5株乳酸菌对MUC2的转录调节机制研究
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摘要
通过构建人MUC2启动子荧光素酶报告基因表达质粒,研究5株乳酸菌对MUC2启动子转录活性的影响。采用RT-PCR及ELISA方法分别在mRNA和蛋白质水平上就乳酸菌对SW480结肠细胞内源性MUC2转录表达的影响情况进行了分析。结果显示,5株乳酸菌的发酵上清液(SN)和菌体裂解液(CL)均能明显增强MUC2基因启动子的转录活性,并上调肠道细胞MUC2转录及表达水平,其中L. acidophilus 1.2686菌体裂解液的作用最为显著。进一步研究显示:TGF-β抑制剂可阻断L. acidophilus 1.2686对MUC2的转录激活作用,TGF-β激动剂协同性增强该作用,说明TGF-β信号通路很可能在乳酸菌增强肠道细胞MUC2基因转录表达过程中发挥着重要作用。
The promoter of human MUC2 gene was cloned and its luciferase reporter assay plasmid was constructed. The influence of five kinds of Lactobacillius to the transcriptional activity of MUC2 promoter was analyzed with the method of luciferase reporter assay. The change of MUC2 expression after the treatment of Lactobacillius in SW480 cells was detected by RT-PCR and ELISA, respectively. The results showed that the fermentation supernatant and cell lysate of Lactobacillius could enhance the transcriptional activity of the MUC2 promoter and induce the transcription and expression of endogenous MUC2 in SW480 cells. Among all the samples, the cell lysate of L. acidophilus 1.2686 exhibited the most apparent abilities. Furthermore, our studies found that this induction function of Lactobacilli on the MUC2 transcription and expression could be blocked by the TGF-β inhibitor, while be synergicallyaugmented by TGF-β, indicating that TGF-β signaling pathway might play an important role in mediating the transactivation of MUC2 in intestinal epithelial cells induced by Lactobacillius.
The promoter of human MUC2 gene was cloned and its luciferase reporter assay plasmid was constructed. The influence of five kinds of Lactobacillius to the transcriptional activity of MUC2 promoter was analyzed with the method of luciferase reporter assay. The change of MUC2 expression after the treatment of Lactobacillius in SW480 cells was detected by RT-PCR and ELISA, respectively. The results showed that the fermentation supernatant and cell lysate of Lactobacillius could enhance the transcriptional activity of the MUC2 promoter and induce the transcription and expression of endogenous MUC2 in SW480 cells. Among all the samples, the cell lysate of L. acidophilus 1.2686 exhibited the most apparent abilities. Furthermore, our studies found that this induction function of Lactobacilli on the MUC2 transcription and expression could be blocked by the TGF-β inhibitor, while be synergicallyaugmented by TGF-β, indicating that TGF-β signaling pathway might play an important role in mediating the transactivation of MUC2 in intestinal epithelial cells induced by Lactobacillius.
引文
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[2]国立东,王丽群,蒋琛,等.乳酸菌调控体内胆固醇代谢综述[J].中国乳品工业,2016,44(2):32-36.
[3]程龙,龚福明,李晓然,等.具有高效ACE抑制活性乳酸菌的筛选及其益生特性研究[J].现代食品科技,2015,31(11):127-134.
[4]Perdigon G,Vinti IE,Alvarez S,et al.Study of the possible mechanisms involved in the mucosal immune system activation by lactic acid bacteria[J].Journal of Dairy Science,1999,82(6):1108-1114.
[5]Satu V,Matti K,Seppo S,et al.Staphylococcus aureus adheres to human intestinal mucus but can be displaced by certain lactic acid bacteria[J].Microbiology,2006,152:1819-1826.
[6]Gill SR,Pop MD,Eboy RT,et al.Metagenomic analysis of the human distal gut microbiome[J].Science,2006,312:1355-1359.
[7]郑瑞珠,何夙旭,杨雅麟,等.益生乳酸菌水产动物消化道黏附机制研究进展[J].中国农业科技导报,2014,16(3):134-142.
[8]郝冉,罗学刚,贾玮,等.6株乳杆菌胃肠道定植能力分析[J].中国生化药物杂志,2012,33(5):555-558.
[9]Candela M,Perna F,Carnevali P,et al.Interaction of probiotic Lactobacillus and Bifidobacterium strains with human intestinal epithelial cells:Adhesion properties,competition against enteropathogens and modulation of IL-8 production[J].International Journal of Food Microbiology,2008,125:286-292.
[10]Caballero-Franco C,Keller K,De Simone C,et al.The VSL#3 probiotic formula induces mucin gene expression and secretion in colonic epithelial cells[J].Am J Physiol Gastrointest Liver Physiol,2007,292:G315-G322.
[11]Manish K,Vanessa KS,Aralia L C,et al.Probiotic mixture VSL#3 reduces colonic inflammation and improves intestinal barrier function in Muc2 mucindeficient mice[J].Am J Physiol Gastrointest Liver Physiol,2017,312:G34-G45.
[12]余晶仪,郝小燕,龙敏,等.MUC2基因表达对益生菌调节肠屏障作用的影响[J].南方医科大学学报,2013,33(2):197-201.
[13]吴英韬,袁杰利.益生菌在肠道疾病治疗中的应用[J].传染病信息,2015,28(4):245-250.
[14]曾子丹,姚朔影.益生菌的研究前景及国内外发展状况[J].食品工业科技,2007,28(8):251-253.
[15]韩俊燕,赵国忠,杨波,等.婴儿源益生性双歧杆菌的筛选及肠道定殖性研究[J].现代食品科技,2014,32(10):14-21.
[16]Winschau F Z,Shelly M D,Leon M T D.Use of the mCherry Fluorescent Protein To Study Intestinal Colonization by Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 in Mice[J].Applied and Environmental Microbiology,2015,81(17):5993-6002.
[17]胡艳艳,刘小伟.肠道杯状细胞结构和功能的研究进展[J].国际病理科学与临床杂志,2013,33(5):424-430.
[18]Mehmet K,Sara K,Raymond J P,et al.Tracheobronchial air-liquid interface cell culture:a model for innate mucosal defense of the upper airways[J].Am J Physiol Lung Cell Mol Physiol,2009,296(1):L92-L100.
[19]Jono H,Shuto T,Xu H,et al.Transforming growth factor-β-smad signaling pathway cooperates with NF-kBto mediate nontypeable haemophilus influenzae induced MUC2 mucin transcription[J].The journal of biological chemistry,2002,277(47):45547-45557.