CN117695048A - Construction method of CAC animal model - Google Patents
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Abstract
The invention belongs to the field of pharmacology, and relates to a construction method of a CAC animal model. The construction method comprises the following steps: (1) on day 1, intraperitoneally administering a solution of AOM to the mouse; (2) on day 5, intraperitoneally administering a solution of AOM to the mouse; (3) day 8-14, the mice are free to drink 1.5% dss solution; (4) day 15-28, mice were free to drink water; (5) repeating the steps (3) - (4); (6) repeating the above steps (3) - (4) again. The construction method has high tumorigenesis rate and low mortality rate, has uniform tumorigenesis form, can greatly improve the construction efficiency of the CAC animal model and obviously reduce the cost.
Description
Technical Field
The invention belongs to the field of pharmacology, and particularly relates to a construction method of a CAC animal model.
Background
Colorectal cancer (colorectal cancer, CRC) is one of the common cancers and is also a major global health burden, and data related to 2020 shows that the incidence of CRC and the mortality rate of disease are the third and second in global malignant tumors, respectively. Inflammatory Bowel Disease (IBD) is an important risk factor for inducing colorectal cancer, and inflammatory bowel disease patients such as Crohn's Disease (CD) and Ulcerative Colitis (UC) are at a higher risk of colorectal cancer than healthy people. The mechanism of the dynamic evolution process of inflammatory cancer with respect to IBD-CRC is a current research hotspot. In the research of pathogenesis of colorectal cancer associated with Colitis (colisis-associated colorectal cancer, CAC), it is necessary to use in vivo models because the tumor microenvironment plays a central role in tumor proliferation, whereas in vitro models lack tissue environment. The pathogenesis of CAC involves multiple stages of normal mucosal epithelium-abnormal crypt foci-adenoma-adenocarcinoma, exhibiting a development law of "inflammation-atypical hyperplasia-canceration". The AOM/DSS induced mouse model is a common experimental tumor model of CAC. It can mimic the non-genetic characteristics of CAC in terms of normal epithelial/adenoma/cancer progression, an important tool to study the underlying mechanisms of colorectal cancer occurrence and development. But AOM/DSS mouse model construction is susceptible to mouse strain, modeling concentration, and mortality and neoplasia rates are not stable. It is therefore necessary to further optimize the AOM/DSS model building method on the basis of the relevant guidelines.
Disclosure of Invention
Aiming at the defects of the prior art, the inventor unexpectedly discovers in experiments that the method for constructing the CAC (Colitis-associated colorectal cancer, colorectal cancer related to colonitis) animal model has high tumorigenesis rate and low mortality rate, can greatly improve the construction efficiency of the CAC animal model and obviously reduce the cost.
Accordingly, it is an object of the present invention to provide a method for constructing a CAC animal model, comprising the steps of:
(1) On day 1, intraperitoneal injection was given to the mouse AOM solution;
(2) On day 5, intraperitoneal injection was given to the mouse AOM solution;
(3) Day 8-14, mice were free to drink 1.5% dss solution;
(4) Day 15-28, mice were free to drink water;
(5) Repeating the steps (3) - (4);
(6) Repeating the steps (3) - (4) again.
Preferably, in the steps (1) and (2), the dosage of the intraperitoneal injection is 10mg.kg -1 。
Preferably, in steps (1) and (2), the concentration of the AOM solution is 1 mg.mL -1 。
Preferably, in steps (1) and (2), the intraperitoneal injection is a single injection.
Preferably, the solvent of the AOM solution is normal saline; the solvent of the DSS solution is water (e.g., double distilled water);
preferably, the mice are C57BL/6J mice.
Preferably, the mice are male, 5-6 weeks old.
Preferably, the molecular weight of the DSS is 36000-50000 Da.
Preferably, the construction method is used for 1-3 weeks as inflammation formation stage, 4-6 weeks as tissue proliferation stage and 7-10 weeks as tumorigenesis stage.
Preferably, during steps (1) - (6), the mice are free to eat.
