五种卵巢储备功能减退大鼠模型的表型比较

doi:10.12300/j.issn.1674-5817.2025.205

摘要/Abstract

摘要：

目的以生理性卵巢储备功能减退（diminished ovarian reserve，DOR）的围绝经期大鼠为参照，观察比较不同病理性DOR大鼠模型的表型。方法将动情周期正常的8周龄雌性SD大鼠，随机分为对照组、环磷酰胺（cyclophosphamide，CTX）单次组、CTX两次组、CTX多次组、雷公藤组和制动+左甲状腺素钠片（levothyroxine sodium tablet，LTS）组，每组6只，分别采用不同剂量CTX腹腔注射、雷公藤灌胃、重复制动应激联合LTS灌胃造模。另取6只11月龄雌性SD大鼠作为围绝经期组（阳性对照组）。造模期间观察大鼠的阴道脱落细胞与体重，造模结束后ELISA法检测大鼠血清促卵泡激素（follicle-stimulating hormone，FSH）、黄体生成素（luteinizing hormone，LH）、雌二醇（estradiol，E₂）和抗苗勒氏管激素（anti-Müllerian hormone，AMH）浓度，计算卵巢、子宫指数，HE染色后观察卵巢组织病理改变与卵泡计数。结果不同造模方法均可造成大鼠体重增长减慢，动情周期紊乱，卵巢髓质萎缩，皮质增生，颗粒细胞减少，排列疏松，甚至空泡，原始、成熟卵泡减少（P＜0.05），而次级卵泡和闭锁卵泡显著增加（P＜0.05）。各模型组大鼠卵巢、子宫重量与指数无明显变化（P＞0.05）。各模型组大鼠血清LH水平都显著升高（P＜0.05），但血清FSH、E₂和AMH水平在不同模型组变化不同，有的显著升高，有的显著降低（P＜0.05）。结论本研究中的造模方法均可成功构建DOR动物模型。同一药物、不同给药剂量与给药频次，或不同药物、不同造模方法损伤卵巢功能的程度与机制可能不同。模型评价需要综合临床表型、卵巢组织病理与血清生化指标。重复制动应激联合LTS灌胃动物模型最接近围绝经期大鼠模型，可能更符合临床大多数DOR患者的发病特点，值得进一步深入研究。

关键词: 卵巢储备功能减退, 环磷酰胺, 雷公藤, 重复制动应激, 左甲状腺素钠片, 大鼠

Abstract:

Objective Using perimenopausal rats with physiological ovarian reserve dysfunction as the reference, the phenotypes of different pathological ovarian reserve dysfunction (diminished ovarian reserve, DOR) rat models were observed and compared. Methods Eight-week-old female Sprague-Dawley (SD) rats with regular estrous cycles were randomly assigned to six groups: control group, single-dose cyclophosphamide (CTX) group, double-dose CTX group, multiple-dose CTX group, Tripterygium wilfordii group, and restraint stress + levothyroxine sodium tablet (LTS) group, with 6 rats per group. Models were established via intraperitoneal injection of CTX at varying doses, intragastric administration of Tripterygium wilfordii, or repeated behavioral stress combined with intragastric LTS. Additionally, 6 eleven-month-old female SD rats were included as a perimenopausal group(positive control group). During the modeling period, vaginal exfoliated cells and body weight were monitored. Post-modeling, serum concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E₂), and anti-Müllerian hormone (AMH) were measured by enzyme-linked immunosorbent assay(ELISA); ovarian and uterine indices were calculated; and ovarian histopathological changes and follicle counts were evaluated by hematoxylin-eosin staining. Results Different modeling methods can all cause a slowdown in the weight gain of rats, disruption of the estrous cycle, atrophy of the ovarian medulla, hyperplasia of the cortex, reduction of granulosa cells, loose arrangement, and even vacuolation. The number of primordial and mature follicles decreased (P<0.05), while the number of secondary follicles and atretic follicles significantly increased (P<0.05). There were no significant changes in the weight and index of the ovaries and uterus in each model group (P> 0.05). The serum LH levels in each model group were significantly increased (P<0.05), but the serum FSH, E₂, and AMH levels were significantly increased in some groups and significantly decreased in others (P<0.05). Conclusions The modeling methods used in this study can all successfully establish DOR animal models. The same drug, different dosages and frequencies of administration, or different drugs and different modeling methods may result in different degrees and mechanisms of ovarian function impairment. Model evaluation requires a comprehensive consideration of clinical phenotypes, ovarian histopathology, and serum biochemical indicators. The repeated animal model of stress combined with LTS gavage is the closest to the perimenopausal rat model and may better conform to the disease characteristics of the majority of DOR patients in clinical practice, which is worthy of further in-depth study..

