环磷酰胺诱导生精障碍小鼠模型不同恢复期的比较研究_马婧威.pdf

实验动物与比较医学 Laboratory Animal and Comparative MedicineApr.2023,43(2)DOI:10.12300/j.issn.1674-5817.2022.167张 丹，中国协和医科大学药理学博士，曾在美国国立卫生研究院（National Institutes of Health，NIH）从事博士后研究工作4年，现为中国医学科学院北京协和医学院药物研究所天然药物活性物质与功能国家重点实验室、新药作用机制研究与药效评价北京市重点实验室研究员，博士生导师。主要研究方向包括：（1）精神、神经系统疾病的机制研究及相关治疗药物的研发；（2）炎症免疫相关疾病的机制研究及治疗药物研发；（3）活性天然产物的生物靶标鉴定与作用机制研究；（4）肠道微生物在多种疾病进程中的机制及药物研发。发表SCI论文17篇，累计影响因子136分，发表期刊包括Acta Pharmaceutica Sinica B、Redox Biol、Neurobiology of Aging、J Med Chem、Brain Behav Immun等；参编专著3部；获批专利11项；获得2013年度“教育部新世纪优秀人才”、2012年北京市委组织部评选的“北京市优秀人才”等称号。环磷酰胺诱导生精障碍小鼠模型不同恢复期的比较研究马婧威,李根,杨杨,臧彩霞,鲍秀琦,张丹(中国医学科学院北京协和医学院药物研究所,天然药物活性物质与功能国家重点实验室,北京 100050)摘要 目的比较并评价环磷酰胺造模后不同恢复期小鼠生精障碍的改善程度。方法将48只45周龄体质量为1820 g的ICR雄性小鼠随机分为3个对照组和3个模型组，每组8只。3个模型组的每只小鼠于实验第17天连续腹腔注射环磷酰胺60 mg/kg，处理结束后分别继续饲养7 d、14 d和21 d。每个模型组均设相应的对照组，3个对照组小鼠腹腔注射对应体积的生理盐水。各组小鼠在相应时间点通过眼眶静脉采血后处死，取睾丸、附睾和精囊，称重后计算其生殖器官指数。HE染色后比较睾丸和附睾组织病理学变化，精子质量分析测定精子相关指标。ELISA法及相关试剂盒检测血清生殖激素含量、睾丸氧化应激水平和睾丸标志性酶活性变化。结果与对照组相比，环磷酰胺处理结束后7 d、14 d和21 d的模型组小鼠睾丸指数均明显下降（P0.01），7 d、14 d的附睾指数和7 d、21 d的精囊指数明显下降（P0.05）；且随着时间延长，模型组睾丸和附睾组织病理学损伤逐渐减轻。环磷酰胺处理结束后 7 d、14 d的模型组精子数量明显减少（P0.01），血清睾酮水平降低（P0.05），睾丸中丙二醛含量明显增多（P0.01），还原型谷胱甘肽含量和超氧化物歧化酶含量明显降低（P0.05），睾丸标志性酶乳酸脱氢酶活力明显下降（P0.05），-谷氨酰转肽酶活力明显上升（P0.05）；其中7 d的模型组小鼠精子活力明显下降（P0.001），精子畸形率明显上升（P0.05），血清促卵泡素和促黄体生成素明显增加（P0.01）。21 d的模型组小鼠精子相关指标、血清生殖激素水平、睾丸氧化应激水平和睾丸标志性酶活性变化均不明显（均P0.05）。结论环磷酰胺60 mg/kg连续腹腔注射7 d，注射结束后7 d所致小鼠生殖毒性较为明显，能够构建较理想的小鼠生精障碍模型；而随着恢复期的延长，至环磷酰胺处理结束后21 d小鼠生精障碍各项指标逐渐恢复，接近正常水平。关键词 环磷酰胺；生精障碍；恢复期；ICR小鼠中图分类号 R332；R698 文献标志码 A 文章编号 1674-5817（2023）02-0112-12Comparative Study on Different Recovery Periods of the Spermatogenic Dysfunction Mouse Model Induced by CyclophosphamideMA Jingwei,LI Gen,YANG Yang,ZANG Caixia,BAO Xiuqi,ZHANG Dan(State Key Laboratory of Natural Products and Functions,Institute of Materia Medica,Chinese Academy of Medical Sciences&Peking Union Medical College,Beijing 100050,China)人类疾病动物模型 Animal Models of Human Diseases基金项目 国家重点研发计划项目（2018YFA0901900）；中国医学科学院医学科学创新基金项目（2021-I2M-1-028）第一作者 马婧威（1998），女，博士研究生，研究方向：新药活性评价及作用机制研究、神经药理学。E-mail：通信作者 张丹（1978），女，教授，博士生导师，研究方向：新药研发与神经退行性疾病发病机制研究。E-mail：。ORCID：0000-0001-9792-4549112实验动物与比较医学 Laboratory Animal and Comparative MedicineApr.2023,43(2)Correspondence to:ZHANG Dan(ORCID:0000-0001-9792-4549),E-mail:ABSTRACT Objective To compare and evaluate the improvement degree of spermatogenic dysfunction mice at different recovery periods after cyclophosphamide modeling.MethodsForty-eight male ICR mice aged 4-5 weeks with the body weight of approximately 18-20 g were randomly divided into three control groups and three model groups,with 8 mice in each group.Each mouse of three model groups was intraperitoneally injected with 60 mg/kg cyclophosphamide continuously from the 1st to 7th day of the experiment,while each mouse of three control groups was intraperitoneally injected with the corresponding volume of normal saline.Then these mice were continued to be fed for another 7,14 and 21 days after cyclophosphamide injection,respectively.A corresponding control group was set for each model group.The mice in each group were sacrificed after blood collection through orbital veins at corresponding time points.Testis,epididymis and seminal vesicle were taken and weighed,and their reproductive organ indexes were calculated.Histopathological changes of testis and epididymis were compared after HE staining.Sperm quality analysis was used to determine sperm-related indexes.Serum reproductive hormone content,testicular oxidative stress level and testicular signature enzyme activity were detected by ELISA and related kits.Results Compared with the control group,on the 7th,14th and 21st day after cyclophosphamide treatment,the testicular index of mice in the model group decreased significantly(P0.01).The epididymis index decreased significantly on the 7th and 14th day,and the seminal vesicle index decreased obviously on the 7th and 21st day(P0.05).And the histopathological damage of testis and epididymis of the model group gradually alleviated over time.On the 7th and 14th day after cyclophosphamide treatment,the sperm count of the model group declined remarkably(P0.01),the serum testosterone(T)level reduced(P0.05),the malonaldehyde(MDA)content of testis increased significantly(P0.01),the content of reduced glutathione(GSH)and superoxide dismutase(SOD)decreased obviously(P0.05),the lactic dehydrogenase(LDH)activity of testis reduced obviously(P0.05),the gamma-glutamyl transpeptidase(-GT)activity increased significantly(P0.05),the latter two of which are important testicular signature enzymes.Therein on the 7th day after cyclophosphamide treatment,the sperm motility decreased significantly(P0.001),the rate of sperm malformation increased obviously(P0.05),the serum levels of follicle-stimulating hormone(FSH)and luteinizing hormone(LH)increased notably(P0.01).Nevertheless on the 21st day after cyclophosphamide treatment,the sperm-related indexes,the content of serum reproductive hormone,the level of testicular oxidative stress