摘要
目的 在体外人神经细胞瘤细胞株 (SK N AS)中 ,以二恶英 (TCDD)、三甲基胆蒽 (3 MC)作为诱导化合物 ,探讨多环芳烃受体 (AHR、ARNT)基因与细胞色素P45 0 1A1/1B1(CYP1A1、CYP1B1)基因表达的调控 ,并研究其剂量反应关系和时间反应关系。方法 用常规的细胞培养方法 ,用二甲基亚砜 (DMSO)、5 0、5 0 0、10 0 0nmol/LTCDD和 0 1、1 0、10 0μmol/L 3 MC处理细胞 12h、2 4h、48h、72h ,利用提纯RNA和合成cDNA的药盒 ,合成cDNA ,然后通过逆转录聚合酶反应(RT PCR)表达AHR、ARNT和CYP1A1、CYP1B1基因 ,以 β actin作为内对照 ,分析不同处理剂量、时间时基因表达的强度。结果 4种基因在SK N AS中都有基本的表达 ,TCDD在 5 0 0nmol/L以上 ,染毒处理 48h、72h ,对AHR、ARNT及CYP1A1基因表达都有上调作用 ;5 0nmol/L组在染毒处理 72h后对AHR基因表达 ,5 0 0和 10 0 0nmol/L组分别在染毒处理 72h和 48h时对CYP1B1基因表达也有上调作用 ;3 MC 1 0、10 0 μmol/L在染毒 72h后对AHR、ARNT、CYP1A1基因表达有上调作用 ,其中 10 0 μmol/L组在染毒 48h后就有上调作用 ,但对CYP1B1基因表达上调仅仅在 10 0 μmol/L染毒 48h组。结论 本研究结果提示 ,TCDD、3 MC两种多环芳烃类化合物在SK N AS中 。
Objective To study the regulation of AHR, ARNT, CYP1A1, CYP1B1 genes expression in the SK-N-AS cell line after treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) and 3-methylcholanthrene(3-MC) and their time and dose-response. Methods The human cell line SK-N-AS was treated with DMSO, 5.0, 50.0, 100.0 nmol/L TCDD and 0.1, 1.0, 10.0 μmol/L 3-MC for 12, 24, 48, 72 hours, respectively. RNA of cell line was purified by RNA kit. The cDNA synthesis kit was used to synthesize cDNA. Then, PCR was conducted to amplify the fragment of AHR, ARNT, CYP1A1, CYP1B1 DNA. Results AHR, ARNT, CYP1A1, CYP1B1 genes were constitutively expressed in the human cell line SK-N-AS. AHR, ARNT, CYP1A1 were up-regulated after 48 hours, 72 hours treated with 50.0, 100.0 nmol/L TCDD and after 72 hours treated with 1.0 μmol/L, 10.0 μmol/L 3-MC, respectively. AHR gene was also up-regulated after 72 hours treated with 5.0 nmol/L TCDD. The up-regulation of CYP1B1 was only observed after 48 hours, 72 hours treated with 50.0, 100.0 nmol/L TCDD and after 48 hours treated with 10.0 μmol/L 3-MC. Conclusion The results showed that the expressions of AHR, ARNT, CYP1A1, CYP1B1 genes could be significantly regulated by TCDD and 3-MC in the SK-N-AS cell line. This cell line should be a good model for further mechanistic studies on AHR and CYP450 expression.
出处
《中国职业医学》
CAS
北大核心
2003年第3期10-13,共4页
China Occupational Medicine