In this application, AOM refers to azoxymethane; DSS refers to sodium dextran sulfate.
The 1.5% DSS solution refers to 1.5g/100mL.
The method for constructing the CAC animal model has the advantages of high tumorigenicity rate, low mortality rate and uniform tumorigenicity form, and can greatly improve the construction efficiency of the CAC animal model and obviously reduce the cost.
Drawings
FIG. 1 shows the grouping of mice and their mode of administration.
Figure 2 is the body weight change during the first cycle DSS dosing of mice.
Fig. 3 shows changes in colon and neoplasia in mice.
FIG. 4 is a graph of colon HE staining of mice.
FIG. 5 shows the change in tumor markers and inflammatory factors in mouse serum.
FIG. 6 is a graph of colon HE and Ki67 staining of mice.
Figure 7 is a graph showing the change in body weight of mice with DSS dosing and free water.
FIG. 8 shows the variation of the proinflammatory factor IL-6 in a three-stage model group.
FIG. 9 shows the variation of the proinflammatory factor IL-1β in a three-stage model group.
FIG. 10 shows the change in the proinflammatory factor TNF-alpha in a three-stage model group.
FIG. 11 shows the changes in the inflammatory factor IL-4 in a three-stage model group.
FIG. 12 shows the changes in the inflammatory factor IL-10 in a three-stage model group.
Fig. 13 is a variation of the three stage model set CA 199.
FIG. 14 shows the changes in the expression of cyclin D1, CDK2, CDK4 proteins in the colon of CAC mice at the stage of tumor formation.
Figure 15 is a bar graph of CDK2 protein expression in the colon of CAC mice at the stage of tumor formation.
FIG. 16 is a bar graph of CDK4 protein expression in the colon of CAC mice at the stage of tumor formation.
FIG. 17 is a bar graph of the expression of CyclinD1 protein in the colon of CAC mice at the stage of tumor formation.
FIG. 18 is a flow cytometry assay for infiltration of T cells (CD4+, CD8+, CD49b+) in CAC mice peripheral blood.
FIG. 19 shows FITC method for detecting colon permeability of CAC mice.
FIG. 20 is a Western blot analysis of the expression of the colon compact junction proteins claudin-1 and occludin.
FIG. 21 is a bar graph of claudin-1 protein expression in the colon of CAC mice at the stage of tumor formation.
FIG. 22 is a bar graph of the expression of occludin protein in the colon of CAC mice at the stage of tumor formation.
Detailed Description
The present invention will be described below by way of examples, but the present invention is not limited thereto. The experimental methods shown in the following examples are conventional methods unless otherwise specified. The reagents and materials shown are all commercially available products.
The percentage concentration of the DSS solution referred to is in g/100mL.
1. Material
1.1 experimental animals: SPF grade male C57BL/6J mice, 5-6 weeks old, 20-21 g in mass, purchased from Beijing vitamin Torilis laboratory animal technology Co., ltd., qualification No. SCXK (Beijing) 2021-0006. Raising in GLP clean animal house, at 23+ -1.5 deg.C and 45+ -15% humidity. The experimental protocol was approved by the ethical committee of the institute of traditional Chinese medicine, academy of Chinese medicine.
1.2 drugs and reagents: AOM (micllin, cat No. C14453858, purity 95%); DSS (dextran sodium sulfate, molecular weight 36000-50000 Da, cat# S7708, america MP Co.); 4% tissue cell fixative (Beijing Soy Bao technology Co., ltd.; cat# 20221220); CA724 (Shanghai enzyme-linked biotechnology Co., ltd.; cat# Sep 2022); CA199 (Shanghai enzyme-Linked Biotechnology Co., ltd.; cat# Oct 2022); CEA (Shanghai enzyme-linked biotechnology Co., ltd., product number: ml 0631107); PBS (Beijing Soy Bao technology Co., ltd., product number: 20220726); fecal occult blood test paper (Pinctada martensii Biotechnology Co., ltd., cat# B211001); IL-6 (Biontech, germany, cat# P334473); TNF-alpha (BioNTech, germany, cat# P342290).
