环境化学物质VCD致卵巢早衰动物模型的构建
|
摘要
研究背景
卵巢早衰病因和机理比较复杂,尚没有合理有效的治疗方法。卵巢早衰可严重影响患者的生活质量,而且直接促进某些严重疾病的发生,如卵巢早衰导致年轻妇女不育、提前闭经等一系列内分泌变化,增加心血管疾病的发生率,导致骨质疏松。因此,进一步探讨卵巢早衰(POF)的病因学及寻求有效的治疗方法是解决这个疾病的关键。鉴于POF人体研究的可行性低,建立与人类相似的生殖内分泌和组织学变化的POF模型对于POF发病机制的研究和进一步治疗具有重要价值。
女性生来具有有限的原始卵泡池,生后不可再生。绝大多数原始卵泡将走向闭锁,只有不到1%的卵泡发育成熟并排卵。因此,暴露于原始卵泡毒性的化学物质中的女性生育能力就会受到损害,也可能使女性提早绝经。有研究发现环境物质能破坏卵泡中的卵母细胞进而影响卵巢功能,由于人们接触到越来越多的环境化学物质制品,职业暴露可能增加不孕及卵巢早衰的危险。因此环境物质对卵巢毒性的影响越来越受到关注。
VCD(4-vinylcyclohexene diepoxide)是VCH(4-vinylcyclohexene)的主要代谢产物之一,VCH产生于生产橡胶轮胎、阻燃剂、杀虫剂、可塑剂及抗氧化剂等的过程中,研究发现VCH和VCD能选择性破坏雌鼠和短尾猴卵巢内的原始卵泡和初级卵泡,最终引起雌鼠卵巢早衰,而VCD是VCH卵巢毒性的主要活性形式。因此本研究选择VCD构建POF动物模型,通过观察阴道涂片及不同时间段卵巢组织学变化等判断造模效果,旨在观察卵巢逐步衰竭的过程及探讨该动物模型的成功率,和是否是能模拟生理性的卵巢早衰,为卵巢早衰的病因和治疗提供研究方法和实验依据。
研究目的
1.建立环境化学物质VCD致卵巢早衰小鼠模型。
2.观察VCD作用下小鼠卵巢功能变化的相关指标。
3.观察VCD用药后卵巢功能逐步衰竭的过程。
4.提高人们对环境因素的关注。
研究内容和方法
1.研究对象：
雌性昆明小鼠60只,28天龄,体重90±10 g,购自山东大学动物实验中心,温度控制在22±2℃,相对湿度为50%-60%,自由饮食,每天光照时间为12h。
2.研究方法：
2.1实验方案
将60只小鼠随机分为VCD组、佐剂对照组、正常对照组和去势组四组,VCD组和芝麻油组分别每天腹腔注射同等量的VCD和芝麻油5ml／kg,注药2周后每天观察小鼠阴道涂片,观察小鼠动情周期变化。分别于注药后不同时间取卵巢和子宫组织做组织学检查,同时计数各级卵泡数目,观察小鼠子宫和卵巢的形态及组织学变化。
2.2分组与用药
将实验动物随机分为4组：
A组：即VCD用药组20只,每天腹腔注射VCD160mg／kg,即5ml／kgVCD溶液(VCD溶于芝麻油中),连续注射15天；
B组：佐剂对照组即芝麻油组16只,每天注射同等剂量芝麻油5ml／kg,连续注射15
天；
C组：即正常对照组20只,不做任何处理；
D组：即去势组4只,于注药第1天行双侧卵巢切除术。
2.3实验观察指标
①小鼠一般状态观察：观察处理后各组小鼠的活动、饮食及体重变化等情况。
②小鼠动情周期的观察：每天取小鼠阴道分泌物涂片并HE染色,观察小鼠动情周期变化情况。
③小鼠卵巢和子宫组织学变化：分别取不同时间段(d18,d37,d80和d120天)小鼠卵巢和子宫组织做病理切片并HE染色,切片厚度为5μm,每隔20μm取一张切片,光镜下观察小鼠卵巢和子宫的组织学改变。
④不同时间段卵泡计数：于注药后d18,d37,d80和d120天分别卵泡计数,为避免重复计数卵泡,原始卵泡和初级卵泡以卵母细胞核作为标记物计数,次级卵泡和窦卵泡以卵母细胞核仁为标记物计数。每只取20张切片分别计数各级卵泡数目。
结果
1.动情周期变化：注药d60天VCD组动情周期不规则数占80%,d80天全部紊乱。芝麻油组和正常对照组动情周期变化较稳定,去势组小鼠均处于动情间期,无动情周期变化
2.卵泡计数和卵巢组织学变化：注药d18天VCD组原始卵泡消失,初级卵泡较少见,次级卵泡和窦卵泡未见明显减少,注药d37天VCD组初级卵泡偶见或消失,次级卵泡和窦卵泡总数减少至正常对照组的10.08%(8.33／82.67),其卵泡总数为正常对照组3.96%
(11／278),d80天VCD组各阶段生长卵泡数严重减少(P<0.01)；d120天VCD组5只小鼠卵巢体积均明显减小(5／5),镜下见卵巢萎缩明显、皮质增厚、结构紊乱,各级卵泡消失或偶见,正常对照组和芝麻油组可见各级发育中的卵泡.
