寒热不同证候大肠癌大鼠细胞外基质特征差异分析

Difference in Pathological Characteristics of ECM between Colorectal Cancer Rats with Han(寒)Syndrome and Re(热)Syndrome

目的:从细胞外基质(ECM)的角度探讨寒热不同证候大肠癌发生方式不同的可能机制。方法:将70只雄性大鼠随机分为正常对照组10只、寒证模型组、热证模型组各30只。采用冰水灌胃(0℃)结合冰水泡浴(10℃)和辣椒素0.9 mg/mL加30%乙醇灌胃的方法建立寒热证大鼠模型,正常对照组给予等体积的生理盐水灌胃。从第6 w开始在寒热证基础上采用1,2-二甲肼(DMH)溶液25 mg/kg颈后皮下注射,1次/w,连续12次诱导大肠癌模型。在造模期间寒热证模型组分别给予冰水灌胃10 mL/kg和30%乙醇溶液一直持续至第38 w。组织病理学分析采用苏木素-伊红(HE)、马森(Masson)和天狼星红染色对造模38 w的结肠组织切片进行观察;采用实时荧光定量聚合酶链式反应(PCR)和免疫组化法对结肠组织I型胶原(Col I)、III型胶原(Col III)、赖氨酰氧化酶相关蛋白-2(Loxl2)的mRNA和蛋白表达进行检测。结果:与正常对照组比较,热证模型组和寒证模型组大鼠结肠组织中的胶原密度明显增加;与热证模型组相比,寒证模型组大鼠结肠组织胶原的密度进一步增加;对胶原纤维化相关指标进行检测结果表明,与正常对照组相比,寒、热证模型组大鼠结肠组织中COL I、COLIII和LOXL2蛋白和mRNA表达明显上调(P<0.05或P<0.01);与热证模型组相比,寒证模型组COL I、COL III、LOXL2蛋白和mRNA表达更高(P<0.05或P<0.01)。结论:寒、热证不同宿体大肠癌发生发展的方式不同可能与胶原纤维化程度的差异有关。

Objective:This study aims to explore the different mechanisms for colorectal cancer in rats with Han(寒)syndrome and Re(热)syndrome from the perspective of extracellular matrix(ECM).Methods:A total of 70 male Wistar rats were randomized into three groups:Re(热)syndrome model group(RSM group,n=30),Han(寒)syndrome model group(HSM group,n=30),and control group(CK,n=10).Han(寒)syndrome was induced by oral administration of ice water(0℃)and ice water(10℃)bath,while Re(热)syndrome by oral administration of 30%ethanol and 0.9 mg/mL capsaicin.Rats in the CK were given(gavage)normal saline of equivalent volume.From the 6 th week,rats in the HSM group and RSM group were treated(posterior cervical subcutaneous injection)with 25 mg/kg 1,2-dimethylhydrazine(DMH)once a week for 12 weeks in a bid to stimulate colorectal cancer,and respectively received 0℃ice water and 30%ethanol to the 38 th week.Then,after hematoxylin-eosin(HE),Masson’s trichrome,and Picrosirius red staining,the colon sections of rats were observed.Immunohistochemistry and real-time fluorescent quantitative PCR were employed to examine the expression of collagen typeⅠ(COLⅠ),collagen typeⅢ(COLⅢ),and lysyl oxidase like-2 protein(LOXL2)in rat colon tissue.Results:The density of the collagen in colon tissue of both HSM group and RSM group was significantly larger than that in the CK,and the density in the HSM group was larger than that in the RSM group.As for the indexes related to collagen fibrosis,the mRNA and protein expression of COLⅠ,COLⅢ,and LOXL2 in rat colon tissue of HSM group and RSM group was higher than that of the CK(P<0.05 or P<0.01)and the expression in HSM group was higher than that in RSM group(P<0.05 or P<0.01).Conclusion:The different in occurrence and development of colorectal cancer between rats with Han(寒)syndrome and Re(热)syndrome may be related to the degree of collagen fibrosis.