阅读说明：本技术 一种溃疡性结肠炎转化动物模型的构建方法 (Method for constructing ulcerative colitis transformation animal model ) 是由 郝海平 曹丽娟 黄海 于 2019-11-22 设计创作，主要内容包括：本发明公开了一种溃疡性结肠炎转化动物模型的构建方法,该方法建立了在DSS急性结肠炎模型中联用艰难梭菌(Clostridium difficile)的炎症转化模型,通过不同的给菌方式,包括腹腔注射、灌胃、灌肠三种手段。该模型优势在于该模型能够显著增加小鼠肠系膜淋巴细胞Th17的比例,并且上调IL-17的分泌；更接近于临床溃疡性结肠炎患者的病理学特征；小鼠通过灌胃给艰难梭菌的效果最好。(The invention discloses a method for constructing an ulcerative colitis conversion animal model, which establishes an inflammation conversion model of Clostridium difficile (Clostridium difficile) combined in a DSS acute colitis model, and adopts three means of intraperitoneal injection, gastric lavage and enema through different bacteria feeding modes. The model has the advantages that the model can remarkably increase the proportion of mouse mesenteric lymphocyte Th17 and up-regulate the secretion of IL-17; more closely resembles the pathological features of clinical ulcerative colitis patients; mice gave the best results to clostridium difficile by gavage.)

1. A method for constructing an ulcerative colitis transformation animal model is characterized in that: after mice are drunk with Dextran Sodium Sulfate (DSS) and then are given to Clostridium difficile (Clostridium difficile) through different ways, observation and biochemical index measurement are carried out, and when the number of Th1 or Th17 in the colon of the mice is increased, the expression of IL-17 in colon tissues is increased and the expression of inflammatory factors is up-regulated, the model construction is successful.

2. The method for constructing an animal model for transforming ulcerative colitis according to claim 1, wherein the method comprises the steps of: the mice used male mice.

3. The method for constructing an animal model for transforming ulcerative colitis according to claim 1, wherein the method comprises the steps of: the different routes are intraperitoneal injection, gastric lavage or enema.

4. The method for constructing an animal model for transforming ulcerative colitis according to claim 1, wherein the method comprises the steps of: the route is intragastric.

5. The method for constructing an animal model for transforming ulcerative colitis according to claim 1, wherein the method comprises the steps of: the mice were first given Dextran Sodium Sulfate (DSS) for 7 days, stopped on day8, and then given clostridium difficile (clostridium difficile).

6. The method for constructing an animal model for transforming ulcerative colitis according to claim 1, wherein the method comprises the steps of: the inflammatory factor is IL-1 beta, IL-6 or TNF-alpha.

7. The method for constructing a transformed animal model of ulcerative colitis according to any one of claims 1 to 6, wherein: the model is used for constructing a DSS and Clostridium difficile (Clostridium difficile) induced acute ulcerative colitis model, a chronic recurrent ulcerative colitis model, a Clostridium difficile (Clostridium difficile) infection related ulcerative colitis model and a colitis cancer transformation model.

8. The method for constructing a transformed animal model of ulcerative colitis according to any one of claims 1 to 6, wherein: the use of the model in preclinical study assessment of acute and chronic ulcerative colitis and colitis cancer conversion-related therapeutic drugs.

Technical Field

The invention relates to a construction method of an animal model, in particular to a construction method of an ulcerative colitis conversion animal model.

Background

Difficile (Clostridium difficile) is a spore-forming, gram-positive, anaerobic bacterium that was identified in 1978 as the bacterium responsible for Antibiotic-associated pseudomembranous colitis [ Antibiotic-associated anaerobic bacterium to toxin-producing bacterium.n.engl.j.med.298, 531-534 ]. Despite the overall decline in infectious disease mortality over the last 35 years, the mortality rate for U.S. diarrheal disease has increased. This trend was attributed to the spread of very virulent strains of Clostridium difficile (Clostridium difficile) in the past decade, including also the epidemic ribosome 027 strain used in the study [ Binary toxin and death after Clostridium difficile infection. emery. Infect. Dis.17, 976-. The american centers for disease control and prevention estimate that clostridium difficile alone causes nearly 500000 infections and 29000 deaths annually in the united states. These reports and others underscore the importance of studying Clostridium Difficile Infection (CDI) and identifying therapeutic targets that reduce the severity of the disease [ emulsifying infection prog c.differential surgery Team (2015.) and Burden of Clostridium differential surgery in the United states n.engl.j.med.372, 2369-.

