实验动物与比较医学 ›› 2024, Vol. 44 ›› Issue (6): 587-596.DOI: 10.12300/j.issn.1674-5817.2024.065

• 人类疾病动物模型 •    下一篇

非哺乳期乳腺炎大鼠模型的建立及评价

殷玉莲(), 马丽娜, 屠思远, 陈玲, 叶媚娜, 陈红风()()   

  1. 上海中医药大学附属龙华医院, 上海 200032
  • 收稿日期:2024-05-08 修回日期:2024-11-13 出版日期:2025-01-04 发布日期:2024-12-25
  • 通讯作者: 陈红风(1964—),女,博士,主任医师,上海市名中医,研究方向:乳腺良恶性疾病的临床及实验研究。E-mail: chhfluk2068@163.com。ORCID:0000-0001-6311-0651
  • 作者简介:殷玉莲, 副主任医师,医学博士,硕士生导师,海派顾氏外科第六代传承人,师从上海市名中医陈红风教授,上海市卫生健康系统“陈红风名中医劳模创新工作室”骨干成员。近5年发表论文33篇,其中英文8篇;授权国家发明专利1项、实用新型专利5项,软件著作权2项;先后入选上海市“医苑新星”青年医学人才培养计划、上海市科委“扬帆计划”,主持并参与国家自然科学基金青年项目、上海卫健委临床专项、上海市中医药发展三年行动计划等多项课题。主要研究方向为中医药防治乳腺炎症性疾病、乳腺癌及其共病 (心脏、骨、脑) 的临床和基础研究。现担任上海市中医药学会中医乳腺病分会青年委员会副主任委员、中华中医药学会精准医学分会青年委员等。
    陈红风,主任医师,二级教授,医学博士,博士生导师,海派顾氏外科第五代传承人,师从中医外科名家陆德铭教授。现为全国先进工作者、上海市名中医、第七批全国老中医药专家学术经验继承工作指导老师、全国优秀中医临床人才、国家中医药管理局重点学科中医乳腺病学学科带头人、上海中医药大学中医外科学学术带头人。主攻中医药防治非哺乳期乳腺炎、转移性乳腺癌、乳腺增生病等的临床和实验研究,主持国家级和省部级科研项目20余项,成果获教育部科技进步二等奖、中华中医药学会科学技术奖一等奖等。主编全国高等中医院校规划教材《中医外科学》《中医外科学临床研究》《实用中医乳房病学》等专著30余部。发表论文近百篇,其中SCI收录20余篇。现任上海市中医药学会乳腺病分会主任委员、中华中医药学会乳腺病分会副主任委员、中国中医药研究促进会乳腺病专业委员会副主任委员、上海市中西医结合学会乳腺病专委会顾问等。
    陈红风,主任医师,二级教授,医学博士,博士生导师,海派顾氏外科第五代传承人,师从中医外科名家陆德铭教授。现为全国先进工作者、上海市名中医、第七批全国老中医药专家学术经验继承工作指导老师、全国优秀中医临床人才、国家中医药管理局重点学科中医乳腺病学学科带头人、上海中医药大学中医外科学学术带头人。主攻中医药防治非哺乳期乳腺炎、转移性乳腺癌、乳腺增生病等的临床和实验研究,主持国家级和省部级科研项目20余项,成果获教育部科技进步二等奖、中华中医药学会科学技术奖一等奖等。主编全国高等中医院校规划教材《中医外科学》《中医外科学临床研究》《实用中医乳房病学》等专著30余部。发表论文近百篇,其中SCI收录20余篇。现任上海市中医药学会乳腺病分会主任委员、中华中医药学会乳腺病分会副主任委员、中国中医药研究促进会乳腺病专业委员会副主任委员、上海市中西医结合学会乳腺病专委会顾问等。E-mail: chhfluk2068@163.com。ORCID:0000-0001-6311-0651
  • 基金资助:
    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)

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

YIN Yulian(), MA Lina, TU Siyuan, CHEN Ling, YE Meina, CHEN Hongfeng()()   

  1. Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China
  • Received:2024-05-08 Revised:2024-11-13 Published:2024-12-25 Online:2025-01-04
  • Contact: CHEN Hongfeng (ORCID:0000-0001-6311-0651), E-mail: chhfluk2068@163.com

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200 μL哺乳期SD母鼠乳汁混合200 μL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28天时略低于对照组(P>0.05);iNOS在末次免疫后第7天显著高于对照组(P<0.001)。 结论 成功构建大鼠NPM模型,其乳房局部具有肿块、脓肿、溃疡等表现以及乳腺导管扩张和浆细胞浸润的典型病理特征,可为该临床疑难疾病的诊疗研究奠定基础。

关键词: 非哺乳期乳腺炎, Wistar大鼠, 催乳素, 免疫炎症

Abstract:

Objective 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, 200 μL of lactating SD rat milk was mixed with 200 μL 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. On the 7th day, ductal dilation, epithelial necrosis and detachment, and pronounced periductal plasma cell and lymphocyte (predominantly T-lymphocytes) infiltration were observed. On the 14th day, there was a proliferation of fibrofatty tissue, small blood vessels, and granulation tissue, with scattered plasma cells in the interstitium. By the 28th day, inflammatory cell infiltration and fibrous tissue proliferation were reduced, with granuloma formation. Serum PRL levels in the model group were significantly increased on the 10th day (P<0.05) and the 20th day (P<0.001). IL-6 and TNF-α levels in mammary tissue were higher in the model group compared to the control group on the 3rd, 7th, 14th, and 28th days (P<0.05). IL-1β levels were higher on the 3rd, 7th, and 14th days compared with the control group (P<0.01) but lower than the control group on the 28th day (P>0.05). iNOS levels were significantly higher on the 7th day after the final immunization (P<0.001). Conclusion A successful NPM model was established in rats, which exhibited typical pathological features such as local mammary masses, abscesses, ulcers, ductal dilation and plasma cell infiltration. This model can serve as a foundation for further research into the diagnosis and treatment of this clinically challenging disease.

Key words: Non-puerperal mastitis, Wistar rats, Prolactin, Immune inflammation

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