Abstract Objective DMH colorectal tumor model improved traditional methods of induction, the establishment of an efficient, stable, practical model of colorectal adenomas, for sporadic adenomas biological behavior and molecular biology mechanism. Methods SPF SD male rats. The inducing agent for of 1,2  dimethylhydrazine (DMH) and PPAR  γ receptor antagonist (GW9662). PPAR  γ agonist is pioglitazone. The experiment was divided into four groups: negative control group, DMH group, DMH + GW9662 group, DMH + GW9662 + pioglitazone ketone group. A total of 12 weeks. The end of the test taken distal colorectal (1/2) of all colorectal with formalin 4 ° C overnight fixed entity under the microscope after the blue staining observed counting polyps, and photographed. All polyps micro uplift mucosa were examined histologically. The results of the negative control group without adenoma occurred. The DMH group found an adenoma adenoma induction success rate was 12.5% ​​(1/8), the number of tumor-bearing 0.125 (1/8). The DMH + GW9662 group found 16 adenomas, inducing a success rate of 87.5% (7/8), the number of tumor-bearing 2.0 (16/8). The DMH + GW9662 + pioglitazone group found five adenomas, inducing a success rate of 28.6% (2/7), the number of tumor-bearing 0.714 (5/7). Conclusions to DMH joint application GW9662 can be induced in the short term success of colorectal adenomas. More practical value of the model than a simple DMH-induced rat model of colon cancer and aberrant crypt foci.

Key words adenomatous polyps; 1,2  dimethyl hydrazine; GW9662; pioglitazone; PPAR γ

 Establishment of Animal Model Sporadic Colorectal Adenoma

    ZHANG Yao  peng, LV Yu  min, LI Jun, JIN Zhu, HAN Ya  jing, LI Chuan  feng, GU Fang

    Department of Digestive Disease, Peking University Third Hospital, Beijing 100083, China

    Corresponding Author: LV Yu  min, E  mail: ddbbol@163.comAbstract: Objective To build an economical, effective and stable new experimental animal model of sporadic colorectal adenoma based on traditional DMH  induced colorectal carcinoma model.The new animal model can be used to investigate the biological behavior and molecular mechanism of adenomas.Methods Male Sprague  Dawley rats were chosen in this study.Carcinogenic agent was DMH.GW9662 was chosen as the antagonist of PPAR  γ.Agonist of PPARγ was pioglitazone.Thirty  seven rats were randomly grouped into four groups, named as negative control group, group DMH, group DMH + GW9662 and group DMH + GW9662 + Pioglitazone.The experiment period was 12 weeks.At the end of experiment, all rats were sacrificed by euthanasia.Half of the distal colorectum was taken and immersed in formalin at 4 ℃ overnight, and then recorded the polyps by anatomic microscope when stained by methylene blue.Photos of polyps were taken with high resolution digital camera and reversal film.All polyps and some flatten risen mucosa were verified by histology.Results There were no polyps found in negative control group.One adenoma was found in Group DMH.The incidence of adenoma was 12.5% ​​(1/8), and the adenoma load was 0.125 (1/8). In Group DMH + GW9662, 16 adenomas were found in all 8 rats.The incidence of adenoma was 87.5% (7/8), and the adenoma load was 2.0 (16/8). The incidence of adenoma and the adenoma load in Group DMH + GW9662 + Pioglitazone were 28.6% (2/7), 0.714 (5/7) respectively.Conclusion The combination of DMH and GW9662 can induce rat colorectal adenoma efficiently in a short experiment period.This animal model can be used in colorectal adenoma and pre  cancerous lesions related researches and have more advantages compared with traditional DMH induced colorectal carcinoma model and ACF model.

    Key words: Adenomatous Polyps; 1,2  Dimethylhydrazine; GW9662; Pioglitazone; PPAR gamma

Traditional main animal models of colon cancer in rodents, the common inducers of 1,2  dimethyl hydrazine (DMH) and the azo oxygen methane (AOM), which is an important means for the study of human colorectal cancer. But there are also a lot of inadequacies, such as the induction of a long time, only reflect the characteristics of the cancer, can not be used for the study of precancerous lesions. Aberrant crypt foci (ACF) as a model of early tumor research to overcome a matter of time, but there are also a number of disadvantages, such as the evaluation index subjectivity, lack of histological evidence.