Key words: Diminished ovarian reserve, Cyclophosphamide, Tripterygium wilfordii, Repeated braking stress, Levothyroxine sodium tablet, Rat

中图分类号:

R-332

李丽美,黄雪萍,陈建清,等.五种卵巢储备功能减退大鼠模型的表型比较[J]. 实验动物与比较医学.. DOI: 10.12300/j.issn.1674-5817.2025.205.

LI Limei,HUANG Xueping,CHEN Jianqing,et al. Comparison of Five Methods for Induction of Model Rat with Diminished Ovarian Reserve[J]. Laboratory Animal and Comparative Medicine. DOI: 10.12300/j.issn.1674-5817.2025.205.

图/表 6

表1 各组大鼠动情周期变化 (n(%)；n=6)

Table 1 Changes of estrous cycle of rats in each group

组别 Group	动情周期 Estrous cycle			χ² 值 χ² value	P值 P value
组别 Group	正常 Regular	紊乱 Dsorder	延长 Delayed	χ² 值 χ² value	P值 P value
对照组 Control group	6(100%)	0(0%)	0(0%)
CTX单次组 Single-dose CTX group	2(33.3%)	3(50.0%)	1(16.7%)	6.00	0.05
CTX两次组 Double-dose CTX group	2(33.3%)	3(50.0%)	1(16.7%)	6.00	0.05
CTX多次组 Multiple-dose CTX group	1(16.7%)	5(83.3%)	0(0%)	8.57	0.003^*
雷公藤组 Tripterygium wilfordii group	1(16.7%)	4(66.7%)	1(16.7%)	8.57	0.014^*
制动+LTS组 Restraint stress +LTS group	2(33.3%)	3(50.0%)	1(16.7%)	6.00	0.05
围绝经期组 Perimenopausal group	1(16.7%)	3(50.0%)	2(33.3%)	8.57	0.014^*

表2 各组大鼠每周体重增长率比较 (

x ˉ ± s

, n=6)

Table 2 Comparison of weekly body weight growth rate in each group

组别 Group	体重增长率/% Growth rate of body mass/%
组别 Group	第1周 Week 1	第2周 Week 2	第2周－第1周 Week 2-week 1
对照组 Control group	9.58±2.67^#●○♥	19.49±5.79^{#●○♥♣}	9.92±7.62^{#●○♥♣}
CTX单次组 Single-dose CTX group	8.07±3.22^#●○♥	17.85±1.85^#○♥♣	9.78±3.84^{#●○♥♣}
CTX两次组 Double-dose CTX group	14.34±4.31^*#♠	9.10±3.50^*#♠♣	-5.24±6.69^*♠
CTX多次组 Multiple-dose CTX group	10.24±4.45^#●○	1.44±6.72^{*●○♠♥}	-8.80±5.05^*#♠
雷公藤组 Tripterygium wilfordii group	16.86±3.60^*#♠♣	9.00±6.65^*#♠♣	-7.86±6.97^*♠
制动+LTS组 Restraint stress +LTS group	17.71±5.02^*#♠♣	12.91±2.88^*#♣	-4.80±7.11^*♠
围绝经期组 Perimenopausal group	1.76±2.23^{*●○♠♥♣}	0.12±0.58^{*●○♠♥}	-1.64±2.18^*♠♣
F值 F value	13.207	15.820	10.655
P值 P value	0.000	0.000	0.000

表2 各组大鼠每周体重增长率比较 ( $x ˉ ± s$ , n=6)