Ulcerative Colitis (ulcerogenic Colitis, UC): chronic inflammatory diseases, characterized by a continuous ulceration of the mucous membrane in the rectum and colon, starting in the rectum, expanding to different extents and spreading to the greatest extent to the cecum, are manifested clinically as diarrhea, intermittent hematochezia or purulent stool, abdominal pain, vomiting, etc. However, the etiology has not been determined so far, and is generally considered to be influenced by factors such as environment, genetics and intestinal microorganisms [ incorporated infection and prediction of the intestinal microorganisms with time, based on systematic review. gastroenterology 2012; 142: 46-54, e42 ]. Where IBD is an independent risk factor for CDI. IBD patients have more severe CDI than non-IBD patients, manifested by a shortened time from admission to CDI, an increased length of hospitalization, an increased number of surgeries and an increased rate of mortality in hospitalization [ inclusion of Clostridium difficile infection in clinical laboratory in hepatol.5, 339 in 344 ].

The common animal models of the existing Ulcerative Colitis (UC) model are mainly: dextran Sodium Sulfate (DSS) model, 2, 4, 6-trinitrobenzenesulfonic acid (TNBS) model. The pathogenesis of DSS-induced colitis model DSS is a sulfated polysaccharide synthesized from sucrose, and its mechanism of inducing colitis may be associated with affecting DNA synthesis, causing activation of T cells, neutrophils, macrophages, etc. through various pathways, causing altered cytokine expression, leading to disruption of the intestinal epithelial barrier [ Temporal and systemic analysis of clinical and molecular parameters in dextran sulfate induced color. e6073 ]. DSS colitis is characterized by superficial, focal inflammation and ulceration, with all colon attacks, with inflammation affecting mainly the mucosal and submucosa. However, the animal mortality rate is high in the manufacturing process of the DSS acute colitis model, and the symptoms naturally recover 8-9 days after the drug is stopped, so that the observation of the curative effect of the drug is influenced, and therefore, the DSS acute colitis model is suitable for the research of pathogenesis. The pathogenesis of TNBS colitis animal model is that after ethanol breaks the intestinal mucosal barrier, TNBS infiltrates the colonic tissue and binds with macromolecular substances to form holoantigens, causing a series of immune and Inflammatory responses of the intestinal mucosa [ a Reviewon Chemical-Induced inflammation incision model in rodents, korean JPhysiol Pharmacol, 2014, 18 (4): 279-288]. However, the TNBS model requires tight dose control, and is more similar to Crohn's Disease (CD) and less similar to ulcerative colitis. Currently, the main problem encountered in drug development for ulcerative colitis is the discovery of drug targets for immune response-related molecules and intestinal immune processes. As the use of TNF- α antibody drugs in UC matures, drugs targeting immune response-related molecules and gut immune processes have attracted attention, but the problem associated with them is the increase in drug resistance. IL-17 levels were higher in colonic mucosa in UC patients [ Interleukins IL-33 and IL-17/IL-17A in tissues with intestinal university. hepatology, 2010, 57 (104): 1442-1444]. This suggests that IL-17 may be a therapeutic target for UC. However, a problem with the classical animal model of DSS-induced colitis is that there is no significant increase in IL-17 levels in the colon of mice. Therefore, there is a need to construct a novel ulcerative colitis transformation model that can mimic the levels of IL-17 in the colonic mucosa of clinical ulcerative colon patients.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a DSS-induced and C.difficile-combined ulcerative colitis transformation animal model.