    Peroxisome proliferator-activated receptor  γ (PPAR  γ) is a member of the nuclear hormone receptor superfamily, play a key role in glucose and lipid metabolism. Many studies have shown that PPAR  γ involved in tumorigenesis, development, colon tumor tissue expression and distribution have significant exception, and phasic changes. From normal tissue to adenoma, adenocarcinoma, PPAR  γ expression gradually increased adenoma tissue distribution of PPAR  γ exception. And in vitro studies, the selective PPAR  γ receptor antagonist GW9662 can block the drug sulindac tumor cells with tumor preventive effect inhibited GW9662 has the potential to promote tumor formation [1  2].

    This study by the combined tumor inducer DMH and has the potential to promote the role of tumor formation selective PPAR  γ receptor antagonist GW9662 the establishment of an economic and practical, stable, efficient and objective model of adenoma, to make up for colon cancer model and ACF Model insufficient.

    1 Materials and methods

    1.1 Animals and grouping

    SPF male Sprague  Dawley rats, the size of 5 to 6 weeks of age, weighing about 200 g, rearing environment SPF Laboratory Animal Center of Peking University, constant temperature and humidity conditions, feeding, daily exposure time of 12 h , feed, water, are not restricted. Animals purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., SPF level of feed and drug-containing special feed provided by a third-Feed Co., Ltd. Beijing Branch Australia and collaborative production.

    37 rats were randomly divided into four groups, DMH group 10; GW9662 group, negative control group, DMH + GW9662 + pioglitazone ketone group 9.

    1.2 of drugs and the use of methods

    Tumor-induced and specific PPAR  γ receptor antagonists GW9662 the drug 1,2  dimethylhydrazine (DMH) were purchased from Sigma (China), PPAR  γ agonist pioglitazone by Hangzhou Sino-US East Pharmaceutical Co. Company kindly. DMH use the first induction dose of intraperitoneal injection of 120mg/kg, 2 weeks after subcutaneous injection of 20mg/kg strengthen once. GW9662 two weeks after the first dose in the DMH, the first dose of intravenous 0.3mg/kg and 0.1mg/kg weekly intravenous. Pioglitazone through the made of a special feed as PPAR  γ receptor agonist administered in a dose of 3.2mg/kg, in DMH 2 weeks after the first administration. The experimental design of 12 weeks.

    1.3 The results of treatment

    Overdose of chloral hydrate test the final period the animals were killed and distal 1/2 colorectal fixed with formalin 4 ° C overnight. 2% methylene blue solution was staining observed under a stereomicroscope after polyp, and record the number and size of photography, entity under microscope drawn, in accordance with the rat intestinal tissue processing method for dewatering, transparent, embedded in paraffin after standby. Pathology experts taken polyps and flat mucosa were examined histologically after routine HE staining of paraffin-embedded specimens sliced, typing, grading and adenomas.

    1.4 statistical methods

    The test data for statistical analysis using SPSS 13.0 software. Measurement data with a ± s. Weight  nutrition index analysis, group one-way ANOVA, P <0.05 line LSD Post Hoc Tests between groups analysis. Group on the adenoma the number using Pearson Chi  Square Test analysis, P <0.05 between groups using Fisher’s exact Chi  Square Test analysis.

    2 Results

    2.1 rat death

    The end of the test period, negative group (n = 9), no death, it is all included the adenoma to form statistical analysis and weight – nutrition index analysis. DMH group of 10 rats total of two rats died eight SD rats were included in adenoma formation statistical analysis, which is a larger neck ulcer in the model of the induction period, healing slower kept separate appear abnormal weight loss until the ulcers heal back pack weight began to increase, but with the same group on the weekly weight gain and final body weight compared to other rats vary greatly, it is not included in the weight – nutrition index analysis. DMH died + GW9662 group of nine rats, a statistical analysis and weight – nutrition index analysis of rat are 8 inclusion adenoma formation. DMH + GW9662 + pioglitazone group of nine rats died two rats are seven statistical analysis and weight – nutrition index analysis included in adenoma formation. Analysis of the time of death from the date of the start of the test were 2,15,16,24,66 days, dead rats showed expansion of the cecum, small bowel dilatation, a small amount of ascites, local necrosis of the ileum, black, and the colon is generally no obvious abnormalities .