Preparing an AOM solution: the 25mg AOM powder was diluted to 1mg/mL with normal saline immediately before use. Dosage for intraperitoneal injection: 10mg/kg;0.1mL/10g.
Preparing a DSS solution: accurately weighing a proper amount of DSS powder, metering the volume of double distilled water to the required concentration (g/100 mL), and preserving at 4 ℃ for later use.
The first-cycle DSS administration comprises: DSS dosing on days 8-14 and free drinking water from mice on days 15-28.
2. Method of
2.1 grouping and administration of mice:
animals were fed adaptively for 7 days, mice were grouped according to a random number table, 10 per group. Except for the normal group, the CAC mouse model is constructed by adopting AOM/DSS combined induction of the other groups. The administration was as follows:
normal group (NC group): the mice were injected intraperitoneally with the same volume of physiological saline, free to drink, a single time on the first day.
3.5% dss gavage group (group A1):
(1) Day 1, 10mg/kg single -1 Intraperitoneal injection to mice 1mg/mL -1 AOM solution of (a);
(2) On days 8-14, mice were given 3.5% DSS solution, 0.2mL/10g each, by gavage twice daily at 8 am and 14 pm;
(3) Day 15-28, mice were free to drink water;
(4) Repeating the steps (2) - (3); (second cycle. Same as below)
(5) Repeating the steps (2) - (3) again. (third cycle. Same as below)
3.0% dss gavage group (group A2):
(1) Day 1, 10mg/kg single -1 Intraperitoneal injection to mice 1mg/mL -1 AOM solution of (a);
(2) On days 8-14, the mice were given 3.0% DSS solution, 0.2mL/10g each time, by gavage twice daily at 8 am and 14 pm;
(3) Day 15-28, mice were free to drink water;
(4) Repeating the steps (2) - (3);
(5) Repeating the steps (2) - (3) again.
2.5% dss intragastric group (group A3):
(1) Day 1, 10mg/kg single -1 Intraperitoneal injection to mice 1mg/mL -1 AOM solution of (a);
(2) On days 8-14, mice were given 2.5% dss solution, 0.2mL/10g each time, by gavage twice daily at 8 am and 14 pm;
(3) Day 15-28, mice were free to drink water;
(4) Repeating the steps (2) - (3);
(5) Repeating the steps (2) - (3) again.
2.5% dss free drink group (group B1):
(1) Day 1, 10mg/kg single -1 Intraperitoneal injection to mice 1mg/mL -1 AOM solution of (a);
(2) Day 8-14, mice were free to drink 2.5% dss solution;
(3) Day 15-28, mice were free to drink water;
(4) Repeating the steps (2) - (3);
(5) Repeating the steps (2) - (3) again.
2.0% dss free drink group (B2 group):
(1) Day 1, 10mg/kg single -1 Intraperitoneal injection to mice 1mg/mL -1 AOM solution of (a);
(2) Day 8-14, mice were free to drink 2.0% dss solution;
(3) Day 15-28, mice were free to drink water;
(4) Repeating the steps (2) - (3);
(5) Repeating the steps (2) - (3) again.
1.5% dss free drink group (group B3):
(1) Day 1, 10mg/kg single -1 Intraperitoneal injection to mice 1mg/mL -1 AOM solution of (a);
(2) Day 8-14, mice were free to drink 1.5% dss solution;
(3) Day 15-28, mice were free to drink water;
(4) Repeating the steps (2) - (3);
(5) Repeating the steps (2) - (3) again.
0.75% dss free drink group (group B5):
(1) Day 1, 10mg/kg single -1 Intraperitoneal injection to mice 1mg/mL -1 AOM solution of (a);
(2) Day 5, 10mg/kg single -1 Intraperitoneal injection to mice 1mg/mL -1 AOM solution of (a);
(3) Day 8-14, mice were free to drink 0.75% dss solution;
(4) Day 15-28, mice were free to drink water;
(5) Repeating the steps (3) - (4);
(6) Repeating the steps (3) - (4) again.
