阴道萎缩大鼠模型的建立及在药效评价中的应用

doi:10.12300/j.issn.1674-5817.2022.014

实验动物与比较医学 ›› 2022, Vol. 42 ›› Issue (6): 531-540.DOI: 10.12300/j.issn.1674-5817.2022.014

阴道萎缩大鼠模型的建立及在药效评价中的应用

杨丽雅¹, 宋涛², 何佳璘³, 郭一鸣³, 亓明康³, 王含必³()(), 王慧萍¹()()

^1.国家卫生健康委科学技术研究所, 北京 100081
^2.潍坊市妇幼保健院, 潍坊 261021
^3.北京协和医院妇产科, 国家妇产疾病临床研究中心, 疑难重症及罕见病国家重点实验室, 中国医学科学院北京协和医学院, 北京 100730

收稿日期:2022-02-11 修回日期:2022-07-26 出版日期:2022-12-25 发布日期:2023-01-04
通讯作者: 王含必（1972—），女，博士，副主任医师，从事妇科内分泌疾病的诊疗研究。E-mail： zhw2005@aliyun.com。ORCID： 0000-0002-7961-1896；
王慧萍（1974—），女，博士，研究员，从事药理学研究。E-mail：wanghuiping@nrifp.org.cn。ORCID：0000-0002-5698-1691
作者简介:杨丽雅（1995—），女，硕士，技师，从事药理学研究。E-mail：yangliya@ nrifp.org.cn
基金资助:
国家卫生健康委科学技术研究所中央级公益性科研院所基本科研业务费专项资金资助：POI发病的网络调控机制及早期干预作用(2021GJZ08)

Establishment of a Vaginal Atrophy Rat Model and its Application in Pharmacodynamic Evaluation

Liya YANG¹, Tao SONG², Jialin HE³, Yiming GUO³, Mingkang QI³, Hanbi WANG³()(), Huiping WANG¹()()

^1.National Research Institute for Family Planning, Beijing 100081, China
^2.Weifang Maternal and Child Health Hospital, Weifang 261021, China
^3.Department of Obstetrics and Gynecology, National Clinical Research Center for Obstetric & Gynecologic Diseases, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100730, China

Received:2022-02-11 Revised:2022-07-26 Published:2022-12-25 Online:2023-01-04
Contact: WANG Hanbi (ORCID: 0000-0002-7961-1896), E-mail: zhw2005@aliyun.com
WANG Huiping （ORCID：0000-0002-5698-1691）, E-mail: wanghuiping@nrifp.org.cn

摘要/Abstract

摘要：

目的建立适宜筛选阴道用制剂药效学的动物模型，给予相应药物并评价监测药物治疗效果的生物学指标。方法采用大鼠双侧卵巢摘除术（ovariectomy，OVX）建模，观察OVX后15～21 d大鼠动情周期、子宫湿重和阴道组织形态学，判断术后阴道萎缩模型建立成功的最佳时间。SD雌性大鼠随机分为正常组、假手术组、模型组、药物1组、药物2组和溶剂对照组，每组10只。药物1组、药物2组和溶剂对照组每日分别阴道给予普罗雌烯软膏、更宝芬和等质量溶剂，连续14 d。末次给药后3 d测定大鼠体质量和子宫湿重，HE染色法分析阴道组织形态学，免疫组织化学法检测雌激素受体蛋白α（estrogen receptors-α，ERα）表达。结果 OVX后21 d已造成明显阴道萎缩，适宜进行后续实验。OVX后17～21 d无动情周期变化，术后21 d阴道上皮厚度显著降低（P<0.05），阴道褶皱变少，无鳞状上皮细胞覆盖；体质量上升（P<0.05），子宫湿重下降（P<0.05）。药物1组和药物2组阴道萎缩症状明显改善，给药组大鼠体质量较模型组下降（P<0.05），阴道组织形态学受损情况得到改善，组织ERα蛋白表达增加（P<0.05），子宫/体质量比上升（P<0.01），但子宫内膜厚度未明显增加（P>0.05）。结论大鼠OVX模型适宜用作筛选阴道用制剂，阴道组织形学态、子宫内膜厚度、子宫/体质量比可作为监测药物疗效的指标。

关键词: 阴道制剂, 阴道萎缩, 去卵巢大鼠模型, 药效评价

Abstract:

Objective To establish an animal model suitable for screening vaginal preparations, administering the corresponding drugs, and evaluating biological indicators for monitoring drug treatment effects. Methods Bilateral ovariectomy (OVX) was used to establish the disease model of rat. The estrous cycle, uterine wet weight, and vaginal tissue morphology were observed from day 15 to day 21 after OVX to determine the optimal time for successful vaginal atrophy model after the OVX operation. Sprague-Dawley female rats were randomly divided into the following groups: normal group, sham group, model group, drug group 1, drug group 2, and solvent control group (n=10). Drug group 1, drug group 2, and solvent control group were treated with promestriene, Colpotrofin?, and solvent control for 14 days, respectively. Three days after the last administration, the body weight and uterus wet weight of the rats were measured, the histological morphology of the vagina was analyzed by HE staining, and estrogen receptors-α (ERα) expression was detected by immunohistochemistry. Results Twenty-one days after OVX, the vaginal atrophy model was established, which was suitable for the follow-up experiments. The change of estrous-cycle wasn't observed from day 17 to 21 after OVX. On 21 days after OVX, the vaginal epithelium thickness reduced (P<0.05), vaginal folds reduced, no squamous epithelium was observed, body weight increased (P<0.05), and uterine wet weight decreased (P<0.05). In drug group 1 and drug group 2, the symptoms of vaginal atrophy improved. Compared with the model group, the body weight of rats in drug group 1 and 2 decreased (P<0.05), the vaginal histomorphology improved, the expression of ERα protein in vaginal tissue up-regulated (P<0.05), and the ratio of uterine wet weight to body weight increased (P<0.01), but the endometrial thickness was not significantly thickened (P>0.05). Conclusion The rat OVX model is suitable for screening vaginal preparations. Vaginal histomorphology, endometrial thickness, and uterine/body weight ratio can be used as indicators to monitor drug efficacy.

Key words: Vaginal preparations, Vaginal atrophy, Ovariectomized rat model, Pharmacodynamic evaluation

中图分类号:

R-332

杨丽雅, 宋涛, 何佳璘, 郭一鸣, 亓明康, 王含必, 王慧萍. 阴道萎缩大鼠模型的建立及在药效评价中的应用[J]. 实验动物与比较医学, 2022, 42(6): 531-540.

Liya YANG, Tao SONG, Jialin HE, Yiming GUO, Mingkang QI, Hanbi WANG, Huiping WANG. Establishment of a Vaginal Atrophy Rat Model and its Application in Pharmacodynamic Evaluation[J]. Laboratory Animal and Comparative Medicine, 2022, 42(6): 531-540.

图/表 7

图1 各组大鼠在双侧卵巢摘除术（OVX）后的阴道涂片（瑞氏染色，×40）（A和B）及动情间期所占比例（C）注：A为正常组大鼠的阴道涂片可见动情周期规律变化（a～d分别显示动情前期、动情期、动情后期和动情间期）。B为模型建立组大鼠OVX后的阴道涂片（a～d分别为18、19、20、21 d，均显示动情间期）。C为模型建立组（21只）以及模型组、药物1组、药物2组和溶剂对照组（每组各10只）大鼠在OVX后7~21 d的动情间期所占比例（n=61）。图中比例尺大小均为50 μm。

Figure 1 Vaginal smears after ovariectomy （OVX） of rats in each group （Wright's staining， ×40）（A and B）and the proportion of the diestrus period （C）Note: A, regular changes in the estrous cycle were observed in the vaginal smears of the normal group (a–d show pre-estrous, estrous, post-estrous, and diestrus periods, respectively). B, vaginal smears after OVX in the model establishment group (a–d was 18, 19, 20, and 21 days after OVX, respectively, showing the diestrus period). C, the proportion of estrous/diestrus on days 7–21 after OVX in the model establishment group (21 rats), model group, drug group 1, drug group 2 and solvent control group (10 rats in each group) (n=61). Scale bars in all panels are 50 μm.

图2 双侧卵巢摘除术（OVX）后大鼠体质量（A）和子宫/体质量比（B）变化注：A为正常对照组和模型建立组大鼠15～21 d的体质量变化；B为模型建立组大鼠OVX后15～21 d的子宫/体质量变化。每日大鼠数量n=3，与模型建立组术后21 d相比，*P<0.05。

Figure 2 Body weight (A) and uterine wet weight/body weight (B) of rats from day 15–21 after ovariectomy (OVX)Note: A, weight changes of rats in the normal control and model establishment groups during days 15–21; B, changes in uterus wet weight/body weight at days 15–21 after OVX in the model establishment group. Daily number of rats n=3. *P<0.05, compared with the model establishment group.