    The 2.2 weight  nutrition index analysis

    Statistical analysis shows that were statistically significant differences in mean terminal body weight, the average weight gain among the four groups, nutrition index. The negative control group in the terminal body weight, body weight change in nutritional indices were higher than the I group, a statistically significant difference. The DMH group in nutritional index other difference was not statistically significant. DMH-induced tumor formation in the process significantly reduces the test animals nutrition index, GW9662, pioglitazone both drugs there is no statistically significant effect on the test animal nutrition index, see Table 1.

    2.3 adenoma formation

    The morphology and histopathology confirmed the negative control group without adenoma. DMH group had an adenoma formation, about the size of 4.0mm, pathological grade Ⅰ ~ Ⅱ of tubular adenomas, the polyps induced a success rate of 12.5% ​​(1/8), the number of tumor-bearing rats (on average, each rat the number of intestinal tumors formed) is 0.125 (1/8). The DMH + GW9662 group found Table 1, the rat body weight change and nutritional indicators table

    16 adenomas. 13 large adenomas entities visible under the microscope, Figure 1a, the largest of about 6.0mm, the smallest about 0.3mm, the average size of approximately 1.3mm. 3 slightly elevated mucosa after histologically proven microadenomas polypoid microscope section where the point of view of only duct constitute, as shown in Figure 1b. 16 adenomas pathological manifestations of tubular adenomas Class I, 9, as shown in Figure 2a. Ⅰ ~ Ⅱ grade adenomas 4, as shown in Figure 2b. Ⅱ ~ Ⅲ grade adenoma 4, as shown in Figure 2c. Adenomas induced a success rate of 87.5% (7/8), the number of rat tumor-bearing 2.0 (16/8). DMH + GW9662 + pioglitazone treated group found five adenomas, maximum 5.0mm minimum 1.0mm, with an average size of about 2.7mm, are tubular adenomas Ⅰ ~ Ⅱ grade adenomas induced a success rate of 28.6% (2/7) The number of murine tumor-bearing 0.71 (5/7). Adenomas induced the success rate and the number of tumor-bearing Pearson Chi  Square Test and one-way ANOVA, and found that DMH + GW9662 group on the success rate and polyps in the total number of induced tumors were greater than the other three groups, a statistically significant difference, was no significant difference between the three groups, are shown in Table 3.

    3 Discussion

    2 adenomas of the the histopathological table since 1970 Druckrey H first reported with 1,2  dimethylhydrazine-induced rodent colon cancer model, the model has been widely used in colorectal cancer development and metastasis The induced effect table *: compared with other three groups, P ≤ 0.016 and molecular level mechanism, as well as tumor chemotherapy and chemical intervention study [3]. Common inducers DMH and AOM. Although different studies use dose, route of administration, the induction period and the total observation period is different, but, overall, has the following characteristics: (1) model for colorectal cancer in adenocarcinoma, adenoma (2) The average number of tumor-bearing 1.08 to 1.95, (3) animal tumors success rate of 70% to 90%, only 10% to 30%; (4) model induction time ranging from 27 weeks to 40 weeks .

    Rubio C, et al [4] in a flat adenoma – carcinoma sequence model of 300 SD rats, DMH-induced and observed for 27 weeks, 278 (92.7%) of which produce tumors in rats, were formed adenoma or adenocarcinoma 358, with an average per rat tumor-bearing capacity of 1.19 only 60 (16.76%), adenomas only the amount of tumor-bearing 0.2 adenoma, these tumors. Medina V [5] study, positive group tumor incidence was 94.7% (18/19) for a period of 8 months, the tumor number 37, each rat tumor-bearing 1.95, but Only one adenoma. In 2000, Ravnik G [6] 150 Wista rats, DMH-induced 15 times, forming 45 tumors, including 24 adenocarcinoma, signet ring cell carcinoma 14, adenoma 7, each rat The average number of tumor-bearing 0.3, the number of adenomatous tumor-bearing is only 0.05. Early intervention role Goeting NLM [7] conducted a on warfarin precancerous lesions AOM, observed for 40 weeks, 27 tumors found in the positive control group, 25 rats, each rat tumor-bearing an amount of 1.08. Such as Kobaek L [8] observed three kinds of inbred rats BDIX / Orllco, F344/NHsd, WAG / Rij intestinal tumor response induced by AOM (15mg/kg wk) found the highest incidence of tumors, the shortest cycle germline is BDIX inbred rats, but the tumors still 23 to 27 weeks, the tumor was only 75%. Melen M et al [9] in the study DMH rat intestinal mucosa proliferation, apoptosis trial, which lasted 21 weeks, were found in 37 cases of tumors, including 27 cases of adenocarcinoma cases of signet ring cell carcinoma, 9 adenomas. Hirose M, et al [10] DMH 40mg/kg subcutaneous injection induced F334 rat tumor anticancer effects in order to observe the green tea extract, which lasted 36 weeks, the positive tumor incidence of 81% (17/21), producing adenoma rats 9, 14 adenocarcinoma, on average, each rat the number of tumor-bearing 1.8, the number of adenomatous tumor-bearing only 0.7.

