摘要
目的 探讨核因子E2p45相关因子2（Nrf2）基因在茶多酚中的主要化学物质epigallocatechin gallate（EGCG）诱导结肠癌细胞尿苷二磷酸葡萄糖醛酸转移酶（UGT）1A及其同工酶UGT1A8、UGTIA10表达中的调节作用。方法EGCG分别作用Caco-2及HT-29结肠癌细胞12h，逆转录聚合酶链反应（RT-PCR）检测基因表达水平的变化，免疫细胞化学及激光共聚焦显微镜观察细胞内Nrf2蛋白定位的改变。此外，利用RNA干扰技术抑制细胞内Nrf2基因的表达，观察阻断前后EGCG对基因表达水平影响的改变。结果（1）Caco-2及HT-29结肠癌细胞系、结肠癌及癌旁组织中UGTlA、1A8、1A10基因表达水平存在差异。（2）EGCG作用Caco-2及HT-29细胞后Nrf2、UGTlA、1A8、1A10表达升高1．8～9．2倍（均P〈0．05），免疫细胞化学及激光共聚焦显微镜检测到Nrf2蛋白向细胞核内聚集。（3）酶切分析和测序证实成功构建了RNA干扰真核表达载体pSilence-Nrf2-A、B、C、D及随机序列对照pSilence-CON。（4）pSilence-Nrf2-B质粒转染可显著抑制Nrf2基因的表达，在caco-2及HT-29细胞中抑制率分别为81．46％±1．68％及84．72％±2．08％（实验重复3次）；稳定筛选建立的Nrf2低表达的细胞系Caco-2-siNrf2、HT-29-siNrf2，不仅基础UGT1A酶的表达水平降低，而且EGCG作用后酶诱导表达的作用消失。结论Nrf2基因参与了EGCG诱导UGT1A及其同工酶表达的过程并在其中起关键作用，为进一步论证EGCG在结肠癌化学预防中的作用及其可能的机制提供了新的论据。
Objective To investigate the role of human transcription factor NF-E2-related factor 2 （Nrf2） in the induction of the gene expression of uridine 5＇-diphosphate-glucuronosyltransferase （UGT） 1A and its isoforms by epigallocatechin gallate （EGCG）. Methods （ 1 ） Human colon carcinoma cells Caco-2 and HT-29 were cultured. Immunocytochemistry, western blotting and confocal laser microscopy were used to detect the protein expression of Nrf2. Twenty samples of colon carcinoma with surrounding normal tissues were collected during endoscopic course. （2） RNA interference expression vector pSilencerTM 3. 1-H1 was used to construct four Nrf2-trageting plasrnids ： pSilence-Nrf2-A, B, C, and D and a control pSilence-CON. Cells were transfected with pSilence-Nrf2 for 48 hours to observe the effects of transient transfection. Cells were stably transfected with pSilence-Nrl2-B for 4 weeks and re-named as Caco-2-siNrf2 and HT-29-siNrf2 （ siNrf2 cells） ,and others stably transfected with blank plasrnid pSilencerTM 3.1-H1 were used as controls. （ 3 ） EGCG was added into the culture fluid of cells before and after the stably transfection. RT-PCR was used to detect the rnRNA expression of Nrf2, UGTIA, UGTIA8 and UGTIA10 in cells and the samples of human colon cancer tissue. Results （ 1 ） The expressions of UGT1A8 and UGT1A10 rnRNA were significantly lower than that in the surrounding healthy rnucosa. （2）The rnRNA expression of Nrf2, UGT1A8, and UGT1A10 increased by 1.8-9.2 times after the addition of EGCG （ all P 〈0. 05 ）. Immunocytochernistry, western blotting and irnrnunofluorescence demonstrated a significant increase of Nrf2 protein expression in the nucleus after treatment with EGCG. （3） Sal I enzyme digestion and DNA sequencing confirmed that pSilence-Nrf2-A, B, C, and D and pSilence-CON were all successfully constructed. The inhibition rate of Nrf2 gene expression was above 80% 48 h after transfection with pSilence-Nrf2-B, and that was no significant difference after transfection with pSilence-CON （ P 〉 0.05 ）. There was specific inhibition of Nrf2 in Caco-2-siNrf2, HT-29-siNrf2 cells （both P 〈0.01 ）. （4） The basal levels of UGT1A8 and UGT1A10 mRNA expression in the Caco-2-siNrf2 and HT-29-siNrf2 cells were lower by 15%-65% in comparison with those in control,and the induction of genes by EGCG was largely attenuated in them （ all P 〉 0.05 ）. Conclusion Nrf2 is localized in the cytoplasm of non-stimulated cells, and EGCG triggered its rapid nuclear accumulation. Suppression of Nrf2 gene expression results in down-regulation of the constructive expression of UGT genes and their induction by EGCG. EGCG induces the expression of UGT1A,UGT1A8 and UGT1A10 genes via a Nrf2-dependent mechanism.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2006年第2期82-87,共6页
National Medical Journal of China
基金
国家自然科学基金资助项目(30370634)
