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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 observe the effect of interleukine 6 (IL 6) and antisense oligonucleotide (ASON) on the expression of IL-6 in RPE cells from human retinal pigment epithelial cells (RPE) , For the prevention and treatment of proliferative vitreoretinopathy looking for new drugs. Methods The cultured RPE cells were stimulated with IL-1β, and the expression of IL-6 protein and mRNA in RPE cells was observed by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry and in situ hybridization. Results Under the condition of IL-1β, the expression of IL-6 protein in RPE cells was time-and dose-dependent. The IL 6 protein content in supernatant of RPE cells in control group was 2.0 × 10 6 g / L after stimulated with IL 1β for 8 h, while IL 6 protein in IL 6ASON group (200 × 10 5 mol / L) Reduced to 5 5 × 10 7 g / L; there was significant difference between the two groups (t = 4 5 18, P 0 01). The results of immunohistochemistry showed that the expression of IL-6 protein in the cytoplasm of RPE cells in the control group was shades of brown, while the staining of IL-6ASON group was lighter. There was significant difference between the two groups (t = 2 32 5, P <0 0 5). In situ hybridization results showed that the expression of IL 6 mRNA in the cytoplasm of RPE cells in the control group was purple-blue, while the staining of IL-6ASON group was lighter. The results of image analysis showed that there was significant difference in gray value between the two groups (t = 3 74 6 , P <0 0 1). Conclusions Under the stimulation of IL 1β, human RPE cells can express IL 6 protein obviously.