Levels of VEGF , IL6, leptin in the aqueous humor of patients with type 2 diabetes
Jun Zhao, FangYi Liu, YongSheng Chen
1Department of Ophthalmology, 2Center of Clinical Laboratory, Peoples Hospital of Linyi City, Linyi 276000, Shandong Province, China
Correspondence to:Jun Zhao.Department of Ophthalmology, Peoples Hospital of Linyi City, Linyi 276000, Shandong Province, [email protected]
Abstract
AIM: To assay the contents of vascular endothelial growth factor (VEGF), interleukin6 (IL6) and leptin in the aqueous humor of patients with type 2 diabetes and to explore their roles in the pathogenesis of diabetic retinopathy (DR).
METHODS: The contents of VEGF and IL6 in the aqueous humor of 70 patients (70 eyes) with type 2 diabetes were determined by ELISA technique, and the content of leptin was determined by radioimmunoassay. According to the fundus examination after mydriasis and fundus fluoresecein angiography, experimental groups include 22 cases (22 eyes) without diabetic retinopathy, 28 cases (28 eyes) with simple diabetic retinopathy and 20 cases (20 eyes) with proliferative diabetic retinopathy. The control group include 20 cases (20 eyes) with senile cataract.
RESULTS: The contents of VEGF in the aqueous humor of three experimental groups were (250.32±26.77), (300.11±58.89), (496.23±91.06)ng/L respectively; the contents of IL6 were (162.81±33.92), (256.76±64.15), (391.27±90.46)ng/L respectively; the contents of leptin were (69.80±21.37), (155.08±32.76), (230.27±56.92)ng/L respectively. In the control group, the contents of VEGF,IL6 and leptin in the aqueous humor were respectively(144.69±26.55)ng/L,(86.71±22.69)ng/L, (43.62±20.02)ng/L. There were statistically significant differences between control group and experimental groups (F=118.62,P<0.01;F=110.53,P<0.01;F=101.22,P<0.01). The levels of VEGF, IL6, leptin in the aqueous humor were increased in turn in control group, nondiabetic retinopathy (NDR)group, background diabetic retinopathy (BDR) group and proliferative diabetic retinopathy (PDR) group. The level of VEGF was significantly correlated with the levels of IL6, leptin in the aqueous humor (r=0.995,P<0.01;r=0.776,P<0.01); the contents of VEGF,IL6,and leptin in the aqueous humor were also significantly correlated with the course of diabetes and severity of diabetic retinopathy (r=0.722,P<0.01;r=0.716,P<0.01)(r=0.869,P<0.01;r=0.865,P<0.01)(r=0.776,P<0.01;r=0.765,P<0.01).
CONCLUSION: VEGF, IL6, and leptin play active roles in the generation and development of DR, and the level of VEGF in the aqueous humor closely correlates with the level of IL6 and leptin in the aqueous humor of diabetic patients.
KEYWORDS: vascular endothelial growth factor; interleukin6; leptin; diabetic retinopathy; type 2 diabetes
