Brink N, Szamel M, Young A R, Wittern K P, Bergemann J
Beiersdorf AG, Paul Gerson Unna Skin Research Center, Hamburg, Germany.
Inflamm Res. 2000 Jun;49(6):290-6. doi: 10.1007/PL00000209.
Ultraviolet (UV) exposure induces local immunosuppression and inflammation in human skin. Cytokines are, in part, responsible for these responses. To investigate the effects of UV-induced gene expression at the molecular level we established a sensitive in vivo/ex vivo method for a comparative quantification of cytokines and receptors involved in the local skin immune reactions.
Specific mRNA levels of human UV-irradiated skin were determined by real time quantification (TaqMan RT-PCR). Highly efficient PCR-reaction conditions were obtained by designing very short PCR-templates (72-87 bp). The most sensitive PCR-conditions were obtained by optimisation of primer and Mn(OAc)2-concentrations, which led to significant PCR signals (C(T)-value) of less than 36 cycles. A strong correlation between PCR efficiency of the internal control (GAPDH) compared to targets (IL-1beta, IL-10, IL-10r, TNFalpha, IL-7) allowed the use of deltadelta C(T)-method to quantify comparable mRNA levels.
Interleukin-1beta (IL-1beta), Interleukin-10 (IL-10), and tumour necrosis factor alpha (TNFalpha) mRNA levels were increased in a time- and dose-dependent manner. Interleukin-1beta induction reached a maximum (approx. 44-fold) 6 h after a UV-dose equivalent to 3 times the minimal erythemal doses just perceptible (MEDjp). Maximal TNFalpha mRNA expression (approx. 14-fold) was also detected 6 h after UV exposure. Interleukin-10 mRNA induction reached a maximum of approximately 14-fold 24 h after UV-irradiation of 3 MEDjp. Time- and dose-dependent changes in Interleukin-7 and Interleukin-10 receptor mRNA levels did not occur after UV-irradiation.
Time-distinct gene induction of IL-1beta, TNFalpha and IL-1beta is involved in UV-induced immune reactions, but no considerable changes were found for IL-10r or IL-7.
紫外线（UV）照射可诱导人体皮肤局部免疫抑制和炎症。细胞因子在一定程度上介导了这些反应。为了在分子水平上研究紫外线诱导的基因表达的影响，我们建立了一种灵敏的体内/体外方法，用于比较定量参与局部皮肤免疫反应的细胞因子和受体。
通过实时定量（TaqMan RT-PCR）测定人紫外线照射皮肤的特定mRNA水平。通过设计非常短的PCR模板（72 - 87 bp）获得高效的PCR反应条件。通过优化引物和Mn(OAc)2浓度获得最灵敏的PCR条件，这导致小于36个循环的显著PCR信号（C(T)值）。与靶标（IL-1β、IL-10、IL-10r、TNFα、IL-7）相比，内对照（GAPDH）的PCR效率之间存在强相关性，这使得可以使用ΔΔC(T)方法来定量可比的mRNA水平。
白细胞介素-1β（IL-1β）、白细胞介素-10（IL-10）和肿瘤坏死因子α（TNFα）的mRNA水平呈时间和剂量依赖性增加。在相当于刚刚可察觉的最小红斑剂量（MEDjp）的3倍的紫外线剂量照射后6小时，白细胞介素-1β的诱导达到最大值（约44倍）。紫外线照射后6小时也检测到最大的TNFα mRNA表达（约14倍）。在3 MEDjp的紫外线照射后24小时，白细胞介素-10的mRNA诱导达到最大值约14倍。紫外线照射后，白细胞介素-7和白细胞介素-10受体的mRNA水平没有出现时间和剂量依赖性变化。
IL-1β、TNFα和IL-1β的时间特异性基因诱导参与紫外线诱导的免疫反应，但未发现IL-10r或IL-7有明显变化。
