中洪博元,动物造模,溃疡性结肠炎 z-bo 2020-07-21 1116

     [Main reagent] 5% 2 ,4 ,6 ,TNBs absolute ethanol

[Method] Rats in each group were fasted for 24 hours 1 day before modeling, and ketamine was injected intraperitoneally to lightly anesthetize 0.15ML/100G. Normal group: A 2MM polypropylene tube was inserted into the upper anus of the rat 8 cm, and 0.85% was injected at a time NaCl 0.6 ml; Model I group: calculate TNBs at 50 mg/kg (approximately equal to 0.09 ml/100 g of TNBs stock solution) plus 0.25 ml of 50% ethanol, insert a polypropylene tube into the upper anus 8 cm, and then inject the mixed reagent at one time ; Model II group: Calculate TN1Bs at 100 mg/kg (approximately equal to 0.18 ml/100 g of TNBs stock solution) plus 0.25 ml of 50% ethanol, insert a polypropylene tube into the upper part of the anus 8 cm, and then inject the mixed reagent at one time. Killed after 1 week Animals were evaluated by histomorphology score and light microscope.

　　 [Result] In the UC model caused by TNBs at a dose of 100 mg/kg, multiple ulcers in the colonic mucosa were visible to the naked eye, and the mucosa was significantly congested and edema. Histological examination revealed a large number of neutrophils, lymphocytes, macrophages, and fibroblasts in the mucosa and submucosa, and the formation of granulation tissue and crypt abscesses. There was a milder injury at a dose of 50 mg/kg. The best modelling dosage is 100 mg/kg.

　　Immune complex/formalin model

[Method] Take fresh colonic mucosal tissues of rabbits, homogenize them with a glass homogenizer, freeze at -20°C for 24h, centrifuge at 4000r/min for 30min after freezing and thawing, and take the supernatant to measure its protein by the biuret method Content, put in the refrigerator for later use. Add the same amount of Freund complete adjuvant to make an antigen emulsifier. The modeled rats in each group were injected with 0.4 mL of antigen emulsifier (containing 4 mg of antigen) into the left plantar of the rat on the 1st day, and injected antigen emulsifier into the right plantar, inguinal, abdominal cavity, and back on the 10th, 17, 24, and 31 days respectively. 0.8mL (containing 8mg of antigen, without adjuvant for the last injection). After confirming that the anti-colon antibody is produced in the rat, the rat is anesthetized with ketamine (100mg/kg), enema with 1.5% formalin 2mL, indwelled for 1h, washed with normal saline and discharged, and then used 2mL of antigen solution (containing 8mg of antigen) ), without adjuvant enema, indwell for 2h, then wash and discharge. On the second day of the above-mentioned treatment, the rats developed soft and loose stools, even pus and blood in the stools, apathetic and arched back. Four rats were sacrificed and their colons were taken. The pathology showed partial erosion of the mucosal surface and a small amount of chronic inflammatory cells in the lamina propria, but no ulcers were seen. This may be related to the improper sampling site.

[Advantages and Disadvantages] Although this method of modeling conforms to the pathogenesis of UC, and achieves the combination of systemic immune abnormalities and local inflammatory lesions, the model preparation process is cumbersome, the cycle is longer, the animal mortality is high, and the reproducibility is not ideal. The degree of pathological changes in the intestine is not in line with the pathological process of human UC, and it does not meet the requirements of scientific research experiments.

　　The establishment of a new UC animal model

　　Methods Firstly, the above-mentioned antigen emulsifier 0.8mL (each containing 8mg of antigen) was injected twice into the foot and inguinal of the rat, with an interval of 2 weeks.On the premise of systemic sensitization and immune abnormality in the rat, TNBS ( (Purchased from Sigma company) and anhydrous ethanol in the same volume ratio as a local enema at a dose of 100 mg/kg to cause local inflammation in the rat intestine