3.子宫形态及组织学变化：注药d120天VCD组子宫体积萎缩、内膜变薄、腺腔小,类似于去势组子宫变化。正常对照组和芝麻油组子宫内膜较厚,固有层腺体丰富.
结论
1.用职业性环境化学物质VCD能成功建立卵巢早衰动物模型,实验方法简便,建模成功率高。
2.本研究结果显示VCD能选择性破坏原始卵泡和初级卵泡,最终引起次级卵泡及窦卵泡的减少至消失,提示VCD作用于卵巢的靶细胞可能是原始卵泡和初级卵泡,卵巢组织学观察并未见炎型反应及坏死结构,其致卵巢早衰的机制可能是通过凋亡途径。
3.用职业性环境化学物质VCD所致的卵巢早衰过程类似于女性生理性的卵巢早衰过程,这对于卵巢早衰的临床研究和治疗提供一定的帮助。
4.保护女性远离卵巢毒性物质,对女性生殖健康至关重要。
Background
The etiology and mechanism of Premature ovarian failure(POF) are complicated,and there're no reasonable and effective methods to cure.POF would affect patient's life quality seriously and lead to certain diseases directly.For example,premature ovarian failure may lead to infertility,ahead of menostasia and osteoporosis.So,to further discuss the etiology of POF and to search an effective method is the key point to solve the problem.In view of the low feasibility of study in human body,establishing a POF model similar to human's reproductive endocrinology and histology changes is very important to investigate the mechanism and therapy of POF.
The females are born with a finite pool of primordial follicles that cannot be regenerated, the vast majority are lost through a degradative process called atresia,less than 1 percent primordial follicles will mature and ovulate. As a result,exposure to chemicals that cause depletion of the primordial follicles can jeopardize the reproductive capacity of females and may cause early menopause.There're reports that environmental chemicals can destroy the oocyte of follicles and affect the ovary function.Because people are exposed to ever-increasing combinations of environmental chemicals, occupational exposure may increase the risk of infertility and POF. So,there is an increased awareness of environmental chemicals'ovotoxity.
4-vinylcyclohexene diepoxide(VCD) is one of the main metabolic products of 4-vinylcyclohexene(VCH), VCH is a product of rubber, flame retardants, pesticide, plastics and antioxidant production.there're reports that VCH and VCD can selectively destroy the primordial and primary follicles of the mice and Macaque. it was determined that VCD is the ovotoxic form of the parent compound VCH, Consequently, POF model study have utilized VCD. By observing vaginal smear,ovarian histology changes in different time points to evaluate the model.The aim is to observe the ovarian failure gradually,investigate the achievement ratio of the model and to judge the ability of simulating physiology POF,serving as a method and foundation for the study of POF etiology and therapy.
Objective
1. To establish the mice model of POF by using enviromental chemical VCD.
2. To observe and check the relevant markers of ovarian function by VCD.
3. To observe the process of ovarian failure gradually.
4. To show more attention to environment agent.
Materials and Methods
1. Experimental subjects:
The experimental subjects were 60 female kunming mice,28 days old,weighted from 80-100 grams,purchased from the animal experiement center of Shandong university, The room temperature was maintained at 20-24℃and the relative humidity is 50%-60%, Animals were given food and water ad libitum, and maintained on a 12-h light/dark cycle.
2. Experimental methods
2.1 Experimental schedule
60 Kunming mice were divided into four groups at random,including VCD group, adjuvant control group,normal group and castrated group, VCD group and adjuvant control group were treated daily with VCD or vehicle at the same dose 5ml/kg i.p separately.vaginal smear is done 2 weeks after dosing to observe the estrous cycle changes.Meanwhile, Ovaries and uterus were removed and processed for histological evaluation at different time points after the onset of dosing and follicle counts are also performed to observe the morphology and histology changes of the uterus and ovaries.
2.2 Grouping and therapy:
A (VCD group):20 mice, treated daily with VCD 160mg/kg (5ml/kg),15 days, i.p separately.