Inventive group (B4 group):
(1) Day 1, 10mg/kg single -1 Intraperitoneal injection to mice 1mg/mL -1 AOM solution of (a);
(2) Day 5, 10mg/kg single -1 Intraperitoneal injection to mice 1mg/mL -1 AOM solution of (a);
(3) Day 8-14, mice were free to drink 1.5% dss solution;
(4) Day 15-28, mice were free to drink water;
(5) Repeating the steps (3) - (4);
(6) Repeating the steps (3) - (4) again.
Throughout the trial, each group of mice was free to eat. The grouping is shown in fig. 1 and table 1.
TABLE 1 animal groups with different modes of administration
2.2 Disease Activity Index (DAI) assessment: mice were observed for body mass, stool traits, and occult blood status and scored. And adding the scores of the quality decline, the stool characters and the stool conditions to obtain DAI of each mouse, and evaluating the disease activity condition.
2.3 colon histopathological examination of mice: HE staining was performed to look at pathological lesions and tumor formation.
2.4 determination of inflammatory factors and tumor markers in serum of CAC mice: ELISA kit detects inflammatory factors IL-6, TNF-alpha and tumor markers CA199, CEA and CA724 in serum.
3. Results
3.1 modeling Condition assessment
DSS intragastric group (including: 3.5% DSS intragastric group, 3.0% DSS intragastric group, 2.5% DSS intragastric group): mice were gavaged 3.5%, 3.0% and 2.5% of DSS were presented with runny stool at day 6; the fecal occult blood test of the mice is positive on day 7; individual eyes feel bloody stool with symptoms such as reduced water intake and lazy movement, but the hair is basically good in color and luster and good in mental condition. During the first cycle DSS dosing period, the weight loss was evident in the 3.0% DSS group and 2.5% DSS group mice, except for the 3.5% DSS group, which did not significantly decrease with prolonged dosing time.
DSS free drink group (including: 2.5% DSS free drink group, 2.0% DSS free drink group, 1.5% DSS free drink group, 0.75% DSS free drink group): on day 4,3 mice with 2.5% DSS and 2.0% DSS were positive in fecal occult blood test, on day 5, mice were soft and bloody at the anus, body mass was suddenly reduced on day 6, more than half of mice were sticky and bloody, and some mice were accompanied with dark hair, listlessness, lazy, and significantly reduced DSS consumption; pus and bloody stool were all visible to the naked eye on day 7. The mice can drink 1.5% of DSS freely, the test of occult blood stool is positive on day 5, and meanwhile, the anus of each individual mouse has blood stain; the mice are positive in most of the blood-occult tests on day 5 when the mice drink 0.75% of DSS freely, meanwhile, the anus of each individual mouse has macroscopic mucopurulent blood-occult, and the symptoms are more serious than those of single AOM intraperitoneal injection mice with the same concentration of free drink.
The invention comprises the following steps: most of the occult blood stool tests of the mice are positive on day 5, and the anus of each individual mouse has mucopurulent blood stool visible by naked eyes; symptoms were worse than single intraperitoneal injections of AOM in mice given free drink at the same concentration. During the first cycle DSS dosing period, mice developed significant clinical symptoms of ulcerative colitis, while the mice did not significantly drop in mass, but rather slowly decreased in body weight with prolonged dosing time (fig. 2), consistent with a higher degree of clinical morbidity.
The DAI scoring results for the DSS intragastric group and the DSS free drink group are shown in table 2 below. At the end of the first cycle DSS dosing, the DAI score was generally higher in the free-drinking group than in the gavage group, with no statistical significance (P > 0.05) for the difference. After 7 days of free drink of the first circulating DSS, the model mice show typical clinical symptoms of ulcerative colitis, the damage degree is continuously improved along with the time, but the clinical symptoms of the gastric lavage group are not obvious, and after 7 days of gastric lavage administration of the DSS, most mice only show sticky soft stool or harder bloody stool, but do not show clinical typical sticky bloody stool.
