The Study On Bifidobacterium CMS-H004 Strain Aggravated The Damage Of Ulcerative Colitis And Preparation Of Its Antibody

ChapterⅠ: The animal experiment of Bifidobacterium CMS-H004strain aggravated the damage of UCObjective:To test the effect of Bifidobacterium strain CMS-H004 and otherBifidobacterium strain on the murine model of ulcerative colitis inducedby DSS.Methods:The BALB/c mice were randomly divided into seven groups (controlgroup, DSS group, NS group, positive group(5-ASA), Bifidobacteriumstrain CMS-HOO4 group, Bifidobacterium strain PFK, Bifidobacteriumstrain JSQ). Mice of groups DSS, NS, 5-ASA, CMS-HOO4, PFK, JSQwere fed with 5% DSS into the drinking water for 7 days to induce colitis,mice of Control just drink distilled solution, and disease activity index(DAI) was calculated every day including the body weight, fecescharacter and occult blood. The colon length were measured then werehandled with HE and histological damage were assessed in colonic tissueat the end of experiment. Then we measured the myeloperoxidase (MPO)of colon tissue and the serum tumour necrosis factor-α(TNF-α) of eachgroup. Results:The mice in group CMS-H004 showed a higher DAI than those ingroup DSS since the third day to the end of experiment. The appearanceof body weight loss, Hemoccult, fecal bleeding and death of CMS-H004group were earlier than other groups. The ratio of fecal bleeding anddeath of CMS-H004 group were highest. The colon length of CMS-H004group were shortest. The histological scores of mucosa damage and MPOof colon tissue of CMS-H004 group were serious. Compared withtreatment before, Lactobacillus of DSS group fecal was reduced, whilebacteroid, Escherichia coli and Staphylococcus aureus were increasedsignificantly. Bifidobacteria and total aerobe had no changes. CMS-H004group was similar to model group, its Bifidobacteria didn't increase.Conclusions:1) On the base of mucosa injury, Bifidobacterium strain CMS-HOO4can aggravate the mucosa damage of UC.2) DSS resulted Lactobacillus reduced and didn't change theBifidobacterium. Using Bifidobacterium CMS-H004 strain also reducedLactobacillus while its Bifidobacterium didn't increase.ChapterⅡ: Mechanism study of Bifidobacterium CMS-H004 strainaggravated the damage of UC.Objective:To research the mechanism of Bifidobacterium CMS-H004 strainaggravated the damage of UC in vivo and in vitra.Methods:The BALB/c mice were randomly divided into three groups (control group, model group, Bifidobacterium strain CMS-HOO4 group). Everygroup divided into three litter groups(the 3,5,7days after drinking DSS).UC was induced by adding 5% DSS into the drinking water for 7 days.We test the expression of cytokine (IL-1β, IL-6, TNF-α) by RT-PCR andwestern blotting, then observed the expression of ZO-1,β-defensin-2 bywestern blotting.Colonic adenocarcinoma HT-29 cells were cultured and divided intofour groups: Control(group C), TNF-a (group T), BifidobacteriumCMS-H004 (group B), suspended in Bifidobacterium PFK (group P). B.CMS-H004 and B.PFK were culture medium with the concentration of 110~9CFU/ml and added into 24 wells respectively. 1 hour later TNF-a(10ng/ml) was added into each well of three groups as T, B, L. The LDHexpression of each group was measured and after 3 hours, nuclear factor-κB(NF-κB) was also examined.Results:1) The mRNA and protein appearance of IL-1β, IL-6, TNF-αinmodel group was at the fifth day after drinking DSS, while CMS-HOO4group appeared at the third day. At the same time their expressing inCMS-HOO4 group was higher than model group.2) ZO-1 protein of control group expressed abundance. At the 7thday after drinking DSS, the expressing of ZO-1 protein in model groupand CMS-HOO4 group reduced significantly. But there were nosignificant differences between them.3) Control group didn't expressβ-defensin-2 protein. Behind thefifth day after drinking DSS, theβ-defensin-2 protein in model group andCMS-HOO4 group appeared expression, however, theβ-defensin-2 inmodel group was higher then CMS-HOO4 group.4) The LDH had no significance differences between all groups afterstimulated by TNF-α. 5) More NF-κB appears in nucleus in group B compared with othergroups after stimulated by TNF-α.Conclusions:1) Bifidobacterium strain CMS-H004 can result inflammatorycytokines IL-1β, IL-6, TNF-αrelease in advance.2) Bifidobacterium strain CMS-H004 can raise NF-κB appears innucleus.3) DSS and Bifidobacterium strain CMS-H004 can destroy the tightinjunction and induced theβ-defensin-2 expression. However, it isn't themain injury mechanism.ChapterⅢ: Identify CMS-H004 strain and its correlated research.Objective:To identify strain CMS-H004 and conserve to China Center for TypeCulture Collection (CCTCC).Methods:To observe CMS-H004 the morphology characteristic through lightand electron microscope. Then research its biochemical event, X-Galcolouration and metabolism product. To determine its (G+C)mol% andplasmid, as well as amplification its 16SrRNA. To research the ability oftolerate to gastric acid and bile and antibiotics sensitive in vitra.Results:1) CMS-H004 strain is Gram staining positive bacillus and itscolony is round, white.2) CMS-H004 can utilize glucose, manicol lactose, cane sugar, maltose, aspen, wood sugar, arabinose, carubinose, aesculin, glycerine,cellobiose, mycose, melezitose, gossypose, D-glucitol, isodulcite. Its APIcoding is: 47754560.3) The main metabolism product of CMS-H004 stain was acetic acidand lactic acid, while its ratio is 2.63: 1. It still product little amber acid.4) The (G+C) mol% of CMS-H004 stain is 58.64% and noplasmid detected.5) CMS-H004 stain can tolerate to gastric acid and bile in vitra. It isvery sensitive to alficetin, piperacillin, ambramycin, cefamandole,minocycline, azithromycin, rifadin and midrange sensitive to cidomycin,while other 33 kinds antibiotics have no bacteriostasis ring.Conclusions:1) CMS-H004 strain is one of Bifidobacterium adolescentis andhave been conserved to the CCTCC.2) because of the ability tolerate to gastric acid and bile, CMS-H004stain can field planting to intestinal tract.3) CMS-H004 strain is very sensitive to alficetin, piperacillin,ambramycin, cefamandole, minocycline, azithromycin and rifadin, whilemidrange sensitive to cidomycin.ChapterⅣ: To preparation and test antibody of CMS-H004 strainObjective:To preparation the polyclonal antibody of CMS-H004 strain.Methods:Use the protein of CMS-H004 strain adding adjuvant as antigen toimmunize rabbit to obtain the antibody. Then test its best working concentration, sensitivity, specificity by ELISA, Western Blotting andimmune-culture. In the end, test CMS-H004 strain in fecal of UC patientand health adult using this antibody.Results:1) CMS-H004 strain can stimulate rabbit product antibody.Accompany to the reduction of antibody, the reaction between antibodyand CMS-H004 strain was descend. There was very weak reaction under0.05μg/ml.2) The reaction between different bacterium and antibody wasdistinct. CMS-H004 strain's reaction was strongest than others.3) Some health adult (4/20) and UC patients (3/12) have been detecta small quantity CMS-H004 strain growth, while other UC patients (9/12)have generous CMS-H004 strain growth.Conclusions:1) CMS-H004 strain can stimulate rabbit product antibody and itsconcentration, sensitivity, specificity were better.2) In small sample research, Bifidobacterium CMS-H004 strainreside in 20% health adult and all UC patients.