摘要
目的探讨肿瘤坏死因子-α(TNF-α)对肠屏障功能的影响及机制。方法体外培养Caco-2细胞,细胞稳定生长后随机分为四组,对照组给予完全培养基培养,低剂量组给予TNF-α100μg/L,中剂量组给予TNF-α120μg/L,高剂量组给予TNF-α140μg/L,培养6 h后,收集细胞、上清及蛋白等。用倒置显微镜观察各组Caco-2细胞形态变化;用CCK-8法检测各组Caco-2细胞的增殖抑制情况;用RT-qPCR法检测各组细胞MUC2 mRNA表达;用Westernblotting法检测各组细胞通路蛋白JNK1和MKK4表达。结果不同剂量TNF-α刺激6 h后,细胞形态发生变化且细胞间紧密连接逐渐消失,细胞活力明显下降,各组差异有统计学意义(P均<0.05)。对照组、低剂量组、中剂量组和高剂量组细胞活力(A_(450))分别为1.071±0.021、0.767±0.028、0.487±0.021、0.249±0.005;与对照组相比,低剂量组、中剂量组和高剂量组Caco-2细胞活力下降(P均<0.05);低剂量、中剂量和高剂量组两两比较差异均有统计学意义(P均<0.05)。对照组、低剂量组、中剂量组和高剂量组MUC2 mRNA相对表达量分别为1、0.451±0.071、0.159±0.040、0.109±0.001;与对照组相比,低剂量组、中剂量组和高剂量组MUC2 mRNA表达逐渐降低,高剂量组较明显,差异均有统计学意义(P均<0.05)。与对照组相比,低剂量组、中剂量组和高剂量组JNK1蛋白表达逐渐升高,高剂量组较明显,差异均有统计学意义(P均<0.05);低剂量组、中剂量组和高剂量组MKK4蛋白表达高于对照组(P均<0.05)。结论TNF-α可能通过JNK信号通路抑制Caco-2细胞增殖并下调MUC2表达,进而引起肠屏障功能障碍。
Objective To investigate the effect of tumour necrosis factor-α(TNF-α)on intestinal barrier function and its mechanism.Methods Caco-2 cells were cultured in vitro,and the cells were randomly divided into four groups after stable growth.The cells in the control group were cultured in complete medium,the cells in the low-dose group were given 100μg/L TNF-α,the cells in the medium-dose group were given 120μg/L TNF-α,and the cells in the high-dose group were given 140μg/L TNF-α.After 6 h of culture,the cells,supernatants and proteins were collected.The effect of different concentrations of TNF-αon the morphology of Caco-2 cells was observed by inverted microscope;the proliferation inhibition of Caco-2 cells was detected by CCK-8;the expression of MUC2 mRNA was detected by RT-qPCR;and the ex⁃pression of pathway proteins JNK1 and MKK4 was detected by Western blotting.Results After 6-hour stimulation with different doses of TNF-α,the cell morphology changed,the tight junctions between cells disappeared gradually,and the cell viability decreased significantly;the difference of each group was statistically significant(all P<0.05).The cell via⁃bility(A450)of the control group,low-dose group,medium-dose group,and high-dose group were 1.071±0.021,0.767±0.028,0.487±0.021,and 0.249±0.005,respectively.Compared with the control group,the viability of Caco-2 cells decreased in the low-dose,medium-dose,and high-dose groups(all P<0.05),and there were significant differences among the low-dose,medium-dose,and high-dose groups(all P<0.05).The relative expression of MUC2 mRNA was 1,0451±0.071,0.159±0.040,and 0.109±0.001 in the control group,low-dose group,medium-dose group,and high-dose group,respectively.Compared with the control group,the expression of MUC2 mRNA decreased gradually in the lowdose,medium-dose,and high-dose groups,with statistically significant difference,and the high-dose group was more obvi⁃ous(all P<0.05).Compared with the control group,the expression of JNK1 protein increased gradually in th
作者
阿曼古丽·莫明
卡依赛尔·阿布都肉苏力
雷泓
张琪
宋云林
Amanguli Moming;Kayisaier Abudurousuli;LEI Hong;ZHANG Qi;SONG Yunlin(Intensive Care Center,The First Affiliated Hospital of Xinjiang Medical University,Urumchi 830054,China;不详)
出处
《山东医药》
CAS
2022年第34期45-48,共4页
Shandong Medical Journal
基金
国家自然科学基金资助项目(81560310)。
