论文部分内容阅读
目的探讨植物化学保护剂莱菔硫烷(SFN)对结肠癌细胞的生长抑制及诱导作用的抗肿瘤机制。方法①体外培养人结肠腺癌细胞株Caco-2,观察10 -100μmol／L的SFN对Caco-2增殖动力学的影响,MTT法检测细胞生长抑制率。②采用RT-PCR及Western blot检测SFN诱导Caco-2细胞葡萄糖醛酸转移酶 UGT1A及其同工型的表达,高效液相色谱法测定UGT1A的催化活性。③扫描电镜观察SFN诱导细胞凋亡的形态变化;流式细胞仪检测凋亡率。结果①30- 100μmol/L的SFN对细胞增殖均有抑制作用,呈剂量和时间依赖效应。②10- 35μmol／L SFN处理组UGT1AmRNA相对系数与对照组比较差异有统计学意义(P <0．05)。UGT1AmRNA表达量与SFN的剂量呈正相关(r=0．79,P<0．01); 2．5μmol／L SFN处理组与对照组之间UGT1Al(P=0．006)、UGT1A8(P=0．017)、 UGT1A10(P=0．008)表达的差异具有统计学意义;10-30μmol／L SFN作用于Caco -2细胞24h,随着浓度的增加,UGT1A蛋白带的灰度值比值增加。与对照组相比,P<0．05;在SFN处理组对杂环胺N-OH-PhIP的葡萄糖醛酸结合能力明显增高。③扫描电镜观察,25μmol／L处理组无明显改变,50μmol／L、75μmol／L、 100μmol/L处理组发生明显的细胞形态结构变化,典型者出现凋亡小体;流式细胞仪检测结果示随着SFN浓度增高,凋亡率增加,与对照组相比差异有显著性意义 (P<0．01)。结论①植物化学保护剂莱菔硫烷对结肠腺癌细胞株Caco-2的生长增殖具有抑制作用,呈剂量和时间依赖效应;②小剂量的莱菔硫烷能诱导 UGT1A及其同工型UGT1A1、1A8、1A10mRNA表达,UGT1A蛋白表达增加,对杂环胺的葡萄糖醛酸结合能力增强;③大剂量的莱菔硫烷诱导结肠腺癌细胞凋亡。
Objective To investigate the antitumor mechanism of phyto-chemical protective agent sulforaphane (SFN) on the growth of colon cancer cells and its induction. Methods ① Human colon adenocarcinoma cell line Caco-2 was cultured in vitro. The effects of 10 -100 μmol / L SFN on the proliferation kinetics of Caco-2 were observed. The cell growth inhibition rate was detected by MTT assay. ② The expression of UGT1A and its isoform of glucuronosyltransferase in Caco-2 cells induced by SFN was detected by RT-PCR and Western blot. The catalytic activity of UGT1A was determined by high performance liquid chromatography. ③ Scanning electron microscopy was used to observe the morphological changes of SFN-induced apoptosis; apoptosis rate was detected by flow cytometry. Results ①30- 100μmol / L SFN inhibited cell proliferation in a dose-and time-dependent manner. ② The relative coefficient of UGT1A mRNA in 10-35μmol / L SFN treatment group was significantly different from that in control group (P <0.05). The expression of UGT1A mRNA was positively correlated with the dosage of SFN (r = 0.79, P <0.01). There was no significant difference in the expression of UGT1A1 between UGT1A8 and UGT1A8 .017), and UGT1A10 (P = 0.008). There was a significant increase in the gray value of UGT1A protein bands with the increasing concentration of 10-30μmol / L SFN on Caco-2 cells. Compared with the control group, P <0.05; in the SFN treatment group, the glucuronic acid binding ability of heterocyclic amine N-OH-PhIP was significantly increased. ③The results of scanning electron microscopy showed that there was no significant change in 25μmol / L treatment group, obvious morphological changes of cells were observed in 50μmol / L, 75μmol / L and 100μmol / L treatment groups, apoptotic bodies were found in most cases, and flow cytometry With the increase of SFN concentration, the apoptosis rate increased, compared with the control group, the difference was significant (P <0.01). Conclusions ① Sulforaphane inhibits the proliferation of colon adenocarcinoma cell line Caco-2 in a dose-and time-dependent manner. ② Sorafenxuryne can induce UGT1A and its isoforms UGT1A1 and 1A8 , 1A10mRNA expression, UGT1A protein expression increased, the glucuronide binding capacity of heterocyclic amines increased; ③ large doses of sulfasalazine induced colon adenocarcinoma cell apoptosis.