　　 Advantages and disadvantages This method not only achieves the coexistence of systemic immune abnormalities and local inflammatory lesions, but also produces clinical symptoms and intestinal lesions that are closer to human UC. On the 5th day after the local enema, the rat's stool frequency increased, and the stool was loose or mucus. The animal was sluggish, the weight loss was obvious, and the rats were weak and lazy, and showed an arched back. The colon tissues of 4 rats were taken at the 3rd, 6th, and 9th weeks after the local enema. Observation showed that the mucosal hyperemia, edema, and thickening of the intestinal wall were observed under the microscope. Inflammatory changes such as neutrophil infiltration; most rats can see symptoms such as loose stools or mucous stools, and listlessness. At the 12th week, the colon tissue of 4 rats was taken. Observation still showed thickening and narrowing of the intestinal wall. The fibrous tissue formation of the intestinal wall was observed under the microscope, and some ulcers, neutrophil infiltration and other inflammatory changes were still observed. The model method has good reproducibility, low mortality, long maintenance time, and is in line with the human UC pathological mechanism. It not only avoids the cumbersome immune complex/formalin model, poor reproducibility, and high mortality, but also avoids three defects. The nitrobenzene sulfonic acid/ethanol model has shortcomings such as simple cellular immune response and short maintenance time, making it an ideal animal model of human UC.

　　 Sodium Peroxide Nitrite Model

The rat ulcerative colitis model induced by sodium peroxide nitrite is currently one of the latest experimental research methods in the world. It was successfully produced by DANIEL et al. in 1993. The Chinese scholar Zheng et al. established it for the first time in rats in 2001. A preliminary exploration was made in methodology. The method is that the rats are anesthetized with ether, insert a silicone tube with an inner diameter of 2MM into the anus 6CM, use a syringe to inject 0.5ML sodium peroxide nitrite of different concentrations, and then inject a length of 0.25 air to clear the syringe and catheter Solution. -Rats were killed and sick after weeks

　　Physical section, observation under light microscope found that sub can effectively induce the production of UC in experimental rats, and the dose is preferably at a moderate concentration of 6.55MM. Pathology showed that the mucosal injury was more serious, congestion and edema were obvious, multiple ulcers were produced, the intestinal cavity became narrow, and the ulcers under the microscope were deeper, reaching the muscle layer. The key to success is to control the appropriate concentration and stability of the drug. Sodium peroxide nitrate is easily decomposed under normal conditions, and the reagent should not be placed for more than 24 hours.

　　 This model has the advantages of simple production, good reproducibility, and low production cost. Its morphological changes are related to the nitrogen oxidation mechanism and can reflect part of the essential mechanism of ulcerative colitis. It is also one of the most advanced models in the world. It is suitable for the research on the prevention and treatment of ulcerative colitis with traditional Chinese medicine, especially how to clarify the anti-oxidation and regulating nitric oxide mechanism of traditional Chinese medicine compound.

　　 oxazolone method

It is a relatively new modeling method that has emerged in recent years. Oxazolone (OXZ) is an important intermediate for dyes, pesticides, and medicines, and is also a classic hapten substance. "It has been discovered by immunological research that induced mice In the colitis model, cytokine secretion is dominated by the overproduction of IL-4, which belongs to the TH-2 subtype inflammatory response, and the main cytokine of the TH-1 response, IFN-in this model, reduces the inflammatory response caused by the eventual secondary The secretion of transforming growth factor 2B (2B) is balanced, and the disease tends to be self-limiting [10]" Method: / male mice, after ether anesthesia, insert a thin catheter 4 through the anus and slowly inject

　　150L (6, dissolved in 50% ethanol), and then the mice were kept upside down for 30", and there was laziness in the model 2! Diarrhea! Bloody stools with naked eyes. Generally, about half of them died within 4, and the rest of the mice got better around the 7th. 10~12 basically healed, so it is recommended to kill the animals three times after modeling, and collect samples. "The macroscopic view of the colon shows that the disease is mainly the distal colon, the intestinal cavity is dilated, the mucosal congestion and edema, the erosion ulcer, and the formation of pseudomembranes." Colorectal mucosal ulcers, pseudomembrane formation, widespread disappearance of ducts, submucosal hemorrhage, severe neutrophils! Lymphocytes! Plasma cell full-thickness infiltration [11] "This method is simple to make, rapid model generation, good repeatability, and pathological changes. Similar to humans, it is especially suitable for research on pathogenesis and new drug development. "The disadvantage is that the disease is maintained for a short time, self-healing, no changes in the chronic phase, and it is not suitable for simulating chronic recurrence studies."

