缺氧诱导因子1α特异性小干扰RNA对髓样细胞表面黏附分子CD18及神经损伤诱导蛋白-1表达的影响

目的 观察早期糖尿病视网膜病变（DR）状态下，以pSUPER为载体的缺氧诱导因子1α（HIF1α）特异性小干扰（siHIF1α）RNA对髓样细胞表面黏附分子CD18及神经损伤诱导蛋白-1（Ninj-1）表达的影响。方法 实验分为健康人血清培养组（A组）、糖尿病血清培养组（B组）、糖尿病血清培养联合pSUPERH1 siHIF1α转染组（C组）及糖尿病血清培养联合pSUPER空载体组（D组）进行。A组采用健康志愿者血清培养人髓样细胞系白血病细胞系（K562）细胞；B、C、D组均采用DR患者血清培养K562细胞。C、D组在加入DR患者血清前24 h分别转染pSUPERH1 siHIF1α及pSUPER空载体。采用流式细胞仪检测各组K562细胞表面的CD18、Ninj-1表达。行细胞黏附实验，检测各组K562细胞与恒河猴视网膜脉络膜血管内皮细胞系（RF/6A）细胞黏附率。结果 A、B、C、D组K562细胞表面CD18表达比较，4组间差异有统计学意义（F=14.33，P=0.01）。组间CD18表达两两比较，B组明显高于A组（P=0.001）；C组明显低于B组（P=0.001）和D组（P=0.02）；C组与A组间无明显差异（95%可信区间=-14.89~2.13，P=0.12）。A、B、C、D组K562细胞表面Ninj-1表达比较，4组间差异有统计学意义（F=39.38，P=0.001）。组间Ninj-1表达两两比较，B组明显高于A组（P=0.00）；C组与B组间无明显差异（P=0.06）；C组与D组间也无明显差异（P=0.49）。A、B、C、D组K562细胞与RF/6A细胞黏附率比较，4组间差异有统计学意义（F=20.62，P=0.00）。组间K562细胞与RF/6A细胞黏附率两两比较，B组明显高于A组（P=0.00）；C组较B组明显下降（P=0.01），较A组明显提高（P=0.002）；B组与D组间无明显差异（P=0.68）。结论 早期DR状态下，以pSUPER为载体的siHIF1α RNA可降低K562细胞表面CD18的表达，但对Ninj-1表达无明显影响。

Hypoxia-inducible factor 1αregulates the expression of CD18 and ninjurin-1

Objective To investigate the effects of hypoxia-inducible factor 1α (HIF-1α) small interfere RNA construct pSUPERH1-stage of diabetic retinopathy. Methods K562 cells were cultured in 4 groups as control group (group A), diabetic group (group B), diabetes and pSUPERH1-siHIF1α transfect group (group C) and diabetes and pSUPER-retro transfect group (group D). The cells in group A were cultured in human serum from age matched healthy control, and in group B, C and D, the cells were cultured in serum from the subjects of early stage of diabetic retinopathy. Twenty-four hours before the cells were cultured by the serum from the subjects of early stage of diabetic retinopathy, the HIF-1α specific siRNA expression vector pSUPERH1-siHIF1α and empty vector pSUPER-retro were transfected into the cells of group C and D, respectively. The percentages of CD18 and ninjurin-1 positive cell on the surface of K562 cells were measured by Flow Cytometry. The adherent rate between K562 and RF/6A was measured by the rose Bengal staining test. Results The percentages of CD18 positive cell in the group A, B, C and D were significantly different (F=14.33, P=0.01). The percentage of group B was significantly higher than that in group A (P=0.001); the percentage of group C was significantly lower than that in group B (P=0.001) and group D (P=0.02); the difference between group C and A was not significant (95%CI=-14.89 2.13, P=0.12). The differences of the percentage of ninjurin-1 positive cell among the group A, B, C and D were significant (F=39.38, P=0.001). The percentage of group B was significantly higher than that in group A (P=0.00); the difference of the percentage between group C and B was not significant (P=0.06), that was also not significant between group C and D (P=0.49). The differences of the adherent rate between K562 and RF/6A (rhesus monkey retinal choroid blood vessel endothelial cell line) among the group A, B, C and D were significant (F=20.62，P=0.00). The adherent rate of group B was significantly higher than that in group A (P=0.00), the adherent rate in group C was significantly lower than that in group B (P=0.01), but it was still significantly higher than that in group A (P=0.002), the difference of adherent rate between group B and D was not significant (P=0.68). Conclusion Under the early stage of diabetic retinopathy, HIF-1α small interfere RNA pSUPERH1-siHIF1α- may significantly suppress the expression of CD18 on the surface of K562 cells, but it may not significantly influence the expression of ninjurin-1 on the surface of K562 cells.