Screening Of Probiotic Lactobacillus And Its Regulation On The Expression Of Intestinal Epithelial Cells HBD-2 Gene

Human beta-defensin-2(hBD-2)is one kind of antimicrobial peptide which can be induced to express in human epithelial cell.hBD-2 plays an important role in maintaining homeostasis and ecological balance of intestinal tract.Probiotic Lactobacillus is the preponderant bacteria in intestinal tract which can stick to the cell surface and interact with them to regulate the physiological function of epithelial cell.This study aimed to screen the probiotic Lactobacillus and explore the regulating effects for the expression of hBD-2 in the epithelial cell of intestinal tract.Total of 73 strains were purified with the MRS plates from different part of gastrointestinal tract of a healthy piglets(colon,ileum,duodenum,stomach).Then 11 probiotic Lactobacillus which have better acid resistance were screen out.The 11 strains were confirmed to be the Lactobacillus by physiology biochemistry experiment : SB16?HB3?HB7?HB9?HB37?WN5?WN8?KB4?KB5?KB8?KB10?Three Lactobacillus which have excellent properties,are screen out by cholate tolerance experiment,thermostability experiment and bacteriostat experiment.The result of 16 S r RNA sequencing shows that three strains is Lactobacillus.The name respectively is Lactobacillus.sp.HB3?Lactobacillus.sp.HB9 and Lactobacillus.sp.KB8.Dose-dependent stimulation experiment and time dependence stimulation experiment to epithelial cell of human were performed for this three probiotics Lactobacillus.The otherness expression of hBD-2m RNA after stimulating was test by FQ-PCR from m RNA level.The results indicate that three Lactobacillus all can up-regulate the expression of hBD-2 m RNA.But the expression of hBD-2 m RNA is different because of different bacteria and different concentration.Among them,the expression of hBD-2 m RNA after stimulating by Lactobacillus.sp.KB8 is the highest.The expression of hBD-2 m RNA reach the peak after8 h stimulating.Four components-whole cell wall(WCW),whole cell wall peptidoglycan(WPG),lipteichoic acid(LTA)and Lactobacilli cultural supernatant(S)of the Lactobacillus.sp.KB8 were separated and their effects on the expression of hBD-2 m RNA of Caco-2 were test by FQ-PCR.The result show that four components can promote the expression of hBD-2 gene to varying degrees,and dose-dependent can be seen.Among them,whole cell wall peptidoglycan has the strongest ability to up-regulate the expression of hBD-2 m RNA.The best concentration and time to stimulating is 50 ?g/m L and 4 h.