Joyce D A, Steer J H, Beilharz M W, Stranger R
Sir Charles Gairdner Hospital, Nedlands, Western Australia.
Genet Anal. 1995 Mar;12(1):39-43. doi: 10.1016/1050-3862(95)00104-2.
Monocyte derived cytokines (monokines) are important mediators in inflammatory diseases and cancer. Control of monokine expression is also a major therapeutic target in autoimmune inflammation. Whole blood cultures permit examination of monokine expression under conditions which emulate the in-vivo environment whilst avoiding many of the artefacts associated with monocyte separation and culture. Here we describe a system for measuring interleukin-1 beta, interleukin-1 alpha, interleukin-6 and tumour necrosis factor-alpha mRNA in stimulated human whole blood ex-vivo, which can be applied to specimens from treated patients. Oligodeoxyribonucleotide probes are designed to allow standardisation of hybridisation and washing procedures. Washing and reprobing of membranes in appropriate sequence permits measurement of each monokine mRNA and mRNA for glyceraldehyde-3-phosphate dehydrogenase in only 7 ml of lipopolysaccharide-stimulated human blood. The method has been used successfully in studies of dexamethasone and methotrexate action on lipopolysaccharide stimulated IL-beta gene expression.
单核细胞衍生的细胞因子（单核因子）是炎症性疾病和癌症中的重要介质。控制单核因子的表达也是自身免疫性炎症的主要治疗靶点。全血培养允许在模拟体内环境的条件下检查单核因子的表达，同时避免许多与单核细胞分离和培养相关的假象。在此，我们描述了一种用于在体外刺激的人全血中测量白细胞介素-1β、白细胞介素-1α、白细胞介素-6和肿瘤坏死因子-α mRNA的系统，该系统可应用于来自接受治疗患者的标本。设计寡脱氧核糖核苷酸探针以实现杂交和洗涤程序的标准化。以适当顺序对膜进行洗涤和重新杂交，仅用7毫升脂多糖刺激的人血就可以测量每种单核因子mRNA以及甘油醛-3-磷酸脱氢酶的mRNA。该方法已成功用于地塞米松和甲氨蝶呤对脂多糖刺激的IL-β基因表达作用的研究。
