| Lactobacillus can interact with the intestinal mucosal immune system,it is play an important role in the inhibition of intestinal pathogenic bacterium,regulating the immune function of organism,maintain the balance of intestinal micro ecological.However,the precondition of Lactobacillus is to adhere to intestinal epithelial cells,which is the first step in the colonization of intestinal tract.The adhesion of Lactobacillus to intestinal epithelial cells contributes to the colonization of the intestinal tract,the enhancement of the exchange between the Lactobacillus and the intestinal cells,the inhibition of the colonization of pathogenic bacterium in the intestinal tract and the improvement of the immunity of the organism.Therefore,the adhesion of Lactobacillus is an important index to evaluate it.The latest study found that in the research of Lactococcus lactic,serine protease lactocepin in epicyte in addition to the hydrolysis of protein,anti-inflammatory and other functions,but also adjust the adhesion of Lactobacillus,mainly through the influence of the change in van der waals force.In order to study the influence of lactocepin on adhesion to Lactobacillus,The Lactobacillus casei was used as the carrier,and the prtP gene which is encoding lactocepin gene was knocked out by λRed system.Lactobacillus casei mutant strain was constructed.The differences between the mutant and the parental strain have been compared,such as biological characteristics,in vitro and in vivo adhesion and intestinal immunity and so on.The main results are as follows:The growth curve,surface hydrophobicity,ability of self-coagulation,ability of inhibition and drug sensitivity of two strains were compared.The results show that the mutation,the growth rate of Lactobacillus,hydrophobic and ability of self-coagulation decreased significantly,antibacterial activity had no obvious change,different sensitivity to different antibiotics,L.paracasei parental strain is resistant to Norfloxacin,Ciprofloxacin,Bolymyxin B and Streptomycin;to Tetracycline and Penicillin G is medium sensitivity;to Rifampicin,Refazolin and Cefradine is sensitive.Lactobacillus paracasei mutant to Norfloxacin,and Polymyxin B is resistant;to Ciprofloxacin,Penicillin G and Cefradine is medium sensitivity;to Rifampicin,Tetracycline,Refazolin is sensitive.This study tested the adhesion ability of two strains of Lactobacillus to mouse macrophages,spleen cells,intestinal epithelial cells and intestinal mucus and set up four kinds of adhesion model in vitro.The experiment measured adhesion to cells of Lactobacillus with microscopic examination,coated plate method.measured adhesion number of Lactobacillus with fluorescence labeling method in vivo.The results show that lactocepin influenced Lactobacillus’s adhesion on different cells.On macrophages,the number of adhesion of parental strain was 27.7±2.52,the number of adhesion of mutant strain of lacking of lactocepin is 19.3±3.06,were significantly higher than that on spleen cells 19.3±2.08,10.7±1.53 and 14.7± 1.53,8.3±1.53 of intestinal epithelial cells.And the adhesion of parental strain to the three kinds of cells were significantly higher than that of the mutant.The adhesion rate of the parental strains to the small intestinal mucus of the mice was 20.07±0.18%,mutant strain was 16.96±0.82%,the parental strain was significantly higher than that of the mutant strain(P<0.01).Flow cytometry was used to analyze the colonization and distribution of two strains of Lactobacillus in the intestine of mice.The results showed that the colonization of two strains tested Lactobacillus in the intestine is different.The colonization of mutant strain in the intestinal tract mainly occurred in early intragastric administration.The colonization of mutant strain in ileum and colon are both most in first day.the colonization of mutant strain in duodenum and jejunum are both most in third day.The colonization of parent strain is most in colon in first day,in other intestinal segments,the most colonization is occurred in later period of intragastric administration.The parent and mutant strains are mainly colonized in the colon and least in duodenum.In different intestinal segments,the adhesion of the parent strain was significantly higher than mutant(P<0.01).The effects of Lactobacillus on immune organs,immune cells and immune factors were studied by CBA technique in vivo and in vitro.The results showed that two strains of Lactobacillus can improve the immune organ index of mice in the middle of the intragastric administration,later period is not obvious.They can both of improve the conversion rate of spleen lymphocytes,but the parental strain is more significant than the mutant strain.Parerntal strain can significantly improve the energy level and the phagocytic capacity of macrophages(P<0.05),the effect of mutant effect is indistinctive.For the immune factors,parental strains and mutant strains can both of increase the expression of INF-a in high dose(P<0.01),and the parental strain is more significant than mutant strains.The parental strain could increase the expression of IL-10(P<0.01),the mutation strain is indistinctive.The parental strain could increase the expression of INF-γ,and it is related with the dose and time.The effect is significant in high dose(P<0.05),while the effect of mutation strain is indistinctive.Two kinds of Lactobacillus could increase the number of CDllc+ CD80+ double positive cells in the whole stage of feeding mice. |
PDF Full Text Request