TABLE 2CAC mice first cycle DAI score
3.2 different concentration DSS mice tumor formation rate and death conditions
The mice show symptoms of colitis at the early stage of free drinking of DSS administration, including diarrhea, somnolence, inappetence, weight loss and the like, and gradually show mucous stool and bloody stool at the later stage, and show death of the mice; after administration of the second cycle of DSS, mice begin to develop some tolerance to DSS, with symptoms that are slightly worse than in the first cycle, during which time no mice die; upon administration of the third cycle DSS, mice with increased hematochezia conditions develop and the high dose DSS group of mice die again as the accumulation of DSS in the body increases. Dissecting the mice and taking out the same-position intestinal segments for comparison, and finding that the normal control group (NC) mice have good colon elasticity, smooth intestinal wall, no hyperplasia and normal intestinal content fecal characteristics; the colon elasticity of the mice in the model group was lost, the intestinal wall was thinned and shrunken, significantly shorter than that in the normal group (table 3), and there were occasionally significant blood filaments in the intestinal wall with massive formed "cauliflower-like" growth, the intestinal content was thin in stool texture and increased in viscosity.
TABLE 3 comparison of colorectal length of CAC mice
The results of the tumorigenesis are shown in FIG. 3 and Table 4, and the tumorigenesis rate of 3.5% DSS intragastric group mice (A1) is 100%, but the mortality rate is 80%;3.0% DSS intragastric group (A2) had a tumor formation rate of 70% with small but few visual observations of tumors; the 2.5% DSS gastric lavage group (A3) has a tumor formation rate of only 20%; the 2.5% dss free drink group (B1) and the 2.0% dss free drink group (B2) were large and evident as tumors, but mortality was 50% and 30%, respectively; 1.5% dss free-drink mice (B3) had a rate of 80% tumor formation and 0% mortality; the tumor formation rate of 0.75% DSS free drink mice (B5) is 70%; whereas the mice (B4) of the present invention had a tumor formation rate of 100% and a mortality rate of 0%.
Therefore, the CAC mouse model is not ideal in the condition of lavage administration of DSS molding, the high-concentration DSS mortality is higher, the low-concentration tumorigenesis rate is lower, and the safe concentration interval is narrow; DSS free drink mice better present the clinical features of ulcerative colitis and inflammatory carcinoma transformation, the higher the concentration of DSS, the better the mouse's tumorigenesis effect, but the mortality increases with it. But 100% of the tumor forming rate can be achieved after the AOM reinforcing injection is adopted, the tumor forming size and shape are uniform, and the death rate is low. Therefore, the model construction method of the invention is an ideal feasibility scheme for constructing CAC mouse inflammatory cancer transformation model.
TABLE 4CAC mice nodulation and mortality
3.3 pathology of colon tissue of CAC mouse model
Taking the colon of a normal control group and a free drink group mouse with different concentration of DSS for HE staining and observing under a microscope (figure 4), finding that the colon tissue of the normal group mouse consists of a mucous membrane layer, a submucosa, an lamina propria and a plasma layer, the gland arrangement rule is regular and takes a long tube shape, a large number of cavitation cup cells exist in the tissue, and the crypt structure is normal; it was observed that DSS free drink tumor mice most deeply reached deep ulcers of the myolayer, disorder of colonic crypt structure, loss of original tubular structure and partial glandular atrophy necrosis, heterogeneous cells and deep nuclear staining appeared, and lumen surface showed high-grade intraepithelial tumor-like changes; at the same time, infiltration of a large number of inflammatory cells in the tissue is accompanied by basal plasmacytoid, a large number of lymphocytes and neutrophilic granulocytes. The pathological damage extent thereof is reduced with the reduction of the concentration of DSS.