Table 2 Comparison of weekly body weight growth rate in each group

组别 Group	体重增长率/% Growth rate of body mass/%
组别 Group	第1周 Week 1	第2周 Week 2	第2周－第1周 Week 2-week 1
对照组 Control group	9.58±2.67^#●○♥	19.49±5.79^{#●○♥♣}	9.92±7.62^{#●○♥♣}
CTX单次组 Single-dose CTX group	8.07±3.22^#●○♥	17.85±1.85^#○♥♣	9.78±3.84^{#●○♥♣}
CTX两次组 Double-dose CTX group	14.34±4.31^*#♠	9.10±3.50^*#♠♣	-5.24±6.69^*♠
CTX多次组 Multiple-dose CTX group	10.24±4.45^#●○	1.44±6.72^{*●○♠♥}	-8.80±5.05^*#♠
雷公藤组 Tripterygium wilfordii group	16.86±3.60^*#♠♣	9.00±6.65^*#♠♣	-7.86±6.97^*♠
制动+LTS组 Restraint stress +LTS group	17.71±5.02^*#♠♣	12.91±2.88^*#♣	-4.80±7.11^*♠
围绝经期组 Perimenopausal group	1.76±2.23^{*●○♠♥♣}	0.12±0.58^{*●○♠♥}	-1.64±2.18^*♠♣
F值 F value	13.207	15.820	10.655
P值 P value	0.000	0.000	0.000

表3 各组大鼠卵巢、子宫重量与卵巢、子宫指数比较 (

x ˉ ± s

,n=6)

Table 3 Comparison of ovary and uterus weight,ovarian and uterine index in each group

组别 Group	卵巢重量/mg Ovarian weight/mg	子宫重量/mg Uterine weight/mg	卵巢指数/%(mg/g) Ovarian index/%(mg/g)	子宫指数/%(mg/g) Uterine index/%(mg/g)
对照组Control group	122.68±5.09	412.87±50.75	56.71±2.48^#	190.01±13.32
CTX单次组Single-dose CTX group	124.35±13.07	408.87±75.22^#	59.19±5.95^#	195.13±39.13
CTX两次组Double-dose CTX group	125.35±10.53	422.52±110.21	61.85±5.18^#	208.29±53.33
CTX多次组Multiple-dose CTX group	112.47±14.05	399.1±114.50^#	62.16±6.05^#	219.57±55.00
雷公藤组Tripterygium wilfordii group	118.67±15.37	358.83±36.25^#	57.30±7.99^#	173.65±22.36
制动+LTS组Restraint stress +LTS group	132.48±15.34	387.55±58.68^#	60.59±6.00^#	177.01±22.03
围绝经期组Perimenopausal group	123.27±29.16	703.03±150.86	27.58±6.46^{*●○♠♥♣}	157.04±30.99
F值F value	0.869	9.430	25.748	2.018
P值P value	0.527	0.000	0.000	0.089

表3 各组大鼠卵巢、子宫重量与卵巢、子宫指数比较 ( $x ˉ ± s$ ,n=6)

Table 3 Comparison of ovary and uterus weight,ovarian and uterine index in each group

组别 Group	卵巢重量/mg Ovarian weight/mg	子宫重量/mg Uterine weight/mg	卵巢指数/%(mg/g) Ovarian index/%(mg/g)	子宫指数/%(mg/g) Uterine index/%(mg/g)
对照组Control group	122.68±5.09	412.87±50.75	56.71±2.48^#	190.01±13.32
CTX单次组Single-dose CTX group	124.35±13.07	408.87±75.22^#	59.19±5.95^#	195.13±39.13
CTX两次组Double-dose CTX group	125.35±10.53	422.52±110.21	61.85±5.18^#	208.29±53.33
CTX多次组Multiple-dose CTX group	112.47±14.05	399.1±114.50^#	62.16±6.05^#	219.57±55.00
雷公藤组Tripterygium wilfordii group	118.67±15.37	358.83±36.25^#	57.30±7.99^#	173.65±22.36
制动+LTS组Restraint stress +LTS group	132.48±15.34	387.55±58.68^#	60.59±6.00^#	177.01±22.03
围绝经期组Perimenopausal group	123.27±29.16	703.03±150.86	27.58±6.46^{*●○♠♥♣}	157.04±30.99
F值F value	0.869	9.430	25.748	2.018
P值P value	0.527	0.000	0.000	0.089

