DSS/DMH复合法诱导Wistar大鼠结肠炎癌转化模型的建立及评价

Establishment and evaluation of colon inflammation-tumor transformation model in Wistar rats induced by DSS/DMH combination

目的:基于葡聚糖硫酸钠(DSS)/二甲基肼(DMH)联用,建立Wistar大鼠结肠炎癌转化模型并进行模型评价。方法:取40只Wistar大鼠,按每只30 mL/d剂量给予4%DSS饮用3 d,建立溃疡性结肠炎(UC)炎症期模型;随机取UC炎症期模型大鼠34只,继续给予DSS至第7天(死亡8只),建立UC活动期模型;随机取UC活动期模型大鼠20只,按每周25 mg/kg剂量颈部注射DMH 17周(死亡4只),建立UC相关性结肠癌(UC-CAC)转化期模型;随机取UC-CAC转化期模型大鼠10只,继续给予DMH至第24周(死亡4只),建立CAC模型,另设正常组和PBS对照组,每组6只。对结肠炎癌转化各时期大鼠和瘤体特征观察,检测结肠长度、壁厚、湿质量指数,对DAI和CMDI评分进行评价,检测各时期模型CRP、ESR、HGB水平,Masson染色观察结肠病理变化,免疫组化检测结肠瘤体MLH1、PMS2、MSH2、MSH6蛋白表达。结果:UC炎症期大鼠表现稀血便,UC活动期大鼠腹泻与血便量多次频;UC-CAC转化期大鼠结肠成瘤率66.67%(4/6),CAC期大鼠结肠成瘤率100.00%(6/6);CAC期大鼠在第11~18周体质量进入代偿期,之后进入失代偿期;各组比较,CAC期结肠长度最低,湿质量指数和壁厚最高;DAI与CMDI评分分别在UC活动期和CAC期最高;在UC活动期CRP水平最高,ESR和HGB在CAC期分别达到最高和最低;UC炎症期可见结肠隐窝扭曲变形,UC活动期可见隐窝脓肿,组织溃烂,UC-CAC转化期表现为管状中高度分化腺瘤;CAC期表现为高分化锯齿状腺瘤;MLH1、PMS2、MSH2、MSH6蛋白检测均为阴性。结论:DSS/DMH复合法可成功建立UC炎症期、UC活动期、UC-CAC转化期和CAC期结肠炎癌转化模型,方法可靠,特异性强,与中医湿热瘀毒发为癌肿的中医病机较契合。

Objective:Based on the combination of dextran sodium sulfate(DSS)/dimethyl hydrazine(DMH),the inflammation-tumor transformation model of colon in Wistar rats was established and evaluated.Methods:Forty Wistar rats were given 4%DSS at a dose of 30 mL/d for 3 days to establish UC inflammatory phase model.Thirty-four UC rat models in the UC inflammatory phase were randomly selected and continued to be given DSS until the 7th day(8 of them died)to establish UC active phase models.Twenty UC active phase rat models were randomly selected,and injected with DMH at a dose of 25 mg/kg per week for 17 weeks(4 of them died)to establish the UC-CAC transformation phase model.Ten UC-CAC transformation phase rat model rats were randomly selected and treated with DMH until 24 weeks(4 of them died)to establish CAC phase model.A normal group and a PBS control group were set and with 6 rats in each group.The characteristics of rats and tumors in different phases of colon inflammation-tumor transformation were observed.Colon length,wall thickness and wet mass index were measured,and DAI and CMDI scores were evaluated.The levels of C-reactive protein(CRP),erythrocyte sedimentation rate(ESR)and hemoglobin(HGB)were detected at each phase.The pathological changes of colon were observed by Masson staining.The protein expressions of MLH1,PMS2,MSH2 and MSH6 were detected by immunohistochemistry.Results:The rats in UC inflammatory phase showed loose bloody stool,and diarrhea and blood stool in UC active phase became frequent.diarrhea and bloody stool aggravated in UC active phase;The tumorigenic rate of colon in rats during UCCAC transformation phase was 66.67%(4/6),while 100.00%(6/6)in CAC phase;The body weight of CAC rats entered the compensatory period at 11th-18th week,and then entered the decompensation phase;The colon length showed the lowest and the wet mass index and wall thickness demonstrated the highest in the CAC phase among the groups;The DAI and CMDI scores showed the highest in UC active phase and CAC phase respectively.The CRP level demonstrated highest in the UC active phase,and ESR and HGB reached the highest and lowest respectively in the CAC phase;Distortion and deformation of colonic crypt could be seen in the UC inflammatory phase,abscess ulceration in the UC active phase,and tubular moderately well-differentiated adenoma in the UC-CAC transformation phase.The CAC phase showed well-differentiated serrated adenoma;MLH1,PMS2,MSH2 and MSH6 proteins were negative in all cases.Conclusion:DSS/DMH combination could successfully establish colon inflammation-tumor transformation mode including UC inflammatory phase,UC active phase,UC-CAC transformation phase and CAC phase.The method is reliable and specific,which is consistent with the traditional Chinese medicine pathogenesis of dampness,heat,blood stasis and toxin developing into cancer.