副干酪乳杆菌X12调控N-二甲基亚硝胺诱导肠黏膜细胞烷基化损伤机理研究

N-nitrosodimethylamine is a kind of foodborne carcinogens, which induces DNA alkylation of intestinal mucosal cells. Some lactobacillus strains have the ability to reduce the toxicity of NDMA, but the regulating mechanism is still not clear, and there is no report about L. paracasei subsp. paracasei. Our previous research had shown that L. paracasei subsp. paracasei X12, which was isolated from traditional fermented cheese, was significantly stronger than other lactobacillus strains in reducing the toxicity of NDMA, and could block cell cycle in G1 phase to accelerate DNA reparation. On the basis of these, we plan to analyze the relationship between the ability of X12 reducing of NDMA toxic effect, regulating of the intestinal cell cycle and repairing of DNA damaged. The DNA damage will be detected by immunofluorescence technique and high performance liquid chromatography tandem mass spectrometry; The expression of cell cycle proteins will be detected by qRT-PCR and western blot; The activity of activating enzyme of NDMA and DNA repairing enzyme will be analyzed by ELISA assay; The experimental results in vitro will be further verified via rat test. This project will confirm the relationship between the ability of X12 reducing of NDMA toxic effect, regulating of the intestinal cell cycle and repairing of DNA damaged, and will reveal the mechanism of X12 exert on intestinal cells during the process of DNA damage induced by NDMA.

N-二甲基亚硝胺（NDMA）是食源性致癌剂，诱导肠道黏膜细胞烷基化损伤。多个乳杆菌菌种有降低NDMA毒性的能力，但作用机制尚不清楚，且没有副干酪乳杆菌的相关报道。课题组前期研究显示从传统发酵奶酪中分离的副干酪乳杆菌X12降低NDMA毒性的能力显著高于其他乳杆菌，并将细胞周期阻滞在G1期以利于DNA自我修复。本项目在此基础上，拟对X12降低NDMA毒性作用能力、对受损肠道细胞周期调控、受损细胞DNA修复之间的关系进行分析。采用免疫荧光、高效液相色谱串联质谱技术检测肠道细胞DNA损伤，采用实时定量荧光PCR和免疫蛋白印迹技术分析细胞周期蛋白表达，采用ELISA法分析NDMA活化酶、DNA修复酶活性的变化，采用大鼠试验进一步验证体外实验结果。本项目旨在明确X12降低NDMA毒性作用能力与其对肠道细胞周期调控、受损DNA修复之间的关系，揭示X12在NDMA致肠道细胞DNA损伤过程中发挥的作用机制。

N-二甲基亚硝胺（NDMA）可诱导肠道黏膜细胞烷基化损伤。从传统发酵奶酪中分离的副干酪乳杆菌X12具有良好的益生功能，可耐受住胃、肠液的消化。副干酪乳杆菌X12还可降低NDMA对大鼠肠道黏膜细胞IEC-6的影响。CCK-8实验和细胞形态学实验结果显示，副干酪乳杆菌X12可显著降低NDMA对IEC-6细胞的毒性损伤。单细胞凝胶电泳实验、DNA ladder及细胞周期实验结果显示，高剂量的副干酪乳杆菌X12可降低NDMA对IEC-6细胞DNA的损伤，并将IEC-6细胞周期阻滞在G1/S期，抑制细胞的增殖，以利于受损的细胞进行DNA自我修复。细胞分子水平实验通过半定量RT-PCR和实时定量荧光PCR检测NDMA活化基因和细胞周期调控/抑制基因的表达，免疫蛋白印迹技术分析NDMA活化蛋白和细胞周期调控蛋白的表达。分子水平实验结果显示，副干酪乳杆菌X12通过对受损IEC-6细胞周期的调控，并通过对受损细胞DNA修复而降低NDMA的毒性。动物实验结果也显示，副干酪乳杆菌X12可降低NDMA对大鼠结肠黏膜的损伤作用，从而阐明副干酪乳杆菌X12调控由NDMA诱导的大鼠肠道黏膜细胞DNA损伤的机制。