摘要
目的比较环磷酰胺(CTX)不同剂量和停药周期构建的小鼠卵巢早衰(POF)动物模型,寻找最佳造模方式,为进一步研究POF提供更为适用的实验模型。方法8周龄动情周期规律的C57BL/6雌性小鼠共40只,随机分成5组:CON(对照组),CTX80(80 mg·kg^(-1)·d^(-1)),CTX100(100 mg·kg^(-1)·d^(-1)),CTX120(120 mg·kg^(-1)·d^(-1)),CTX150(150 mg·kg^(-1)·d^(-1)),连续腹腔注射10 d,CON组注射生理盐水,其余组注射不同剂量CTX。记录小鼠体重、动情周期和卵巢脏器系数等,比较得出CTX诱导POF模型最佳暴露剂量。使用最佳CTX暴露剂量重新造模,随机分为模型组和对照组,分别在停药后第1、2、3、4周,检测血清性激素,计数各级卵泡等,比较得出CTX诱导POF模型最佳停药周期。结果(1)研究最佳造模剂量时,CTX150死亡2只,而CTX80小鼠仍有完整的动情周期,说明这两种剂量并非最佳。CTX各组在暴露过程中体重呈进行性下降,停止暴露后体重开始回升,但至停药第7天各组体重仍显著低于CON组(P<0.05)。停药第7天各CTX组卵巢脏器系数均显著低于CON组(P<0.01),CTX100最低(P<0.001),但各CTX组间差异无统计学意义(P>0.05)。考虑到尽量缩小药物对实验动物伤害的伦理要求,得出CTX诱导卵巢早衰模型的最佳暴露剂量为100 mg·kg^(-1)·d^(-1)×10 d。(2)研究最佳停药周期时,模型组停药后第1、2、3、4周血清抗苗勒管激素(AMH)水平、雌二醇(E_(2))水平、原始卵泡、初/次级卵泡均显著低于同期对照组(P<0.05),卵泡刺激素(FSH)水平在第1、3、4周均显著高于同期对照组(P<0.05),而第1、2、4周的闭锁卵泡计数均显著高于同期对照组(P<0.05),均表现出POF典型特征。随CTX停药周期延长,模型组小鼠原始卵泡、初/次级卵泡、成熟卵泡计数呈进行性下降,闭锁卵泡计数升高(P<0.05),CTX对卵巢功能损害呈进行性加重。比较得出CTX停药1周是诱导POF模型最佳成模周期。结论CTX100 mg·kg^(-1)·d^(-1)连续暴露10 d是建立CTX诱导小鼠POF模型的最佳造模剂量,最佳成模周期为停药后1周。
Objective:To compare the animal models of premature ovarian failure(POF)in mice constructed by cyclophosphamide(CTX)with different doses and withdrawal cycles for exploring the best model method,so as to provide a more suitable experimental model for further research on premature ovarian failure.Methods:Female C57BL/6 mice with a regular estrous cycle in 8-week-old were randomly divided into five groups:CON group(100 mg·kg^(-1)·d^(-1)),CTX80 group(80 mg·kg^(-1)·d^(-1)),CTX100 group(100 mg·kg^(-1)·d^(-1)),CTX120 group(120 mg·kg^(-1)·d^(-1)),CTX150 group(150 mg·kg^(-1)·d^(-1)).The mice underwent intraperitoneal injection for 10 days,those of the CON group were injected with saline and those in other groups were injected with CTX.The body weight,estrous cycle and ovarian index of the mice were recorded,and the optimal exposure dose of CTX in the model of CTX-induced POF was determined by comparison.Subsequently,female C57BL/6 mice with a regular estrous cycle in 8-week-old that were remodeled with the optimal exposure dose of CTX and randomly divided into the model group and the control group.In the 1st,2nd,3rd,and 4th weeks after drug withdrawal,serum levels of sex hormones were measured,and follicles at all levels were counted by HE staining of the ovaries,which were compared to derive the optimal withdrawal cycle for the CTX-induced POF model.Results:(1)Two mice in CTX150 group died during the modeling process,those in CTX80 group still had a full estrous cycle,which indicating that these two doses were not optimal.Mice in other CTX groups showed a progressive decrease in body weight during the exposure process.The body weight of the mice began to rebound after the cessation of the exposure,but they were still lower than that of CON group on the 7th day of the cessation of cyclophosphamide(P<0.05).The ovarian indexes in CTX groups were significantly lower than that of CON group(P<0.01)and that of CTX100 was the lowest on day 7 of drug cessation(P<0.001),but the differences among CTX groups were not statistically significant(P>0.05).The ethical requirement of minimizing the harm of the drug to the experimental animal was taken into account,which made a conclusion that the optimal cyclophosphamide exposure dose for the cyclophosphamide-induced premature ovarian failure model was 100 mg·kg^(-1)·d^(-1)×10 d.(2)Compared with the control group,serum levels of anti-Müllerian hormone,estradiol,primordial follicle,and primordial/secondary follicle in the model group were significantly lower than that in the control group in the same period at the 1st,2nd,3rd,and 4th weeks after discontinuation of the drug(P<0.05).The levels of follicular stimulating hormone were significantly higher(P<0.05)in the 1st,3rd,and 4th weeks than those of the control group in the same period,while atretic follicle counts were significantly higher(P<0.05)in the 1st,2nd,and 4th weeks than those of the control group in the same period,which all showed the typical characteristics of POF.With the prolongation of CTX withdrawal cycle,the counts of primordial follicles,primary/secondary follicles and mature follicles in the model group of mice showed a progressive decrease,and the counts of atretic follicles increased(P<0.05),indicating that the damage of ovarian function caused by cyclophosphamide was progressively aggravated.The results showed that cyclophosphamide cessation in the 1st week was the optimal modeling cycle for inducing premature ovarian failure.Conclusions:The evidence suggests that 100 mg·kg^(-1)·d^(-1) of cyclophosphamide using for 10 d is the optimal modeling dose to establish a cyclophosphamide-induced premature ovarian failure model in mice,and the optimal modeling cycle is the 1st week after rescission of the cyclophosphamide.
作者
莫金桦
胡鸿
颜秋霞
李鹏东
陈彩蓉
MO Jin-hua;HU Hong;YAN Qiu-xia;LI Peng-dong;CHEN Cai-rong(Department of Reproductive Medicine Center,the Qingyuan Affiliated Hospital of Guangzhou Medical University(Qingyuan People’sHospital),Qingyuan 511500;Department of Laboratory Center,the Qingyuan Affiliated Hospital of Guangzhou Medical University(Qingyuan People’s Hospital),Qingyuan 511500)
出处
《生殖医学杂志》
CAS
2024年第3期365-372,共8页
Journal of Reproductive Medicine
基金
清远市人民医院开放课题项目(202301-104)
清远市人民医院医学科研基金项目(201904-10)
广东省医学科学技术研究基金项目(B2023064)。
关键词
卵巢早衰
动物模型
环磷酰胺
Premature ovarian failure
Animal model
Cyclophosphamide
