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目的建立抑制肠上皮细胞基因组中Muc2基因表达的CRISPR/Cas9系统并探索粘蛋白Muc2在鼠李糖乳杆菌GG株(LGG)抑制大肠杆菌K1(Escherichia coli,E.coli k1)株E44黏附和侵袭肠上皮中的作用机制。方法设计2个长20~25 bp的sg RNA分别靶向Muc2,合成sg RNA寡核苷酸序列并构建CRISPR表达载体,转染野生型人结肠癌Ht29细胞,蛋白免疫印迹法检测抑制效率及通过MTT法检测其细胞活力及生长情况后,竞争性排斥分析验证粘蛋白Muc2在益生菌抑制E44黏附侵袭肠上皮中的作用。结果目的sg RNA寡核苷酸双链成功插入酶切后的lenticrisprv2质粒载体中且序列正确;稳定抑制Muc2表达的细胞株筛选成功;Muc2基因沉默后,与空白对照组相比,其表达水平明显降低,抑制率可达81%(P<0.01);黏附侵袭实验中E44相对黏附率为72.23%(P<0.01),相对侵袭率为81.49%(P<0.01),益生菌LGG的抑制E44黏附和侵袭作用明显下调。结论Muc2基因下调明显降低益生菌抑制E44黏附和侵袭肠上皮细胞的能力,提示益生菌刺激Muc2表达上调可能是其强化加固肠粘膜屏障和拮抗致病菌功能的关键性机制之一,并可为肠道细菌性感染疾病的预防治疗提供了一个新手段。
OBJECTIVE: To establish a CRISPR / Cas9 system that inhibits Muc2 gene expression in intestinal epithelial cells and to explore the role of mucin Muc2 in the adhesion and invasion of Escherichia coli K1 strain E44 in Lactobacillus rhamnosus GG strain (LGG) Intestinal Epithelium in the mechanism of action. Methods Two sg RNAs of 20 ~ 25 bp in length were designed to target Muc2 respectively. The sg RNA oligonucleotide sequence was synthesized and the CRISPR expression vector was constructed. The wild-type human colon cancer Ht29 cells were transfected and the inhibitory efficiency was determined by Western blot After the cell viability and growth were examined by MTT assay, competitive exclusion analysis was performed to verify the role of mucin Muc2 in probiotic inhibition of E44 adhesion invasion to the intestinal epithelium. Results The sg RNA oligonucleotide double strand was successfully inserted into the digested lenticrisprv2 plasmid vector and the sequence was correct. The cell lines stably inhibiting the expression of Muc2 were screened successfully. After Muc2 gene silencing, the expression level of Muc2 gene was significantly higher than that of the blank control group (P <0.01). The relative adhesion rate of E44 was 72.23% (P <0.01) and the relative invasion rate was 81.49% (P <0.01). The probiotic LGG inhibited the adhesion of E44 And invasion was significantly reduced. Conclusions The down-regulation of Muc2 gene significantly reduces the ability of probiotics to inhibit the adhesion and invasion of E44 in intestinal epithelial cells, suggesting that up-regulation of Muc2 by probiotics may be one of the key mechanisms of enhancing the intestinal mucosal barrier and antagonizing pathogenic bacteria, and may be The prevention and treatment of intestinal bacterial infections provide a new means.