　　 Iodoacetamide method

　　 Iodoacetamide () is a sulfhydryl blocking agent, which can

　　 completely denature the protein molecules containing disulfide bonds and maintain the reduced state

　　 state, as a protease inhibitor! Sulfhydryl blocking reagent for biological mass spectrometry

　　Analysis! Protein molecular structure analysis and two-dimensional gel electrophoresis sample preparation

　　 Bei et al. "Iodoacetamide is mainly used in the establishment of animal colitis models

　　 mechanism is: iodoacetamide can denature cell proteins and destroy cells

　　 Normal structure and function, thereby impairing the integrity of the intestinal mucosa, leading to inflammation

　　 syndrome occurs; in addition, iodoacetamide can also inhibit creatine phosphokinase

　　(), which affects mitochondrial function and causes intestinal epithelial cells

"Abnormal function" method: male rats are anesthetized and given 0.23%

"Iodoacetamide (dissolved in 1% methylcellulose) enema" model

　　 After 24, animals appear lazy! Diarrhea! Bloody stools, etc., inflammation

　　 The highest peak is reached about 1 week after the model, and the mucosa begins to regenerate in about 2 weeks, the disease tends to be self-limited, and it is cured in about 3 weeks. "The colon is generally visible in the mucosa

　　 Congestion and edema, the erosion is granular, most small ulcers occur, may be sticky

　　Lian" under the microscope can see scattered ulcers on the mucosal surface, accompanied by a large number of inflammatory cells infiltration

　　 Run, crypt damage, duct damage, epithelial integrity damage [12213]"

　　 The method is simple to make, in addition to inducing colitis, it can also induce CD

"The ideal model of 　　" The disadvantage is that it is self-healing and not suitable for chronic

　　Stage disease research"

　　 dextran sodium sulfate method

　　Dinitrochlorobenzene method

　　 acetic acid method

　　 carrageenan

　　 antigen induced muramyl dipeptide

Ulcerative colitis induced by 　　 compound method (2,4 dinitrochlorobenzene + acetic acid combined modeling method)

　　 fetal rat colon implantation method

　　 Rat colon bacterial strain method

　　 indomethacin colitis model

　　 peptidoglycan polysaccharide (PG-PS) colitis

　　Experience of UC animal model

　　 There are a large number of antigenic substances in the gastrointestinal tract, such as pathogenic bacteria, normal flora, inflammatory bowel disease animal and bacterial toxins, viruses, metabolic chemicals, beverages and food, etc. Therefore, the intestinal immune system has two functions: it must protect the intestinal mucosa and resist the invasion of pathogenic factors; it must also absorb nutrients and tolerate normal intestinal flora. All of these intestinal contents may be potential sources of immunity, but when the body has normal regulatory capabilities, inflammation will not occur. The destruction of the mucosal barrier of intestinal epithelial cells creates conditions for the ingestion of large amounts of intestinal antigens. Uncontrolled immune response and imbalance of immune regulation are the immunological characteristics of ulcerative colitis. The above modeling method reflects an important link in the pathogenesis, that is, ethanol is the initiating factor and plays a role in destroying the intestinal mucosal barrier, so that the infused TNBS acts as a hapten to trigger a local immune injury response in the intestine, combined with rats The imbalance of systemic immune regulation causes the inflammatory process to be amplified step by step, and finally causes tissue damage, pathological changes and clinical manifestations of colitis.

　　 But this kind of model making method still lacks the characteristics of spontaneity and relapse, that is, it lacks the characteristics of human UC remission and relapse alternately. In future model studies, the author believes that the following two issues should be considered: The first is the maintenance time of UC animal models. Although the immune method can make UC animal models to maintain colon lesions for a longer period of time, it is due to the intestines of rats. The repair ability of the tract is strong, so how long it can maintain to meet the human UC is a key consideration in the future; the second is how to solve the problem of clinical remission and recurrence. For the UC model, whether the drug can be administered will relieve the disease and stop the drug. Post-inflammation reappears is what we need to explore and study in the future. I believe that with the continuous efforts and innovations of many researchers in this field, a more ideal UC animal model will gradually conform to the pathological process of human UC, and open up a wider range of systemic immune abnormalities and local inflammatory lesions for the drug treatment of ulcerative colitis. The coexistence of human beings can produce a new world with human beings.