Rieckmann P, Albrecht M, Ehrenreich H, Weber T, Michel U
Department of Psychiatry, University of Göttingen, Germany.
Res Exp Med (Berl). 1995;195(1):17-29. doi: 10.1007/BF02576770.
An easy, reproducible and semi-quantitative, non-radioactive method for the analysis of mRNA expression for various cytokines, (i.e., Interleukin (IL)-1 beta, IL-4, IL-6, tumor necrosis factor (TNF)-alpha, lymphotoxin (LT), transforming growth factor (TGF)-beta, interferon (IFN)-gamma and endothelin-1 (ET-1)) in cells from cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMC) has been established. By means of polymerase chain reaction primers that cover a splice junction, amplification of contaminating DNA was omitted. Densitometric scanning of ethidium bromide-stained agarose gels proved to be very sensitive for semiquantitative analysis of PCR products. Serial tenfold dilutions of cDNA revealed a log-linear regression from 10(6) to 10(2) cells under optimal cycle conditions. The intra- and inter-assay variability of the method was below 10%. With this assay, the cytokine expression pattern of as few as 10(4) mononuclear cells from blood or CSF was determined. This method made it possible to detect differences in the cytokine gene expression pattern of mononuclear cells from patients with different neurological diseases. CSF cells from 43 patients with various neurological diseases were analyzed. TNF-alpha, LT, and IL-1 mRNA were prominent in the CSF cells of most patients with bacterial meningitis. TNF-alpha, LT, IFN-gamma and IL-6 mRNAs were detected in patients with active multiple sclerosis, whereas TNF-alpha, IL-6, and endothelin-1 mRNA expression was found frequently in patients with HIV encephalitis. Pro-inflammatory cytokines were rarely detected in CSF cells from patients with non-inflammatory diseases of the central nervous system. In blood mononuclear cells from patients with multiple sclerosis, TNF-alpha mRNA expression was associated with disease activity. The sensitivity, specificity, velocity and reliability of this assay considerably facilitates the analysis of cytokine production in mononuclear cells even in conditions where only a limited number of cells is available for analysis.
已建立一种简便、可重复且半定量的非放射性方法，用于分析脑脊液（CSF）细胞和外周血单核细胞（PBMC）中多种细胞因子（即白细胞介素（IL）-1β、IL-4、IL-6、肿瘤坏死因子（TNF）-α、淋巴毒素（LT）、转化生长因子（TGF）-β、干扰素（IFN）-γ和内皮素-1（ET-1））的mRNA表达。通过覆盖剪接连接点的聚合酶链反应引物，省略了污染DNA的扩增。溴化乙锭染色琼脂糖凝胶的光密度扫描被证明对PCR产物的半定量分析非常敏感。在最佳循环条件下，cDNA的系列十倍稀释显示出从10(6)到10(2)个细胞的对数线性回归。该方法的批内和批间变异性低于10%。通过该检测方法，可确定来自血液或CSF的低至10(4)个单核细胞的细胞因子表达模式。该方法使得检测不同神经疾病患者单核细胞中细胞因子基因表达模式的差异成为可能。对43例患有各种神经疾病的患者的CSF细胞进行了分析。大多数细菌性脑膜炎患者的CSF细胞中TNF-α、LT和IL-1 mRNA显著。活动性多发性硬化症患者中检测到TNF-α、LT、IFN-γ和IL-6 mRNA，而HIV脑炎患者中经常发现TNF-α、IL-6和内皮素-1 mRNA表达。中枢神经系统非炎性疾病患者的CSF细胞中很少检测到促炎细胞因子。在多发性硬化症患者的血液单核细胞中，TNF-α mRNA表达与疾病活动相关。该检测方法的敏感性、特异性、速度和可靠性极大地促进了单核细胞中细胞因子产生的分析，即使在仅能获得有限数量细胞用于分析的情况下也是如此。