图3 卵巢摘除术（OVX）后大鼠阴道组织病理变化（HE染色，×10）和阴道上皮厚度注：A为模型建立组大鼠OVX后15～21 d和正常组大鼠的阴道组织HE染色结果（图中比例尺大小为100 μm）；B为模型建立组大鼠OVX后15～21 d的阴道上皮厚度（n=3，与术后21 d相比，*P<0.05）。

Figure 3 Histopathological changes of vaginal in rats after ovariectomy (OVX) and the thickness of vaginal epitheliumNote：A, HE staining of vaginal tissues of rats in the model establishment and normal groups days 15–21 after OVX (scale bar is 100 μm); B, the thickness of the vaginal epithelium in the model establishment group days 15–21 after OVX (n=3, compared with 21 days after OVX, *P<0.05).

图4 用药后各组大鼠体质量（A）、子宫湿重（B）和子宫/体质量（C）变化注：药物1组、药物2组和溶剂对照组每日分别阴道给予普罗雌烯软膏、更宝芬和等质量溶剂，连续14 d。每组大鼠n=10。与假手术组相比，***P<0.001；与模型组相比，#P<0.05，##P<0.01，###P<0.001。

Figure 4 Body weight (A), uterine wet weight (B), and uterine wet weight/ body weight (C) of rats in each group after drug treatmentNote：Drug group 1, drug group 2, and solvent control group were treated with promestriene, Colpotrofin?, and solvent control for 14 days, respectively.In each group, n=10. ***P<0.001, compared with the sham group; #P<0.05, ##P<0.01, and ###P<0.001, compared with the model group.

图5 用药后各组大鼠子宫组织病理变化（HE染色， ×2）（A）和子宫内膜厚度（B）注：a为正常组；b为假手术组；c为模型组；d为溶剂对照组；e为药物1组（普罗雌烯软膏）；f为药物2组（更宝芬）。图中比例尺大小为1 mm。每组大鼠n=10。与假手术组相比，***P<0.001。

Figure 5 Histopathological changes of uterus tissue（HE, ×2）(A) and thickness of the endometrium (B)Note：a, normal group; b, sham group; c, model group; d, solvent control group; e, drug group 1（promestriene）; f, drug group 2 （Colpotrofin?）. The scale bar of the panel is 1 mm. In each group, n=10. ***P<0.001, compared with the sham group.

图6 用药后各组大鼠阴道组织病理变化（HE， ×20）（A）和阴道上皮厚度统计（B）注：a为正常组；b为假手术组；c为模型组；d为溶剂对照组；e为药物1组（普罗雌烯软膏）；f为药物2组（更宝芬）。图中比例尺大小为100 μm。与假手术组相比，*P<0.05， **P<0.01；与模型组相比，###P<0.001。

Figure 6 Histopathological changes of vaginal tissue (HE, ×20)(A) and thickness of the vaginal epithelium (B)Note：a, normal group; b, sham group; c, model group; d, solvent control group; e, drug group 1（promestriene）; f, drug group 2 （Colpotrofin?）. The scale bar of the panel is 100 μm. *P<0.05, and **P<0.01, compared with the sham group; ###P<0.001, compared with the model group.

图7 大鼠阴道组织中ERα蛋白表达的免疫组织化学染色结果（DAB染色，×20）（A）和积分光密度值分析（B）注：ERα：雌激素受体蛋白α。a为正常组；b为假手术组；c为模型组；d为溶剂对照组；e为药物1组（普罗雌烯软膏）；f为药物2组（更宝芬）。图中比例尺大小为20 μm。与假手术组相比，*P<0.05；与模型组相比，#P<0.05。

Figure 7 Immunohistochemistry (DAB, ×20）(A) and integral optical density (B) of ERα protein expression in rat vaginaNote：ERα, estrogen receptors-α. a, normal group; b, sham group; c, model group; d, solvent control group; e, drug group 1（promestriene）; f, drug group 2 （Colpotrofin?）. The scale bar of the panel is 20 μm. *P<0.05, compared with the sham group; #P<0.05, compared with the model group.

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阴道萎缩大鼠模型的建立及在药效评价中的应用

Establishment of a Vaginal Atrophy Rat Model and its Application in Pharmacodynamic Evaluation

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