    With the progress of the study of early colorectal cancer and precancerous lesions, especially drug in colorectal cancer early chemical intervention role gradually become a research focus, such as NSAIDs, green tea extract, garlic extract. More obvious, the drawbacks of traditional colon cancer model can not meet the research needs.

    Since 1987, Ranjana Bird, etc. [11] first reported the AOM-induced rat model of aberrant crypt foci (Aberrant crypt foci, ACF), and more study further confirmed the ACF ACF model can replace the colon cancer model for early tumor lesions and early cancer chemo-prevention study, thus greatly reducing the experimental period, saving a lot of manpower, material and financial resources. ACF as a precancerous lesion, however, has been surviving controversy, including the ACF can develop adenomas or how to develop adenomas, ACF and adenomas how ACF-adenoma – carcinoma model whether the same applies to human . Thus ACF model can replace a colon cancer model even human colon cancer for some scientific research surviving controversial [12  13].

    Our previous study found that ACF as an animal model there are many shortcomings: (1) evaluation is subjective, despite Elizabeth AML [14] in 1991 described in detail the evaluation of ACF, but when observed due to dyed effect, and between normal and AC single crypts and a large number of very difficult to achieve the objective, accurate judgment and counting; (2) pathological analysis is difficult and grading mere ACF quantity in number hundred, and containing from 1 to 3 of AC small ACF pathological analysis, it is impossible for each ACF, to observe the extent of its profiled, whether it really has pathology of atypia, and even select a larger ACF drawn from pathological, it is difficult to find during biopsy atypia crypts. Slices can see atypia crypt, the pathological grading are mostly mild atypia, thus pathological analysis is not meaningful; (3) ACF tumors was observed during the initial screening with a tumor chemical intervention effect of the drug have a certain value, but, due to the lesion or tissue mucosa is difficult to selectively cut, is not conducive to the analysis of the tumor occurs at the early stages of molecular biology. changes and study mechanism of drug action and the role of the target protein or target gene. Microdissection technique can selectively cut single duct, but in a limited period of time sufficient molecular biology research organization or cut a certain degree of difficulty, although some use this technical analysis of small adenomas in the APC gene mutation, the majority of still focused on the morphological [15  16].

    The test through a joint application knot rectal cancer inducer DMH and PPAR  γ receptor antagonist GW9662 in the short term (12 weeks) successfully induced colorectal sporadic adenomas. The adenomas induced success rate and the number of tumor-bearing, respectively, 87.5% and 2.0, significantly higher than a separate application DMH 12.5% ​​of the experimental group and 0.13. And through the use of PPAR  γ receptor agonist pioglitazone PPAR  γ receptor antagonist GW9662 to antagonize adenomas induced success rate and the number of tumor-bearing dropped to 28.6% and 0.71 respectively. That PPAR  γ receptor antagonist GW9662 did play a role in promoting tumor formation by PPAR  γ pathway. PPAR  γ pathway an important role in the formation and development of colon tumors and further instructions.

    Experiments to select only the left half of colorectal polyps count. Lamont JT, et al. [17] Summary of animal models of colorectal cancer, the left and right colon cancer incidence with inducers dose and duration of action are closely related, but the overall the colon tumor animal model for the left and right colon The incidence is similar, suggesting that DMH + GW9662 group rats adenoma number in absolute value increase more. Since this experiment is a preliminary study of colorectal adenoma model, GW9662 [18] as a PPAR  γ receptor antagonists currently only used in the in vitro study of cytology, has not been reported in vivo pharmacokinetic characteristics, body The study is small, and thus subject to further study the pharmacokinetics, route of administration and dose.

    Comprehensive analysis of the test established colorectal adenoma model with traditional colon cancer model and the ACF model compared with economic, practical, efficient and objective advantages, you can make up for the inadequacies of the two traditional model. The adenoma model induced a short time, a higher rate of adenoma tumor-bearing, objective evaluation and a certain amount of tissue, bring convenience for tumor mechanisms, treatment and research.