Kipar A, Leutenegger C M, Hetzel U, Akens M K, Mislin C N, Reinacher M, Lutz H
Institute for Veterinary Pathology, Frankfurter Strasse 96, Justus-Liebig University Giessen, D-35392 Giessen, Germany.
Vet Immunol Immunopathol. 2001 Feb 10;78(3-4):305-15. doi: 10.1016/s0165-2427(01)00240-9.
Real-time PCR systems were developed to quantitate cytokine expression in short-time cultivated feline monocytes. Feline-specific interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha) primers as well as TaqMan probes were designed and were adapted to a quantitative PCR system which had been previously established for feline IL-10 and IL-12 p40. Quantitative analysis of cytokine messenger RNA (mRNA) transcription based on the comparison of the cytokine with the housekeeping gene feline glyceraldehyde-3-phosphate dehydrogenase (GAPDH), providing universally expressed mRNA. GAPDH mRNA was readily detectable in cDNA prepared from short-time cultivated peripheral blood monocytes. Cytokine mRNA was demonstrated in all samples at variable amounts. IL-1beta and TNF-alpha mRNA was constitutively expressed whereas IL-6, IL-10 and IL-12 p40 mRNA was generally expressed at a lower level and was occasionally not detected. There was a great variability of cytokine production between individual cats and at different time points in the same cat.
实时荧光定量PCR系统被开发用于定量短时间培养的猫单核细胞中的细胞因子表达。设计了猫特异性白细胞介素-1β（IL-1β）、IL-6和肿瘤坏死因子-α（TNF-α）引物以及TaqMan探针，并将其应用于先前已建立的用于猫IL-10和IL-12 p40的定量PCR系统。基于细胞因子与管家基因猫甘油醛-3-磷酸脱氢酶（GAPDH）的比较，对细胞因子信使核糖核酸（mRNA）转录进行定量分析，GAPDH提供普遍表达的mRNA。在短时间培养的外周血单核细胞制备的cDNA中很容易检测到GAPDH mRNA。在所有样品中均检测到不同量的细胞因子mRNA。IL-1β和TNF-α mRNA组成性表达，而IL-6、IL-10和IL-12 p40 mRNA通常表达水平较低，偶尔未检测到。不同个体猫之间以及同一猫的不同时间点细胞因子产生存在很大差异。
