白细胞介素-6反义寡核苷酸抑制人视网膜色素上皮细胞表达白细胞介素-6

Inhibition of IL-6 antisense oligonucleotide on IL-6 expression by human retinal pigment epithelium in vitro

目的 观察人视网膜色素上皮 (retinalpigmentepithelium ,RPE)细胞表达白细胞介素 6(interleukine 6 ,IL 6 )和IL 6反义寡核苷酸 (antisenseoligonucleotide ,ASON)从基因水平阻断RPE细胞表达IL 6的效应 ,为增生性玻璃体视网膜病变的防治寻找新的药物。方法 将培养的人RPE细胞以IL 1β刺激后 ,用酶联免疫吸附试验 (enzymelinkedimmunosorbentassay ,ELISA)、免疫组化染色及原位杂交等方法 ,观察RPE细胞表达IL 6蛋白质和mRNA的情况。结果 在IL 1β介导下 ,RPE细胞对IL 6蛋白质的表达呈时间和剂量反应关系。用IL 1β刺激 8h ,对照组RPE细胞培养上清液中IL 6蛋白质含量为 2 0 0× 10 6g/L ;IL 6ASON (浓度为 2 0 0× 10 5mol/L)组的IL 6蛋白质含量明显降低 ,为 5 5 0×10 7g/L ;两组比较差异有显著意义 (t=4 5 18,P <0 0 1)。免疫组化染色结果显示 ,对照组RPE细胞胞浆中IL 6蛋白质的表达呈深浅不一的棕黄色 ,IL 6ASON组染色则较浅 ;两组比较差异有显著意义(t=2 32 5 ,P <0 0 5 )。原位杂交结果表明 ,对照组RPE细胞胞浆中IL 6mRNA的表达呈紫蓝色 ,而IL 6ASON组染色较淡 ;图像分析结果显示 ,两组灰度值差异有显著意义 (t=3 74 6 ,P <0 0 1)。结论在IL 1β刺激下 ,人RPE细胞可明显表达IL 6蛋白?

Objective To investigate the inhibition of IL 6 antisense oligonucleotide (ASON) on IL 6 expression by retinal pigment epithelium (RPE) cells on the basis of previous study Methods Cultured human RPE cells was treated with IL 1 β and IL 6 ASON IL 6 mRNA and protein expression were detected by enzyme linked immunosorbent assay (ELISA), immunohistochemistry and in situ hybridization histochemistry (ISH) Results It was demonstrated that the IL 6 expression by RPE cells was dose and time dependent after the stimulation of IL 1 β IL 6 in the conditioned media or in the control group was 2 00×10 6 g/L cells after the exposed to IL 1 β for 8 hours IL 6 ASON (2 00×10 5 mol/L) obviously inhibited IL 6 (5 50×10 -7 g/L cells, t =4 518, P <0 01) production Immunohistochemistry study showed dark blue staining in RPE cytoplasm after stimulation with IL 1 β , while the cells treated with IL 6 ASON showed light staining in RPE cytoplasma ISH displayed that there was a marked reduction in mRNA expression in IL 6 ASON treated group compared with that in the control group ( t =3 746, P <0 01) Conclusion Cultured RPE cells express IL 6 protien and mRNA in dose and time dependent manners when RPE cells are stimulated with IL 1 β The expressions of IL 6 protein and mRNA can be significantly inhibited by IL 6 ASON in cultured human RPE cells