[HT-29细胞中Toll途径的分子表达及调控研究]

[Study on the molecular expression and regulation of toll pathway in HT-29 cells].

作者信息

Chen Xi, Ouyang Qin

机构信息

Department of Gastroenterology, West China Hospital, Sichuan University, Chengdu 610041, China.

出版信息

Sichuan Da Xue Xue Bao Yi Xue Ban. 2010 Jul;41(4):581-5.

PMID:20848773

Abstract

OBJECTIVE

To investigate the changes of TLR4, MD2 and HBD2 in transcription and protein expression levels in HT-29 colon cancer cell line after inducing by cytokines TNF-alpha, IL-1beta, IFN-gamma and LPS, and its relationship with NF-kappaB activation.

METHODS

HT-29 cells were divided into 8 groups, by adding RPMI 1640, TNF-alpha (20 ng/mL), IL-1beta (20 ng/mL), IFN-gamma (20 ng/mL), LPS (50 ng/mL), TNF-alpha (20 ng/mL) + LPS (50 ng/ mL), IL-1beta (20 ng/mL) + LPS (50 ng/mL), IFN-gamma (20 ng/mL) + LPS(50 ng/mL) respectively for intervention. ELISA was applied to detect the IL-8 in the supernatants of each group. The level of TLR4, MD2 and HBD2 mRNA were assayed by RT-PCR. The expressions of TLR4 and NF-kappaB protein of each group were determined by western blot.

RESULTS

IL-8 expressions in supernatant of cytokines and cytokine plus LPS group were higher than that of control (P < 0.01). Pre-incubation with cytokines, following by LPS stimulation, HT-29 cells further augmented IL-8 secretion (P < 0.01). Increased levels of TLR4 and MD2 mRNA in HT-29 cells with Cytokines and cytokine plus LPS stimulating were observed (P < 0.01). The level of HBD2 mRNA were elevated in IL-1beta and LPS plus (TNF-alpha, IL-1beta, IFN-gamma) groups (P < 0.05). Elevated expressions of TLR4 and NF-kappaB protein in HT-29 cells with cytokine and cytokine plus LPS were also observed (P < 0.01). Pre-incubation with cytokines, following by LPS stimulation, HT-29 cells further up-regulated NF-kappaB protein expression (P < 0.05).

CONCLUSIONS

Cytokines (TNF-alpha, IL-1beta, IFN-gamma) can enhance TLR4 and MD-2 expressions and promote the reaction with LPS in intestinal epithelial cells (IEC), which leads to over activity of IEC with commensal bacteria and initiation or aggravation of intestinal inflammation. Inflammatory stimuli (IL-1beta, LPS + TNF-alpha, LPS + IFN-gamma) may induce the transcription and expression of HBD2 gene in IEC, which leads to enhancing intestinal adaptive immune responses and affects the progress of intestinal inflammation.

摘要

目的

探讨细胞因子肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、干扰素-γ（IFN-γ）和脂多糖（LPS）诱导后，HT-29结肠癌细胞系中Toll样受体4（TLR4）、髓系分化蛋白2（MD2）和β-防御素2（HBD2）在转录水平和蛋白表达水平的变化，及其与核因子-κB（NF-κB）激活的关系。

方法

将HT-29细胞分为8组，分别加入RPMI 1640、TNF-α（20 ng/mL）、IL-1β（20 ng/mL）、IFN-γ（20 ng/mL）、LPS（50 ng/mL）、TNF-α（20 ng/mL）+LPS（50 ng/mL）、IL-1β（20 ng/mL）+LPS（50 ng/mL）、IFN-γ（20 ng/mL）+LPS（50 ng/mL）进行干预。采用酶联免疫吸附测定法（ELISA）检测各组细胞上清液中白细胞介素-8（IL-8）水平。采用逆转录-聚合酶链反应（RT-PCR）检测TLR4、MD2和HBD2 mRNA水平。采用蛋白质免疫印迹法检测各组TLR4和NF-κB蛋白表达。

结果

细胞因子组及细胞因子加LPS组上清液中IL-8表达高于对照组（P<0.01）。细胞因子预孵育后再用LPS刺激，HT-29细胞IL-8分泌进一步增加（P<0.01）。细胞因子及细胞因子加LPS刺激的HT-29细胞中TLR4和MD2 mRNA水平升高（P<0.01）。IL-1β组及LPS加（TNF-α、IL-1β、IFN-γ）组HBD2 mRNA水平升高（P<0.05）。细胞因子及细胞因子加LPS刺激的HT-29细胞中TLR4和NF-κB蛋白表达升高（P<0.01）。细胞因子预孵育后再用LPS刺激，HT-29细胞NF-κB蛋白表达进一步上调（P<0.05）。

结论

细胞因子（TNF-α、IL-1β、IFN-γ）可增强肠道上皮细胞（IEC）中TLR4和MD-2表达，并促进其与LPS反应，导致IEC与共生菌过度激活，引发或加重肠道炎症。炎症刺激（IL-1β、LPS+TNF-α、LPS+IFN-γ）可能诱导IEC中HBD2基因转录和表达，从而增强肠道适应性免疫反应，影响肠道炎症进程。