Zhao J, Liu FY, Chen YS. Levels of VEGF , IL6, leptin in the aqueous humor of patients with type 2 diabetes. Int J Ophthalmol
摘要：目的:检测2型糖尿病患者眼房水中血管内皮生长因子(vascular endothelial growth factor,VEGF)、白细胞介素6(interleukin6,IL6)和瘦素（leptin）的含量,并探讨其临床意义。方法:对70例70眼2型糖尿病患者房水中VEGF和IL6的含量采用双抗体夹心酶联免疫吸附法检测,leptin含量采用放射免疫法检测。根据散瞳眼底检查和眼底荧光素血管造影检查,将实验组分为:无糖尿病性视网膜病变组22例22眼、单纯型糖尿病性视网膜病变组28例28眼、增生型糖尿病性视网膜病变组20例20眼,对照组为健康的老年性白内障患者20例20眼。结果: 3组房水VEGF的含量分别为(250.32±26.77)，(300.11±58.89)，(496.23±91.06)ng/L,IL6含量分别为(162.81±33.92)，(256.76±64.15)，(391.27±90.46)ng/L,leptin含量分别为(69.80±21.37)，(155.08±32.76)，(230.27±56.92)ng/L,对照组房水VEGF含量为(144.69±26.55)ng/L、IL6含量为(86.71±22.69)ng/L,leptin含量为(43.62±20.02)ng/L，对照组与实验组比较差异均有统计学意义(F=118.62,P<0.01;F=110.53,P<0.01;F=101.22,P<0.01)。对照组、NDR，BDR与PDR组房水VEGF，IL6，leptin含量有依次增加的趋势。房水中VEGF与IL6，leptin含量有相关性(r=0.995,P<0.01；r=0.776,P<0.01);房水中VEGF，IL6，leptin含量与糖尿病患者的病程及糖尿病性视网膜病变的严重程度有相关性(r=0.722,P<0.01;r=0.716,P<0.01)(r=0.869,P<0.01;r=0.865,P<0.01)(r=0.776,P<0.01;r=0.765,P<0.01)。结论:VEGF，IL6，leptin在糖尿病性视网膜病变的形成过程中有重要作用,且VEGF与IL6，VEGF与leptin之间有相关性。
关键词:2型糖尿病;视网膜病变;血管内皮生长因子;白细胞介素6;瘦素
0引言
糖尿病性视网膜病变(diabetic retinopathy,DR)是糖尿病常见的严重并发症,是发达国家成年人中主要的致盲性疾病之一。学者们对DR的发病机制作了大量研究，认为新生血管形成是增生型糖尿病性视网膜病变 (proliferative diabetic retinopathy，PDR)的主要标志。许多细胞因子参与了PDR的形成，如血管内皮生长因子(vascular endothelial growth factor,VEGF)、白细胞介素6(interleukin6,IL6)、白细胞介素8、瘦素（leptin）等。本研究测定糖尿病患者房水中VEGF，IL6，leptin的含量,旨在探讨它们在DR发生过程中的作用。
1对象和方法
1.1对象
房水标本90例(90眼),全部采自我院眼科200608/200706行白内障超声乳化手术的部分患者。实验组70例,均为内科确诊为2型糖尿病患者,因合并白内障而行手术治疗,术前血糖均控制在8.3mmol/L以下,无其他眼部疾病史。根据患者术后眼底检查及眼底荧光血管造影结果,按照1987年中华医学会糖尿病性视网膜病变分期标准,将实验组分为无糖尿病性视网膜病变组(diabetic patients without retinopathy，NDR)、单纯型糖尿病性视网膜病变组(background diabetic retinopathy，BDR)、增生性糖尿病视网膜病变组(proliferative diabetic retinopathy，PDR)3组。其中,NDR组22例,年龄48～76(平均69)岁,男10例(10眼),女12例(12眼),糖尿病病程0.3～11(平均6.5)a;BDR组28例,男13例(12眼)，女15例(15眼),年龄55～81(平均72)岁,糖尿病病程3～16(平均10)a;PDR组20例,男9例(9眼),女11例(11眼),年龄56～80(平均69)岁,糖尿病病程8～30(平均14)a。对照组20例(20眼),男10例(10眼),女10例(10眼),年龄60～85(平均74)岁,均为成熟期或近成熟期的老年性白内障患者,也无其它眼部疾病史。各组年龄和性别差异无统计学意义。
1.2方法
开睑后,以无菌1mL一次性塑料针管接9号针头,在角膜缘内1mm行前房穿刺,2～5s内从中央瞳孔区抽吸,不触及虹膜、晶状体及角膜内皮,获得未稀释前的房水0.2mL,标本移至已消毒并硅化的0.5mL Eppendorf管,放入液氮中,然后移至80℃的冰箱中。采用ELISA法检测VEGF和IL6的含量，放射免疫法检测leptin含量。从冰箱中取出标本,置于室温下10min待测。人VEGF、IL6的ELISA试剂盒均购自深圳晶美生物工程有限公司(灵敏度为7ng/L,5ng/L),测量仪器为Thermomuliskan MK3型酶标仪。根据试剂盒说明,将标准品用稀释液定比稀释,混合均匀。将酶标板各孔依次编号,然后在相应孔加入不同浓度标准品或标本100μL,用封板胶纸封住反应孔,37℃孵箱孵育90min,再用稀释好的洗涤液洗板4次,充分甩干。在各孔中加入生物素化抗体工作液100μL,用封板胶纸封住反应孔,37℃孵箱孵育60min,洗板4次。每孔再加入酶结合物工作液100μL,37℃孵箱孵育并封住板孔,37℃孵箱孵育30min。然后洗板4次,在每孔中加入显色剂100μL,37℃避光孵育10～15min后，在每孔中加入终止液100μL,混匀,酶标仪450nm处读数,在5min内测定各孔吸光度值(A值),根据定比稀释标准品的A值绘制标准曲线,根据样品A值和标准曲线,算出标本中VEGF的含量(ng/L)。房水中IL6的测定步骤和VEGF的相同。Leptin RIA kit由北京华英放射免疫技术研究所提供，仪器为西安262厂生产的XH6010全自动放免γ分析仪,操作均按说明书进行。
统计学处理：各组房水中的VEGF，IL6和leptin的含量以±s表示,采用SPSS11.5统计软件包对实验结果进行单因素方差分析和直线相关性分析，以P<0.05为差异有统计学意义。
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