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Building method of ulcerative colitis transformation animal model

A technology of ulcerative colitis and animal models, applied in the direction of pharmaceutical formulations, organic active ingredients, medical preparations containing active ingredients, etc., can solve the problems of increased IL-17 levels, and achieve the effect of increasing expression and increasing the number

Pending Publication Date: 2020-01-14
CHINA PHARM UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the problem with the classic DSS-induced colitis animal model is that the level of IL-17 in the mouse colon does not increase significantly

Method used

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  • Building method of ulcerative colitis transformation animal model
  • Building method of ulcerative colitis transformation animal model
  • Building method of ulcerative colitis transformation animal model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The construction method of the ulcerative colitis transformation animal model of the present embodiment comprises the following steps:

[0038] 1. Experimental materials

[0039] 1. Experimental animals: 40 male C57BL / 6J mice, SPF grade, age 5wk, body weight 18-20g, purchased from Shanghai Slack Animal Center.

[0040] 2. Strains: C.difficile ATCC 43255 purchased from American type culture collection (ATCC)

[0041] 3. Main experimental reagents

[0042] Dextran sodium sulfate (DSS) (purchased from MP Biomedicals), FITC-CD4 antibody, PerCP-Cy5.5-CD3e antibody, PE-IFN-γ antibody, Fixation Buffer (both purchased from Biolegend); APC -IL-17A antibody (both purchased from ebosicence company); PMA (purchased from Sigmaaldrich company), Ionomycin ionomycin (purchased from aladdin company), 5% FBS 1640 medium (purchased from Gibco company), mouse IL-17A ELISA Kit (purchased from Shanghai Jitai Yikesai Company), erythrocyte lysate (purchased from Beyontian Company), PBS buff...

Embodiment 2

[0064] Example 2: Observation indicators

[0065] Observe and record the daily body weight changes of the mice given 2% DSS from the 1st day to the 10th day, and record the colon length on the 11th day.

Embodiment 3

[0066] Embodiment 3: Determination of biochemical indicators

[0067] 1.1 Determination of Th1 and Th17 ratios of lymphocytes in spleen and mesenteric lymph nodes by flow cytometry

[0068] 1) Take out the mesenteric lymph nodes and spleen tissues placed in PBS with 2% FBS, transfer them to a 40um blue sieve, place a sterile six-hole plate under the sieve, grind the mesenteric lymph nodes and spleen respectively with a 2.5ml syringe rubber stopper, And wash the screen with 4ml of PBS containing 2% FBS, transfer the filtrate to a 10ml sterile centrifuge tube, and centrifuge at 800g*20min*4°C.

[0069] 2) After the centrifugation, discard the supernatant, and resuspend the spleen in 1ml erythrocyte lysate, lyse on ice for 10 minutes and centrifuge at 800g*5min*4°C; directly resuspend the lymph nodes in 500ul PBS containing 2% FBS, temporarily at 4°C live.

[0070] 3) After the centrifugation of the spleen, discard the supernatant, resuspend in 500ul PBS containing 2% FBS, temp...

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Abstract

The invention discloses a building method of an ulcerative colitis transformation animal model. By using the method, an inflammatory transformation model combining with clostridium difficile in a DSSacute colitis model is built; and different bacterium feeding modes, including three measures of intraperitoneal injection, gastric perfusion and enema are used. The model has the advantages that theproportion of mouse mesenteric lymphocytes Th17 can be obviously increased by the model; the secretion of IL-17 is increased; the result is more similar to the pathological features of clinic patientswith ulcerative colitis; and the clostridium difficile feeding effect on the mice through gastric perfusion is best.

Description

technical field [0001] The invention relates to a method for constructing an animal model, in particular to a method for constructing a transformed animal model of ulcerative colitis. Background technique [0002] Clostridium difficile is a spore-forming, Gram-positive, anaerobic bacterium that was identified in 1978 as the cause of antibiotic-associated pseudomembranous colitis due to toxin-producing clostridia .N.Engl.J.Med.298, 531-534]. Despite an overall decline in infectious disease mortality over the past 35 years, diarrheal disease mortality is still increasing in the United States. This trend has been attributed to the spread of supervirulent strains of Clostridium difficile over the past decade, including the prevalent ribosomal 027 strain used in the study [Binary toxin and death after Clostridium difficileinfection.Emerg.Infect.Dis .17, 976-982]. The U.S. Centers for Disease Control and Prevention estimates C. difficile causes nearly 500,000 infections and 29,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/716A61K35/742A01K67/027
CPCA61K31/716A61K35/742A01K67/027A01K2267/035
Inventor 郝海平曹丽娟黄海
Owner CHINA PHARM UNIV
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