图1 大鼠卵巢HE染色（×100）：A.对照组 B.CTX单次组 C.CTX两次组 D.CTX多次组 E.雷公藤组 F.制动+LTS组 G.围绝经期组
注：图A中箭头所示为次级卵泡；图B中箭头所示为初级卵泡；图C中箭头所示为成熟卵泡；图D中箭头所示为原始卵泡；图F中箭头所示为窦状卵泡；图G中箭头所示为闭锁卵泡；

Figure 1 HE staining of rat ovary （×100）： A. control group B. single-dose CTX group C. double-dose CTX group D. multiple-dose CTX group E. tripterygium wilfordii group F. restraint stress +LTS group G. perimenopausal group
Note：In Figure A， the arrow indicates the secondary follicle； in Figure B， the arrow indicates the primary follicle； in Figure C， the arrow indicates the mature follicle； in Figure D， the arrow indicates the primordial follicle； in Figure F， the arrow indicates the antral follicle； in Figure G， the arrow indicates the atretic follicle.

表4 各组大鼠卵巢各级卵泡计数比较 (

x ˉ ± s

,n=3)

Table 4 Comparison of ovarian follicle count at all levels in each group

组别 group	原始卵泡数 Number of primordial follicles	初级卵泡数 Number of primary follicles	次级卵泡数 Number of secondary follicle	成熟卵泡数 Number of mature follicles	闭锁卵泡数 Number of atretic follicles
对照组 Control group	10.67±0.58^{#○♠♥♣}	8.33±0.58^a	8.33±1.16^abceff	1.67±1.16	3.67±1.16^{#●○♠♥♣}
CTX单次组 Single-dose CTX group	5.67±1.53^*#	11.33±0.58ⁿ	13.67±2.31^*#	0.33±0.58	7.67±1.16^*●○♣
CTX两次组 Double-dose CTX group	4.67±0.58^*	11.33±3.22	14.00±4.36^*#	0.33±0.58	9.67±1.16^*●○
CTX多次组 Multiple-dose CTX group	6.33±0.58^*#	10.33±1.16	14.00±3.61^*#	0.67±1.16	12.00±1.73^*#♠
雷公藤组 Tripterygium wilfordii group	6.33±0.58^*#	13.33±5.03	13.00±1.73^#	2.00±0.00	13.00±1.73^*#♠♥
制动+LTS组 Restraint stress +LTS group	6.33±1.16^*#	11.00±1.73	14.67±2.31^*#	0.33±0.58	13.33±0.58^*#♠♥
围绝经期组 Perimenopausal group	3.00±1.73^{*●○♠♣}	6.33±4.93	1.00±1.73^{*●○♠♥♣}	0.00±0.00	8.67±1.53^*●○♣
F值 F value	14.375	1.692	10.449	3.333	19.614
P值 P value	0.000	0.195	0.000	0.030	0.000

表4 各组大鼠卵巢各级卵泡计数比较 ( $x ˉ ± s$ ,n=3)

Table 4 Comparison of ovarian follicle count at all levels in each group

组别 group	原始卵泡数 Number of primordial follicles	初级卵泡数 Number of primary follicles	次级卵泡数 Number of secondary follicle	成熟卵泡数 Number of mature follicles	闭锁卵泡数 Number of atretic follicles
对照组 Control group	10.67±0.58^{#○♠♥♣}	8.33±0.58^a	8.33±1.16^abceff	1.67±1.16	3.67±1.16^{#●○♠♥♣}
CTX单次组 Single-dose CTX group	5.67±1.53^*#	11.33±0.58ⁿ	13.67±2.31^*#	0.33±0.58	7.67±1.16^*●○♣
CTX两次组 Double-dose CTX group	4.67±0.58^*	11.33±3.22	14.00±4.36^*#	0.33±0.58	9.67±1.16^*●○
CTX多次组 Multiple-dose CTX group	6.33±0.58^*#	10.33±1.16	14.00±3.61^*#	0.67±1.16	12.00±1.73^*#♠
雷公藤组 Tripterygium wilfordii group	6.33±0.58^*#	13.33±5.03	13.00±1.73^#	2.00±0.00	13.00±1.73^*#♠♥
制动+LTS组 Restraint stress +LTS group	6.33±1.16^*#	11.00±1.73	14.67±2.31^*#	0.33±0.58	13.33±0.58^*#♠♥
围绝经期组 Perimenopausal group	3.00±1.73^{*●○♠♣}	6.33±4.93	1.00±1.73^{*●○♠♥♣}	0.00±0.00	8.67±1.53^*●○♣
F值 F value	14.375	1.692	10.449	3.333	19.614
P值 P value	0.000	0.195	0.000	0.030	0.000