3.4 expression of tumor markers and inflammatory factors in serum of CAC mice
Tumor markers are substances which are produced by tumor cells themselves or by the body's response to tumor cells, and which react with the presence and growth of tumors, and which are present during the occurrence, proliferation and spread of tumors. CA199, CEA and CA724 are common indicators for early diagnosis and prognosis of colorectal cancer clinically at present. Tumor markers in the serum of CAC mice were significantly elevated in both CA199 and CEA content, except CA724, compared to the normal group (fig. 5), suggesting tumor development and formation in CAC mice. Inflammatory factor assays found that the serum IL-6 content was significantly higher in mice from the CAC model group than in the normal group, and TNF- α content was higher than in the normal group, but without statistical differences (fig. 5). In combination with the massive inflammatory cell infiltration observed in colon pathology, the rise of inflammatory factors in mouse serum suggests that the formation of CAC mouse tumors is accompanied by the characterization of inflammatory development, which also characterizes to some extent the transformation process of inflammatory-cancers.
3.5 verification of the modeling method
In order to further verify the feasibility of the modeling method of the invention, and observe the tumor formation condition of CAC mice at the same time, the inventors selected 90 mice, 30 of which are blank mice, and 60 of which were modeled by adopting the CAC animal model construction method of the invention. 3 mice were sacrificed weekly from DSS first week dosing, and the colon of the mice was stained with HE and Ki67 (fig. 6) to dynamically observe the inflammatory-cancerous transformation process with pathological lesions and colonic cell proliferation. After the mice are given with the DSS for one week in the first cycle, the mice have obvious inflammatory reaction, inflammatory cytokines are gathered in a large amount, the inflammation is spread after free drinking for one week, the inflammatory reaction starts to be improved in the second week of free drinking, and at the moment, the Ki67 staining does not have obvious positive signals; after the mice DSS is given for one week in the second cycle, the glands of the mice in the model group are obviously destroyed, the inflammation is repeated and aggravated, and meanwhile, the Ki67 staining positivity is increased, and obvious proliferation occurs; after one week of administration of DSS to mice in the third cycle, crypt crowding and atrophy or dysplasia, tumor epithelial cells are arranged in a swelling, lump-like structure, glands merge with each other to form a sieve, nuclei are enlarged, chromatin is coarse, and deep nuclear staining occurs, luminal surfaces show high-level intraepithelial tumor-like changes, a large positive brown signal of Ki67 suggests further exacerbation of proliferation, and a large number of tumor cells are clustered in a nest-like manner in the following two-week free drink, and tumor characteristics are apparent. Meanwhile, the CAC inflammatory cancer transformation process was divided into inflammatory formation (3 weeks), proliferation (6 weeks), and tumor formation stage (7-10 weeks) according to HE pathology and Ki67 proliferation dynamic observation results. Mice were bled from the eye, assayed for inflammatory factors and CA199, and examined for mouse neoplasia and cyclin CyclinD1, CDK2, CDK4 expression during the neoplastic phase. The results showed that 34 mice had a 100% tumor formation rate and a 94.4% survival rate. The body weight of the mice fluctuated with the transition between DSS administration and free water (fig. 7). The three-stage model group had elevated proinflammatory factors IL-6, TNF-alpha, IL-1β, and in particular, the IL-6 elevation was evident (FIGS. 8, 9, 10). The anti-inflammatory factors IL-4 and IL-10 were decreased (FIGS. 11 and 12). CA199 was significantly elevated during the tumor formation phase (fig. 13). The significantly increased expression of cyclin d1, CDK2, CDK4 proteins in the colon of CAC mice at stage of tumor formation (fig. 14, 15, 16, 17), suggesting that the colon cell cycle of CAC mice is blocked.
3.6CAC mouse immune function and intestinal Barrier assessment
The T lymphocyte subpopulation plays an important role in the immune system of the organism, and in the peripheral blood of CAC mice, the percentage of cd4+ T cells is significantly reduced from 50% in the normal group to 40% in the CAC group. In contrast, the normal group cd8+ T cell infiltration rate (50.5%) was higher than the CAC group (56.0%). This resulted in an imbalance in the cd4+/cd8+ cell ratio, a significant decrease in the CAC group (fig. 18). A low CD4+/CD8+ cell ratio suggests immunodeficiency, suggesting dysimmunoregulatory function in CAC mice.
