非哺乳期乳腺炎大鼠模型的建立及评价  

Establishment and Evaluation of a Rat Model of Non-Puerperal Mastitis

作　　者：殷玉莲[1] 马丽娜 屠思远 陈玲 叶媚娜[1] 陈红风[1] YIN Yulian;MA Lina;TU Siyuan;CHEN Ling;YE Meina;CHEN Hongfeng(Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China)

机构地区：[1]上海中医药大学附属龙华医院,上海200032

出　　处：《实验动物与比较医学》2024年第6期587-596,共10页Laboratory Animal and Comparative Medicine

基　　金：国家自然科学基金青年项目“从促进巨噬源性泡沫细胞胆固醇跨膜逆转运探讨九一丹祛腐生肌的作用机制研究”(82104854);上海申康医院发展中心第二轮促进市级医院临床技能与临床创新三年行动计划(2020-2022年)重大临床研究项目“顾氏特色化腐清创法治疗难愈性非哺乳期乳腺炎的多中心临床研究”(SHDC2020CR2051B)

摘　　要：目的通过模拟高催乳素血症及免疫炎症状态,建立非哺乳期乳腺炎(non-puerperal mastitis,NPM)大鼠模型,评价其乳腺局部炎症特征,为乳腺疑难疾病的诊疗研究提供依据。方法选用SPF级Wistar雌性大鼠12只,均分为对照组和模型组。实验期间,对照组未接受任何实验性操作和药物处理;模型组连续7 d每日皮下注射1次盐酸甲氧氯普胺注射液100 mg/kg,在注射首日后第10、20、30天分别采用ELISA法检测血清催乳素水平。注射7 d后,将200μL哺乳期SD母鼠乳汁混合200μL完全弗氏佐剂制备为油包水乳剂,多点皮下注射至模型组大鼠背部进行首次免疫。首次免疫7 d后,再将油包水乳剂经多点皮下注射至大鼠第3、4、5对乳房进行末次免疫。末次免疫后连续观察乳房外观28 d,计算乳腺结节大小;并于第3、7、14、28天,通过HE染色分析乳腺组织形态学,免疫组织化学法检测CD138表达水平,ELISA法测定乳腺组织中白细胞介素(interleukin,IL)-6、IL-1β、肿瘤坏死因子(tumor necrosis factor,TNF)-α和诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)含量,以综合评价造模情况。结果模型组大鼠可见乳腺皮肤破溃,并于破溃处闻及恶臭味,触诊及超声下可见乳腺结节形成。HE染色显示,末次免疫后第3天大鼠乳腺组织中正常导管及小叶结构消失,并见大量浆细胞浸润;第7天可见乳腺导管扩张,导管上皮坏死脱落,管周浆细胞、淋巴细胞(以T淋巴细胞为主)浸润明显;第14天时可见大量纤维脂肪组织、小血管及肉芽组织增生,间质内可见散在浆细胞;第28天时炎性细胞浸润、纤维组织增生现象减轻,肉芽肿形成。大鼠在造模成功后第10天、第20天催乳素水平逐渐升高(P<0.05,P<0.001);模型组大鼠在末次免疫后第3、7、14、28天时,乳腺组织中IL-6和TNF-α含量均较对照组升高(P<0.05);IL-1β含量在末次免疫后第3、7、14天时较对照组升高(P<0.01),第2Objective This study aims to establish a non-puerperal mastitis(NPM)rat model by simulating hyperprolactinemia and immune-inflammatory states,and to evaluate its local inflammatory characteristics in the mammary gland,thereby laying the foundation for research on the diagnosis and treatment of this clinically challenging disease.Methods Twelve SPF-grade Wistar female rats were evenly divided into a control group and a model group.During the experiment,the control group received no experimental treatment or medication.The model group received daily subcutaneous injections of 100 mg/kg metoclopramide hydrochloride for 7 consecutive days.Serum prolactin(PRL)levels were measured using ELISA on the 10th,20th,and 30th days after the first injection.After 7 days of injections,200μL of lactating SD rat milk was mixed with 200μL of complete Freund's adjuvant to prepare an oil-in-water emulsion,which was administered by multiple subcutaneous injections into the back of the Wistar rats for the initial immunization.Seven days after the initial immunization,the emulsion was injected subcutaneously into the third,fourth,and fifth mammary glands for the final immunization.After the final immunization,the rats were observed for 28 days for changes in mammary gland appearance,and the size of mammary nodules was calculated.On the 3rd,7th,14th,and 28th days,hematoxylin-eosin(HE)staining was used to analyze mammary tissue morphology.Immunohistochemistry was employed to detect CD138 expression levels.ELISA was used to measure the levels of interleukin(IL)-6,IL-1β,tumor necrosis factor(TNF)-α,and inducible nitric oxide synthase(iNOS)in mammary tissue to comprehensively assess the model.Results Rats in the model group exhibited mammary skin ulceration and foul odor at the ulcer sites.Palpation and ultrasound revealed the formation of mammary nodules.HE staining showed that on the 3rd day after the final immunization,normal ductal and lobular structures in the mammary glands disappeared,with significant infiltration of plasma cells.

关 键 词：非哺乳期乳腺炎 WISTAR大鼠 催乳素 免疫炎症 

分 类 号：S857.26[农业科学—临床兽医学] Q95-33[农业科学—兽医学]