缺氧诱导因子1α对早期糖尿病视网膜病变状态下人粒细胞系白血病细胞表面黏附分子CD18表达的调节

Hypoxia inducible factor-1α regulates the expression of CD18 in the early stage of diabetic retinopathy

目的 观察在早期糖尿病视网膜病变（DR）状态下,缺氧诱导因子1α（HIF-1α）对细胞表面黏附分子CD18表达以及白细胞和血管内皮细胞黏附的影响.方法 收集早期DR患者及年龄匹配的健康人外周血血清,用于体外培养人粒细胞系白血病细胞（HL60）和恒河猴视网膜血管内皮细胞系细胞（RF/6A）.分为糖尿病血清培养组（B组）、糖尿病＋HIF-1α反义寡核苷酸组（ASODN）（C组）、糖尿病＋HIF-1α正义寡核苷酸组（SODN）（D组）,健康人血清培养细胞为正常对照组（A组）.以流式细胞仪和Real-time PCR分别检测HL60细胞表面CD18蛋白阳性细胞比例和CD18 mRNA的表达水平,以虎红染色法观察HL60细胞和RF/6A细胞的黏附率.结果 A、B、C、D组CD18阳性细胞比例分别为17.06±6.01、42.23±2.60、25.33±3.05、32.40±10.57,各组之间差异有统计学意义（F=36.47,P〈0.001）.和A组比较,B、C、D组CD18 mRNA水平分别是A组的21.05±2.07、2.23±0.96、25.07±2.27倍,各组之间差异有统计学意义（F=180.34,P〈0.001）.A、B、C、D组细胞黏附率分别为0.06±0.002、0.09±0.10、0.05±0.007、0.07±0.01,各组之间差异有统计学意义（F=13.06,P=0.002） 结论在体外培养条件下,糖尿病血清可以促进HL60细胞表达CD18蛋白和CD18mRNA,促进HL60细胞和血管内皮细胞的黏附,HIF-1α表达被抑制后,这种促进作用减弱.HIF-1α对糖尿病状态下细胞表面黏附分子CD18表达及白细胞和血管内皮细胞之间的黏附具有调节作用.

Objective To observe the effect of hypoxia inducible factor-1α（HIF-1）to the expression of cell surface adhesion molecules CD18 and the adhesion ability of leukoeytes and vascular endothelial cells under early stage of diabetic retinopathy condition. Methods The human promyeloeytic leukemia cell line HL60 and the rhesus choroid-retina vascular endothelial cell line RF/6A were cultured in RPMI 1640 medium-10% human serum, which was collected from the subjects of early stage of diabetic retinopathy and age-matched healthy control. The cells were cultured in 4 groups as control group （group A）, diabetic group （group B）, HIF-1 anti-sense oligonucleotides （ASODN） group （group C） and HIF-1 sense oligonueleotides （SODN） group （group D）. The percentages of CD18 positive cell in the HL60 cell were measured by flow cytometry and mRNA in the HL60 cell by real-time reverse transcription-polymerase chain reaction（RT-PCR）. Results The percentage of CD18 positive cell in the group A, B, C and D was 17.06±6.01, 42. 23±2.60, 25.33±3.05 and 32.40±10.57, respectively, the differences among them were significant （F=36.47,P〈0. 001）. Compared to the group A, the expression of CD18 mRNA in the group B, C and D was increased about 21. 05±2. 07,2. 23±0.96 and 25.07±2.27 times, respectively, the differences among them were significant （F=180.34, P〈0. 001）. The adherent rates of HL60 to RF/6A in group A, B, C and D was 0. 06±0. 00, 0. 09±0. 10, 0. 05 ±0. 00 and 0.07 ± 0. 01, respectively, the differences among them were significant（F=13.06,P=0. 002）. Conclusion In vitro, HIF-1 could regulate the expression of CD18 by HL60, and the adhesion of HL60 to RF/6A when the cells were exposed to diabetic serum. The effects of human serum weaken with the inhibition of HIF-1 expression. HIF-1 play regulatory role in the expression of CD18 and adhesion of leukoeytes and vascular endothelial cells under early stage of diabetic retinopathy condition.