复合多因素法对血瘀型肝纤维化大鼠模型的建立和评价

Establishment and Evaluation of Compound Multifactor-induced Liver Fibrosis Rat Model with Blood Stasis Syndrome

目的:本研究在中医学辨证思维指导下,根据血瘀证理论探索和改良造模方法,采用多因素复合法建立血瘀型肝纤维化病证结合大鼠模型。方法:通过二甲基亚硝胺、小牛血清白蛋白和去甲肾上腺素注射液联合注射,外加乙醇灌胃及高脂低蛋白饲料喂养,多因素联合构建血瘀型肝纤维化大鼠模型。造模结束观察大鼠的死亡率,评价中医血瘀证证候,进行证候等级评分,观察肝脏病理学大体形态,检测肝功能改变,采用免疫组化法观察Ⅰ型和Ⅲ型胶原、α-平滑肌肌动蛋白(α-SMA)3种胶原蛋白的表达情况、放射酶免法血清学检测大鼠肝纤维化4项——HA、P3NP、LN、CⅣ的含量。将以上结果与传统四氯化碳(CCl4)单因素造模法进行比较。结果:血瘀证组:①大鼠的死亡率为20%;②大鼠出现瘀斑、舌紫暗、便溏等典型的中医血瘀证证候,血瘀证证候等级评分高;③病理学大体形态观察发现肝脏出现表面暗白、密布灰白结节、质硬脆等纤维样变化;④血清学肝功能检测发现模型大鼠的ALT和AST,以及TBIL、DBIL及IBIL的含量均显著增加;⑤肝脏Ⅰ型和Ⅲ型胶原、α-SMA 3种胶原蛋白的表达水平显著增高;⑥大鼠的血清肝纤维化4项——HA、P3NP、LN、CⅣ的含量均显著增加。与CCl4模型组相比,多因素复合法所致血瘀证模型组以上各项结果(除了死亡率及肝功能外)更为明显,结果有统计学差异(P<0.05)。结论:多因素复合造模方法能有效地改善CCl4造模法的高死亡率缺点;模型动物同时具备了疾病与证候双重特征,既符合中医血瘀证证候的基本特点,又与西医肝纤维化病理特征相一致。

Under the guidance of traditional Chinese medicine（TCM） syndrome differentiation,the liver fibrosis rat model with blood stasis syndrome induced by multifactor method was established according to the theory of blood stasis syndrome.The multifactor method,which included the injection of dimethyl nitrosamine,bovine serum albumin and norepinephrine,ethanol lavage and high-fat,low-protein feed,was used in the establishment of liver fibrosis rat model with blood stasis syndrome.After the establishment of rat model,the rat mortality was observed for the evaluation of TCM blood stasis syndrome.The syndrome rank was also scored.The pathology gross morphology of the liver was observed.Changes of liver function were detected.Immunohistochemical method was used to observe the expression levels of three collagens——the type Ⅰ and type Ⅲ collagen,and α-SMA.The radioactive enzyme immunoassay was used in the detection of the contents of HA,P3 NP,LN and C Ⅳ in liver fibrosis rats.Finally the results were compared to the traditional model establishment by the CC14 single factor method.The results showed that in the blood stasis syndrome group,the rat mortality was 20%.The rat presented with typical symptoms of TCM blood stasis syndrome,such as ecchymosis,dark purple tongue,loose stool and etc.The score of blood stasis syndrome ranked with high grade.The observation of pathology gross morphology of the liver showed fibrillation which included dark white surface,pale nodules,hard brittle texture,and etc.The serological liver function tests showed that contents of ALT,AST,TBIL,DBIL and IBIL were obviously increased in the model rat.The expression levels of type Ⅰ and type Ⅲ collagen,and α-SMA were obviously increased.The contents of HA,P3 NP,LN and C Ⅳ were obviously increased.Compared with the CCl4 model group,different results（except the rat mortality and liver function） of the compound multifactor-induced blood stasis syndrome model were more obvious with statistical differences（P〈0.05）.It was concluded that the compound multifactorinduced model can effectively improve the shortage of high mortality in CCl4 model establishment method.The animal model acquired both the characteristics of disease and syndrome,which was in accordance with the basic features of TCM blood stasis syndrome and the pathological features of liver fibrosis in modern medicine.