The technical scheme is as follows: the invention provides a method for constructing an animal model for transforming ulcerative colitis, which comprises the steps of firstly drinking Dextran Sodium Sulfate (DSS) by a mouse, then feeding Clostridium difficile (Clostridium difficile) by different ways, and then observing and measuring biochemical indexes, wherein when the number of Th1 or Th17 in the colon of the mouse is increased, the expression of IL-17 in colon tissues is increased and the expression of inflammatory factors is up-regulated, the model is successfully constructed.

Further, the mice use male mice, and male sex mice are less affected by endocrine factors and have better body weight uniformity and molding stability than female mice.

Further, the different routes are intraperitoneal injection, intragastric administration or enema. Bacterial infection was performed by each of the above routes.

Further, the route is preferably intragastric.

Further, the mice were allowed to drink Dextran Sodium Sulfate (DSS) for 7 days, and stopped on day8, and then given Clostridium difficile (Clostridium difficile). Under a DSS acute colitis model, the number of Th17 cells in the intestinal tract of a mouse is obviously increased on the third day of modeling, while the number of Th17 cells in the intestinal tract of the mouse is gradually reduced in the later stage of enteritis, which is not consistent with the pathological characteristics of patients with clinical ulcerative colitis, and the pathological characteristics of the clinical ulcerative colitis can be better simulated by up-regulating the number of Th17 in the intestinal tract of the mouse by administering Clostridium difficile (Clostridium difficile) in the later stage of enteritis. Mice were given Clostridium difficile (Clostridium difficile) on day8, which could be rooted.

Further, the inflammatory factor is IL-1 beta, IL-6 or TNF-alpha.

Further, the model is used for constructing a DSS and Clostridium difficile (Clostridium difficile) induced acute ulcerative colitis model, a chronic recurrent ulcerative colitis model, a Clostridium difficile (Clostridium difficile) infection-related ulcerative colitis model and a colitis cancer transformation model. The use of the model in preclinical study assessment of acute and chronic ulcerative colitis and colitis cancer conversion-related therapeutic drugs.

The preferred method of the present invention is as follows:

step one, experimental grouping and model building

Male C57BL/6 mice of 5 weeks of age were selected, weighing 18-20g, and were fed adaptively for one week, and were randomized into groups according to body weight.

Group setting: healthy control group (n ═ 8), enteritis transformation model group (n ═ 8)

Preparing bacterial liquid: culturing to growth log phase (48h) C.difficil bacterial solution, 6000rpm 5min 4 deg.C, re-suspending with pre-reduced physiological saline for 24h in anaerobic workstation, and collecting 100uL bacterial solution for OD₆₀₀Reading to determine bacterial count, and adjusting bacterial concentration to 2.5 x 10⁹cfu/ml。

Healthy control group: free drinking water and feed for every day

DSS enteritis transformation model group: free food intake, free drinking of 2% DSS solution for 7 days, free drinking of normal water for 3 days, and simultaneous administration of C.difficile bacteria via the digestive tract, once a day for 3 consecutive days. The specific administration modes of the C.difficile bacterial liquid include the following two modes:

① on days 8 to 10, the model mice were fasted at 8 am, and were gavaged at 10 am with 0.2ml of C.difficile bacteria solution, and were fed after 2h, and the parallel healthy control mice were gavaged with physiological saline of the same volume.

② enema is carried out every day from 8 th to 10 th days, the mice are anesthetized by intraperitoneal injection with 3% chloral hydrate according to 0.2ml/20g, 0.2ml of bacteria liquid is extracted by a micro-syringe connected with an enema needle, the needle is carefully inserted into the anus of the mice to a depth of 3-4cm, the intestinal wall is prevented from being injured by the needle, the bacteria liquid is slowly pushed in, the mice are fixed on a plane inclined at an angle of 45 degrees with the head facing downwards to ensure that the sensitizing liquid and the intestinal tract fully act, the mice are placed back into a cage after standing for 30min to wake up, and the parallel healthy control group mice are rectally administered with physiological saline with the same volume in the same way.

