遺伝子発現を利用した照射効果測定

用人结肠癌HT-29细胞研究X射线照射对MUC_1产生的影响。使用单克隆抗体（mAb）MY.1E12鉴定MUC 1。免疫显微镜下，6戈伊照射后第4天，MUC 1阳性细胞率为52%，而未照射细胞为26%。通过流式细胞术分析，6戈伊照射的细胞上的MUC 1在第1天升高，到第4天达到平台。Westem blot分析显示，X射线照射后，MUC 1基因500 kDa和390 kDa条带均增加。通过瞬时表达MUC 1-CAT报告质粒来分析MUC 1基因的转录活性，所述MUC 1-CAT报告质粒含有与细菌氯霉素乙酰转移酶（CAT）基因融合的MUC 1基因的5 '侧翼序列。6戈伊X射线照射后，CAT活性的升高与MUC 1的表达平行。此外，为了研究在体内的影响，我们开发了一个稳定的高表达绿色荧光蛋白（GFP）转染人结肠癌HT-29细胞。将表达GFP的细胞（HT-29/GFP）移植到裸鼠的后肢。40 d后，分别以0、6、10、20、30、50戈伊剂量照射荷瘤裸鼠。4天后，Westem印迹分析显示，X射线照射后肿瘤来源的HT-29/GFP细胞的500 kDa和390 kDa条带均增加。这些发现表明，MUC 1在辐射HT-29细胞中的表达增强是由于MUC 1转录的上调。

The effect of X-irradiation on production of MUC1 was studied with human colon carcinoma HT-29 cells. Monoclonal antibody (mAb) MY.1E12 was used to identify MUC1. Immunomicroscopically, the percentage of MUC1 positive cell was 52% in 6 Gy irradiated cells at day 4, compared with 26% in unirradiated cells. By flow cytometric analysis, MUC1 on 6 Gy irradiated cells was elevated at day 1, reaching plateau by day 4. Westerm blot analysis showed both 500 kDa and 390 kDa bands corresponding to two polymorphic MUC1 alleles were increased after X-irradiation. The transcriptional activity of the MUC1 gene was analyzed by transient expression of MUC1-CAT reporter plasmid containing the 5'-flanking sequence of the MUC1 gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. Elevation of CAT activity was observed in parallel with the expression of MUC1 after 6 Gy of X-irradiation. Moreover, to investigate in vivo effects, we developed a stable high-expression green fluorescent protein (GFP) transfectant of human colon carcinoma HT-29 cells. GFP-expressing cells (HT-29/GFP) were transplanted into the hind rimb of nude mice. After 40 days, tumor-bearing nude mice were irradiated of 0, 6, 10, 20, 30, 50 Gy. After 4 days, Westem blot analysis showed both 500 kDa and 390 kDa bands were increased of tumor derived HT-29/GFP cells after X-irradiation. These findings indicate that enhanced expression of MUC1 in irradiated HT-29 cells was due to upregulation of the MUC1 transcription.