The increase in serum FITC-D levels (P < 0.05) in CAC mice compared to normal group (fig. 19) suggests an increase in intestinal permeability in inflammatory carcinoma transformed mice. The expression of the intestinal canal zonulin Occludin, claudin-1 protein was significantly reduced (P < 0.05) (FIG. 20, FIG. 21, FIG. 22). The CAC mice were prompted for damage to the intestinal mucosal barrier.
The AOM/DSS induced mouse model is a common experimental tumor model for the development of colonic cancer associated with colitis. In particular, it can mimic the non-genetic characteristics of CRC in terms of normal epithelial/adenoma/cancer progression. In addition, it is an important tool to study the underlying mechanisms of colorectal cancer occurrence and development, as well as a valuable and efficient model for evaluating new therapeutic regimens. In previous studies, researchers have spent time finding the best conditions for inducing tumor development using AOM and DSS, including dosages of AOM and/or DSS. Unfortunately, however, literature studies on AOM and DSS synergistically induced tumor development in mice are not extensive and detailed. Suzuki et al reported that 4 inbred mice had colorectal tumorigenesis sensitivity sequences of Balb/C > C57BL/6 > C3H/HeN > DBA/2N, C3H/HeN and DBA/2N mice were not found adenocarcinoma. Melgar et al compared the difference in progression of DSS colitis in two common strains, balb/C, C57BL/6, with 1 cycle post-withdrawal from DSS, balb/C mice recovered from colitis symptoms, while C57BL/6 mice continued in inflammatory response, with colitis progressing to chronic phase; it is believed that C57BL/6 mice have reduced tolerance to DSS compared to Balb/C mice, and the inflammatory process continues for a long period of time, eventually progressing from acute inflammation to chronic colitis. The active product of the cancerogenic substance AOM enters the intestinal tract after being metabolized by the system, and is decomposed into methyl carbonium ions by flora in the intestinal tract to induce DNA alkylation, and the pro-inflammatory agent DSS causes a long-term inflammation background by destroying the intestinal mucosa barrier, so that the CAC process from inflammation to dysplasia to tumorigenesis can be completely reproduced by combining the two. The C57BL/6 mouse is used for constructing a genetically modified animal model, can ensure the high stability on genetic background and the consistency of experimental data, and is widely applied to the construction of an AOM/DSS model. For this purpose, the inventors selected C57BL/6 mice for model construction. The inventor researches and discovers that once every week, six continuous weeks of mice simply injected with AOM can generate tumors; for this reason, the inventors selected the usual AOM dosing of 10mg/kg, set different AOM injection times, and simultaneously selected different DSS concentrations to investigate the optimal tumorigenic conditions in both gastric lavage and free drinking. The intragastric administration of DSS in the CAC mouse model is not ideal, and intragastric DSS does not cause significant symptoms of ulcerative colitis. Meanwhile, the high-concentration DSS of the gastric lavage administration has higher mortality, the low-concentration tumorigenesis rate is not good, and the safe concentration interval is narrow; DSS free drink mice better show the clinical characteristics of ulcerative colitis and inflammatory cancer transformation, and the higher the concentration of DSS, the better the tumor forming effect of the mice, but the mortality rate is increased. The free drink concentration of 1.5% DSS has good tumorigenic effect and low death rate, and the AOM can reach 100% tumorigenic rate after the intensive injection, therefore, the intensive injection of the AOM once is adopted, and the establishment of the CAC mouse inflammatory cancer transformation model by adopting the concentration of 1.5% DSS is an ideal feasible scheme.
