胆囊胆固醇结石湿热证小鼠造模方法的研制与评价

Development and Evaluation of Modeling Method of Mice with Damp-heat Syndrome of Cholesterol Stone in Gallbladder

目的通过建立三种胆囊胆固醇结石湿热证小鼠模型,分析并评价模型间差异,基于中医基础理论探索合适的胆囊胆固醇结石湿热证动物模型。方法将24只C57BL/6J小鼠,随机分为空白对照组(K组)、内因模型组(M组)、内外因模型A组(A组)、内外因模型B组(B组)。K组采用普通饲料喂养并给予正常饮用水;M组采用高脂高胆固醇致石饲料喂养,给予20%蔗糖水饮用;A、B组给予高脂高胆固醇致石饲料和20%蔗糖水并置于湿热环境中,造模周期为12周,其中A组全程处于湿热环境,B组造模后4周置于湿热环境。观察并记录各组小鼠造模前后的一般状态,进行旷场实验;造模结束后取材并进行相关指标检测。结果与K组相比,M、A、B三组出现不同程度的湿热证的中医临床证候表现,体视镜下小鼠胆囊可见不同程度浑浊,HE染色表明肝脏、小肠、舌组织出现胆结石和湿热证相关的病理性改变,血清碱性磷酸酶(ALP)、γ-谷氨酰基转移酶(GGT)、总胆红素(TBIL)和间接胆红素(IBIL)也不同程度的增高,胆汁中胆固醇(TC)、胆汁酸(TBA)含量升高;综合评价以上结果,B组更显著地体现了胆囊胆固醇结石湿热证候。结论给予高脂高胆固醇致石饲料和20%蔗糖水12周加湿热环境4周的综合造模方法,可复制质量良好的胆囊胆固醇结石湿热证动物模型。

Objective To analyze and evaluate the differences between the models,and explore suitable animal models of gallbladder cholesterol gallstone damp-heat syndrome based on the basic theories of Chinese medicine,by establishing three mouse models of gallbladder cholesterol gallstone damp-heat syndrome.Methods Twenty-four C57BL/6J mice were randomly divided into blank control group(group K),internal factor model group(group M),internal and external factor model group A(group A),internal and external factor model group B(group B).Group K was fed with ordinary feed and given normal drinking water;group M was fed with high-fat and high-cholesterol lithogenic feed and given 20%sucrose water for drinking;group A and B were given high-fat,high-cholesterol lithogenic feed and20%sucrose water.In a hot and humid environment,the modeling cycle was 12 weeks.Group A was placed in a hot and humid environment for the whole process,and group B was placed in a hot and humid environment 4 weeks after modeling.We observed and recorded the general state of each group of mice before and after modeling,and conducted an open field experiment;after modeling,materials were taken and related indicators are tested.Results Compared with the group K,the groups of M,A,and B showed different degrees of damp-heat syndrome in TCM clinical symptoms.The mouse gallbladder could be seen to be turbid to different degrees under the stereoscope.HE staining showed pathological changes related to gallstones and damp-heat syndrome in the liver,small intestine,and tongue tissues.Serum alkaline phosphatase(ALP),γ-glutamyl transferase(GGT),total bilirubin(TBIL)and indirect bilirubin(IBIL)also showed increase to varying degrees.Cholesterol(TC)and total bile acid(TBA)in bile increased.Comprehensive evaluation of the above results showed that group B more significantly reflected the damp-heat syndrome of cholesterol gallstones.Conclusion The comprehensive modeling method of giving high-fat and high-cholesterol calculus feed and 20%sucrose water for 12 weeks in a humidified heat environment for 4 weeks can replicate a good-quality animal model of gallbladder cholesterol calculus damp-heat syndrome.