表5 各组大鼠血清FSH、LH、E₂、AMH比较 (

x ˉ ± s

,n=6)

Table 5 Comparison of serum FSH, LH, E₂ and AMH in each group

组别 Group	雌二醇ρ/(pg·mL^-1) E₂ρ/(pg·mL^-1)	黄体生成素ρ/(pg·mL^-1) LHρ/(pg·mL^-1)	促卵泡激素ρ/(pg·mL^-1) FSHρ/(pg·mL^-1)	抗苗勒氏管激素ρ/(ng·mL^-1) AMHρ/(ng·mL^-1)
对照组 Control group	310.02±36.80^#	353.33±64.79^#●♣	10.11±0.83^○♥	1.16±0.32^#●♠
CTX单次组 Single-dose CTX group	304.40±28.63^#	473.64±101.95^#●	11.35±4.80	1.65±0.58^*○♥♣
CTX两次组 Double-dose CTX group	321.28±36.98^#	448.60±103.37^#●	7.08±0.97^*	0.97±0.35^#●♠
CTX多次组 Multiple-dose CTX group	325.62±25.30^#	508.77±130.94^*	8.18±1.19	0.90±0.47^#●♠
雷公藤组 Tripterygium wilfordii group	310.43±36.89^#	499.03±122.67	8.24±0.83^*	1.13±0.31^#●♠
制动+LTS组 Restraint stress +LTS group	271.99±42.61^♥♣	631.27±200.25^*♠♥	8.78±1.62	1.92±0.43^*
围绝经期组 Perimenopausal group	250.93±30.33^{*○♠♥♣}	634.50±149.51^*♠♥	8.73±1.93	1.81±0.34^*
F值 F value	3.827	2.484	3.497	6.232
P值 P value	0.005	0.042	0.008	0.000

表5 各组大鼠血清FSH、LH、E₂、AMH比较 ( $x ˉ ± s$ ,n=6)

Table 5 Comparison of serum FSH, LH, E₂ and AMH in each group

组别 Group	雌二醇ρ/(pg·mL^-1) E₂ρ/(pg·mL^-1)	黄体生成素ρ/(pg·mL^-1) LHρ/(pg·mL^-1)	促卵泡激素ρ/(pg·mL^-1) FSHρ/(pg·mL^-1)	抗苗勒氏管激素ρ/(ng·mL^-1) AMHρ/(ng·mL^-1)
对照组 Control group	310.02±36.80^#	353.33±64.79^#●♣	10.11±0.83^○♥	1.16±0.32^#●♠
CTX单次组 Single-dose CTX group	304.40±28.63^#	473.64±101.95^#●	11.35±4.80	1.65±0.58^*○♥♣
CTX两次组 Double-dose CTX group	321.28±36.98^#	448.60±103.37^#●	7.08±0.97^*	0.97±0.35^#●♠
CTX多次组 Multiple-dose CTX group	325.62±25.30^#	508.77±130.94^*	8.18±1.19	0.90±0.47^#●♠
雷公藤组 Tripterygium wilfordii group	310.43±36.89^#	499.03±122.67	8.24±0.83^*	1.13±0.31^#●♠
制动+LTS组 Restraint stress +LTS group	271.99±42.61^♥♣	631.27±200.25^*♠♥	8.78±1.62	1.92±0.43^*
围绝经期组 Perimenopausal group	250.93±30.33^{*○♠♥♣}	634.50±149.51^*♠♥	8.73±1.93	1.81±0.34^*
F值 F value	3.827	2.484	3.497	6.232
P值 P value	0.005	0.042	0.008	0.000

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