression of VEGF and ICAM-1 ,while significantly promoted cell apoptosis （P 〈 0.05 ）. Conclusion TKLK participate in the progression of DR via interaction with VEGF and ICAM-1.