Furthermore, the onset of colon cancer involves multiple stages of normal mucosal epithelium-abnormal crypt foci-adenoma-adenocarcinoma, exhibiting a development rule of "inflammation-atypical hyperplasia-canceration". The inventor finds that the DSS free drink mice have typical pathological characteristics of ulcerative colitis in the first cycle through dynamic material drawing and related index detection of the mice, and Ki67 staining proliferation is obvious in the second cycle until tumor formation in the third cycle. From this, it was clarified that the inflammatory phase (3 weeks), the tissue proliferation phase (6 weeks) and the tumorigenic phase (7-10 weeks) are changes in the progress of conversion of colitis cancer. Inflammation can increase the risk of cancer by providing bioactive molecules to cells infiltrating the tumor microenvironment, such as oncogenic programs that maintain continuous proliferation of cells, inhibit apoptosis signals, cell cycle disorders, promote proliferation and differentiation of cells, and reprogramming of energy metabolism. In the course of transformation of inflammatory cancer, inflammatory factors such as TNF-alpha, IL-6, IL-1 beta and the like are increased in three stages, and particularly IL-6 is remarkable. And analyzing genetic damage of colon mucous membrane caused by long-term inflammatory stress caused by IL-6 inflammatory factors and the like, so that self tissues are over-repaired, intestinal epithelial cells are abnormal in proliferation, and finally colon cancer is caused. Cd4+ and cd8+ T cells are of paramount importance in the immunomodulation process, the lower the ratio of cd4+/cd8+ the lower the immune response capacity of the body. In the model group, CD4+, CD49b+ (NK tumor killer cells) and CD4+/CD8+ are reduced, CD8+ T lymphocytes are increased, which is probably related to tumor cytokines in CAC mice, the tumor cells can synthesize and secrete immunosuppressive factors in a large amount, the amount of CD4 antigen is reduced, obvious inhibition effects on proliferation and differentiation of T lymphocytes and the like are generated, the reactivity of CD8+ cells is obviously increased, the CD4+ T lymphocytes are further limited, the amount of the CD4+ T lymphocytes is obviously reduced, and the proportion of CD4+/CD8+ is unbalanced. It has been reported that lymphocyte subsets are altered in tumor patients, resulting in immunosuppression, which promotes tumor development and progression. The reduction of the immune function of the organism leads the bacteria to have good living environment, and causes the massive synthesis of inflammatory mediators such as IL-6, TNF-alpha, IL-1 beta and the like. IL-6 is thought to be a missing link between inflammation and tumors. Clinical reports demonstrate that IL-6 is elevated in the serum of colon cancer patients. It has also been shown that cytokines such as IL-6, TNF-alpha, IL-1 beta, etc. promote the growth of colon cancer epithelial cells, and that IL-6, TNF-alpha activate STAT3 and NF- κB, which persist inflammatory responses, and play an important role in colon cancer production, where IL-6 expression is positively correlated with tumor size.
Based on the above description of the invention, one skilled in the art could fully apply the invention, and all such modifications as are intended to be included within the scope of the present invention.
Claims (10)
1. A method of constructing a CAC animal model, the method comprising the steps of:
(1) On day 1, intraperitoneal injection was given to the mouse AOM solution;
(2) On day 5, intraperitoneal injection was given to the mouse AOM solution;
(3) Day 8-14, mice were free to drink 1.5% dss solution;
(4) Day 15-28, mice were free to drink water;
(5) Repeating the steps (3) - (4);
(6) Repeating the steps (3) - (4) again.
2. The construction method according to claim 1, wherein in steps (1) and (2), the dosage of the intraperitoneal injection is 10 mg-kg -1 。
3. The construction method according to claim 1, wherein in steps (1) and (2), the concentration of the AOM solution is 1 mg.mL -1 。
4. The method of claim 1, wherein in steps (1) and (2), the intraperitoneal injection is a single injection.
5. The method of claim 1, wherein the solvent of the AOM solution is physiological saline; the solvent of the DSS solution is water.
6. The method of claim 1, wherein the mouse is a C57BL/6J mouse.
7. The method of claim 1, wherein the mice are male, 5-6 weeks old.
8. The construction method according to claim 1, wherein the molecular weight of the DSS is 36000-50000 Da.
9. The method of claim 1, wherein the method comprises inflammatory formation phase for 1-3 weeks, tissue proliferation phase for 4-6 weeks, and tumorigenesis phase for 7-10 weeks.
10. The method of claim 1, wherein during steps (1) - (6), the mice are free to eat.
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