诱导Caco-2细胞凋亡的海洋源植物乳杆菌B-27胞外多糖结构特征及合成基因的研究

The exopolysaccharide produced by lactic acid bacteria is important in the application for food industry. It is necessary for assessment of the function and application that the structure and activity of the exopolysaccharide produced by lactic acid bacteria is clarified. The exopolysaccharide produced by the strain Lactobacillus plantarum B-27, which was isolated from fan shell in Dalian sea area, exerted antiproliferative effect on colon cancer cell line Caco-2. But the molecule structure, the structure and activity relationship and the key gene of the exopolysaccharide is not clear. The purpose of the project is to study the primary structural characteristic of polysaccharide composition including determining the monosaccharide component and the linkages of glycosidic bond; study the advanced structral characteritic of polysaccharide composition including the symmetry and screw pitch; study the effects of different polysaccharide complsition with different chemical structure on the protein expression related to the mitochondrial pathway and the EGFR pathway during apoptosis of Caco-2 cells, and analyse the difference of Caco-2 cell apoposis induced by different polysaccharide composition, determine the key polysaccharide composition having anticancer activity; determine the key genes which involve in the EPS synthesis by the bioinformatics analysis of Lactobacillus plantarum B-27 genome and gene knockout techniques.

乳酸菌胞外多糖在食品及功能食品领域有广阔的应用前景，阐明乳酸菌胞外多糖的化学结构及关键生物合成基因，将为胞外多糖构效关系的研究及开发利用提供理论依据。本课题组从大连海域扇贝中分离获得产胞外多糖的海洋源植物乳杆菌B-27，前期研究发现该菌株胞外多糖可诱导人结肠癌细胞Caco-2凋亡，但该菌株胞外多糖的化学结构、构效关系及合成基因簇尚不明确。本项目拟对该菌株胞外多糖进行分离纯化，获得多糖单一组分，研究多糖组分的单糖组成、糖苷键连接方式、分子内成键方式，确定多糖分子对称性、螺距、溶液构象，明确其一级和高级结构；研究多糖单一组分对Caco-2细胞凋亡途径相关蛋白表达的影响，分析多糖组分化学结构与诱导细胞凋亡的关系，确定具有诱导细胞凋亡的多糖功能基团；对植物乳杆菌B-27进行生物信息学分析和基因敲除实验，定位可诱导肿瘤细胞凋亡的多糖单一组分合成基因簇，分析基因序列并进行功能注释。

本课题研究了一株植物乳杆菌所产胞外多糖（EPS）的结构特征及其抑制肿瘤细胞增殖、缓解结肠炎小鼠、结肠癌小鼠症状的功能。植物乳杆菌B-27（-12）EPS纯化为4种单一组分，分子量分别为35391Da，95566 Da，77647 Da和120974 Da。组分L12-W结构为→4)-Glcp-(1→、Glc-(1→、Manp-(1→、→4,6)-Glcp-(1→、→6)-Glcp-(1→、→2-Man-1→和→6)-Manp-(1→等残基组成。.植物乳杆菌B-27（-12）基因组全长3.31Mb，由1个3.25Mb的染色体和1个53,541bp的质粒构成。染色体的GC含量为44.6%，质粒的GC含量为38.7%，全基因组GC含量为44.49%；含有三个胞外多糖合成相关的基因簇，分别为cps2、cps3和cps4，分别编码了10、8和10个基因。.植物乳杆菌B-27（-12）EPS可抑制人结肠癌细胞系HT-29增殖，HT-29细胞内ROS、Bax、细胞色素C的表达量随着胞外多糖作用浓度的增加而增加，而Bcl-2和PCNA则随着EPS作用浓度的增加而减少，细胞内的caspase蛋白酶被活化，进而促进细胞凋亡。.植物乳杆菌B-27（-12）灌胃结肠炎小鼠，可减缓结肠炎小鼠体重下降、结肠缩短等症状，显著降低（p<0.05）结肠炎小鼠血清中促炎因子白细胞介素8（IL-8）及肿瘤坏死因子（TNF-α）的表达量，提升抗炎因子白细胞介素10（IL-10）的表达量，在一定程度缓解结肠炎小鼠的炎症反应。灌胃植物乳杆菌B-27（-12）提高结肠炎小鼠肠道菌群多样性，促进有益菌群的生长，有效抑制肠道内致病菌生长；抑制结肠炎小鼠结肠内Tnfaip8、Bax等促炎蛋白的产生，刺激MUC2等肠粘膜蛋白及HSP70等应激蛋白的产生，从而缓解结肠炎。.植物乳杆菌B-27（-12）及其EPS灌胃结肠癌小鼠，小鼠结肠肿瘤明显减少，阻止结肠缩短（p<0.05），显著提高了结肠癌小鼠免疫能力（p<0.05），显著减少小鼠的炎症细胞因子IL-8、IL-1β以及TNF-α的浓度（p<0.05），从而减轻结肠癌小鼠炎症。