Step two, collecting samples

On day 11, after fasting for 4h, mice were sacrificed by cervical dislocation, the abdominal cavity was cut, spleen and mesenteric lymph node were collected, placed in PBS containing 2% FBS, and placed on ice for flow-assay of immune cell clustering; dissociating colon, taking out the whole colon segment from the position 1cm from the upper end of anus to the tail end of cecum, splitting along the longitudinal axis of mesentery, repeatedly washing with ice-precooled PBS, and then sucking water with clean filter paper. After macroscopic examination and scoring, 1cm of the proximal and distal colon tissues were cut, fixed in 4% paraformaldehyde solution, embedded in normal paraffin, and sectioned to be left for histopathological examination and immunohistochemical staining. The colon tissue is stored in an ultra-low temperature refrigerator at minus 80 ℃ for detection such as RT-PCR, ELISA and the like.

Step three, observing the indexes

Daily body weight changes were observed and recorded for mice given 2% DSS from day 1 to day 10, and colon length was recorded on day 11.

Step four, biochemical index determination

Detecting the content changes of splenic and mesenteric lymph node lymphocytes Th1 and Th17 by flow; ELISA is used for detecting the content of IL-17 in serum and colon; RT-qPCR detects the expression of inflammatory factors (IL-17, IL-1 beta, IL-6, TNF-alpha) in colon tissue.

The method is characterized in that the combined use mode of DSS-induced ulcerative colitis and C.difficile is adopted, and different approaches (intraperitoneal injection, gastric lavage and enema) for clostridium difficile are adopted to observe the weight change, the colon length, the histological damage of the colon and the macroscopic manifestation of the mouse; measuring the quantity change of lymphocyte Th17, measuring the IL-17 content in colon tissue and serum, detecting the expression of inflammatory factor, and exploring a more clinical ulcerative colitis model.

Has the advantages that: the method can obviously increase the proportion of the intestinal immune system Th17 and simultaneously up-regulate the content of IL-17; and by different routes to clostridium difficile (intraperitoneal injection, gavage, enema). The model method combines a DSS-induced ulcerative colitis model with colitis caused by clostridium difficile infection, establishes an animal model more suitable for clinical ulcerative colitis transformation, and can more effectively simulate the IL-17 level in a patient with clinical ulcerative colitis. The model has application value that based on the obvious increase of the IL-17 level in a patient with clinical ulcerative colitis, the number of Th17 in the intestinal immunity of a mouse can be obviously increased by combining DSS colitis and clostridium difficile, and the expression of IL-17 in colon tissues is obviously increased; meanwhile, the expression of inflammatory factors (IL-1 beta, IL-6 and TNF-alpha) is also obviously up-regulated. Based on the analysis of experimental results, the reproducibility of experiments, the difficulty of operation and the like, the fact that the clostridium difficile is given to the mice by the gastric lavage is determined to construct a better ulcerative colitis transformation animal model.

Drawings

FIG. 1: schematic diagram of model building of DSS model by administering mouse with difficile via different ways (intraperitoneal injection, gastric lavage and enema).

FIG. 2: difficile administered to mice by different routes (intraperitoneal injection, gavage, enema) affected the weight (a) and colon length (B) of DSS model mice. P < 0.05, p < 0.01, p < 0.001, vs Control;

FIG. 3: c, performing different ways (intraperitoneal injection, gastric lavage and enema) on the mouse clostridium difficile to perform HE staining analysis on the proximal colon and the distal colon of the DSS model mouse;

FIG. 4: difficile administered to mice by different routes (intraperitoneal injection, gastric lavage and enema) has influence on the mRNA level of inflammatory factors of mice in the DSS model. P < 0.05, p < 0.01, p < 0.001, p < 0.0001, vs Vehicle;

FIG. 5: difficile administered to mice by different routes (intraperitoneal injection, gastric lavage and enema) has an effect on the number of lymphocytes Th1 and Th17 in spleen (A) and mesenteric lymph node (B) of the mice in the DSS model. P < 0.05, p < 0.01, p < 0.001, vsDSS;

FIG. 6: the effect of different ways (intraperitoneal injection, gastric lavage and enema) of administering clostridium difficile to mice on the IL-17mRNA level (A), the serum IL-17 content (B) and the colon tissue IL-17(C) of DSS model mice. P < 0.05, p < 0.01, p < 0.001, vs DSS.

Detailed Description