本計畫為兩年計畫，目的為探討CYP1B1是否可作為為肺癌易感性指標，第一年建立The Real-time RT-PCR assay以定量人類週邊血液中CYP1B1基因表現程度，接著探討影響CYP1B1基因表現程度及被誘導程度之因素，包括Aryl hydrocarbon receptor(AhR), Aryl hydrooarbon receptor nuclear translocator (Arnt)基因表現、性別及抽煙習慣之相關性。另外以Western immunoblot and immunohistostaining method檢測正常肺及肺腫瘤中CYP1B1表現之位置及程度。我們發現性別是影響人類週邊血液中CYP1B1基因表現重要因素，非吸煙女性之AhR and CYP1B1基因表現程度比非吸煙男性高，但是CYP1B1被誘導性在女性較低，經控制性別及抽煙習慣後，CYP1B1與AhR基因表現呈正相關，而CYP1B1被誘導性與Arnt呈正相關。接著我們以Western immunoblot於正常肺及肺腫瘤中均偵測到CYP1B1蛋白，接著以組織免疫螢光染色法確定CYP1B1蛋白位於Smooth muscle cells之細胞質，而Pneumocytes並未表現CYP1B1，3分之19的正常肺組織的正常氣管細胞表現CYP1B1蛋白。第二年計畫將比較正常人及肺癌病人之血液中CYP1B1基因表現程度，另外繼續以組織免疫螢光染色法檢測CYP1B1蛋白於NSCLC組織中表現情形，並且探討血液中及肺組織中CYP1B1與AhR表現之相關性。 The relationships between gene expression of aryl hydrocarbon receptor (AhR), aryl hydrocarbon receptor nuclear translocator (Amt), cytochrome P4501B 1 (CYP 1B1) were determined in 32 cultivated human lymphocytes. Cytochrome P450 induction was performed by lymphocytes with benzanthracne. The relative gene expression levels were determined by quantitative real-time RT-PCR assay. We found gender is an important confounding factor for gene expression. AhR and CYP1B1 levels in non-induced lymphocytes were significantly higher in female nonsmokers than in male nonsmokers (p<0.05). Nevertheless, CYP1B1 inducibility was lower in female nonsmokers. After controlling for gender and cigarette smoking, AhR levels positively correlated with CYP1B1 levels (p<0.01). Arnt levels also correlated with CYP1B1 levels in induced lymphocytes (p<0.01). However, AhR levels were negatively correlated with CYP1B1 inducibility. These data indicate that CYP1B1 expression levels were correlated with AhR and Arnt levels in cultivated lymphocytes. We further examined the expression and localization of CYP lB1 in normal lungs and non-small cell lung cancers (NSCLC). CYP1B1 was detectable in microsomes of normal lungs and NSCLC with Western immunoblotting. Utilizing immunohistochemical staining, we found that CYP1B1 was constitutively expressed in the cytoplasm of smooth muscle cells, but not in pneumocytes. Bronchiolar epithelia from 3 out of 19 normal lungs expressed CYP1B1. In the next year, you will quantify CYP1B1 gene expression in peripheral blood from healthy subjects and lung cancer patients, and examine CYP1B1 protein expression in NSCLC.