B (adjuvant control group,sesame oil group):16 mice, treated daily with sesame oil 5ml/kg, 15 days, i.p separately.
C (normal group):20 mice,not be treated.
D (castrated group):4 mice,ovaries were removed on the first day of injecting.
2.3 The outcome measurement
2.3.1 Common conditions observing the mice's activity,diet and weight changes after treating.
2.3.2 Estrous cycles:vaginal discharging smear and HE dyeing to observe the estrous cycle changes.
2.3.3 Ovaries and uterus'histologic changes:Ovaries and uterus were removed and processed for histological evaluation at different time points(on day18,37,80 and 120) after the onset of dosing, the slice thickness is 5 um, we got one slice every 20 um.
2.3.4 Follicle counts at different time points:Follicles were counted on day 18,37,80,120 after the onset of dosing.To avoid double counting,primordial and primary follicles were counted when we saw the oocyte neclei,and secondary and antral follicles'oocyte nucleolus. We counted 20 sections per ovary.
Results
1. Estrus cycles changes:Eighty percent of VCD group mice displayed disruptions in estrous cyclicity on day 60,and all treated mice were in disturbance on day 80.there's a stable estrous cycles of sesame oil and normal group mice and all in diestrus of castrated group.
2. Follicle counts and histology changes of the ovary:On day 18 following onset of treatment, primordial follicles of VCD group disappeared,primary follicles is decreased while larger follicles were unaffected compared with controls; On day 37, primary follicles disappeared or seen by chance and secondary and antral follicles were reduced to 10.08%(8.33/82.67) of normal group,the total number of follicles is 3.96%(11/278) of normal group;On day 80,there was a substantial reduction in the number of secondary and antral follicles (P<0.01);On day 120,the growing follicles disappeared or seen by chance,the ovaries and uterus showed apparently atrophy(5/5),cotex thickened and structure disturbance. There're kinds of follicles in different stages in normal and sesame oil group.
3. The mophology and histology changes of the uterus:On day 120,VCD group showed uterus atrophy,endomembrane thinned and gland dicreased,similar to the castrated group.while in normal and sesame oil group,there'er thicker endomembrane and plentiful gland in lamina propria.
Conclusion
1. Anminal model of POF can be successfully established by using environmental chemical VCD, the experimental method is simple and achievement ratio is high.
2. This study showed that VCD. can selectively destroy the primordial and primary follicles,and finally induced the secondary and antral follicles decreased or disappeared, suggested that the target of VCD may be primodial and primary follicles,the histologic observing showed no inflammation or necrosis,suggesting the mechanism of POF model is through apoptosis.
3. The POF model by using VCD is similar to female physiological POF process,which is useful to clinical study and therapy of POF.
4. To protect female humans from ovotoxity chemicals is of vital importance to female's reproductive health.
卵巢早衰病因和机理比较复杂,尚没有合理有效的治疗方法。卵巢早衰可严重影响患者的生活质量,而且直接促进某些严重疾病的发生,如卵巢早衰导致年轻妇女不育、提前闭经等一系列内分泌变化,增加心血管疾病的发生率,导致骨质疏松。因此,进一步探讨卵巢早衰(POF)的病因学及寻求有效的治疗方法是解决这个疾病的关键。鉴于POF人体研究的可行性低,建立与人类相似的生殖内分泌和组织学变化的POF模型对于POF发病机制的研究和进一步治疗具有重要价值。
女性生来具有有限的原始卵泡池,生后不可再生。绝大多数原始卵泡将走向闭锁,只有不到1%的卵泡发育成熟并排卵。因此,暴露于原始卵泡毒性的化学物质中的女性生育能力就会受到损害,也可能使女性提早绝经。有研究发现环境物质能破坏卵泡中的卵母细胞进而影响卵巢功能,由于人们接触到越来越多的环境化学物质制品,职业暴露可能增加不孕及卵巢早衰的危险。因此环境物质对卵巢毒性的影响越来越受到关注。
VCD(4-vinylcyclohexene diepoxide)是VCH(4-vinylcyclohexene)的主要代谢产物之一,VCH产生于生产橡胶轮胎、阻燃剂、杀虫剂、可塑剂及抗氧化剂等的过程中,研究发现VCH和VCD能选择性破坏雌鼠和短尾猴卵巢内的原始卵泡和初级卵泡,最终引起雌鼠卵巢早衰,而VCD是VCH卵巢毒性的主要活性形式。因此本研究选择VCD构建POF动物模型,通过观察阴道涂片及不同时间段卵巢组织学变化等判断造模效果,旨在观察卵巢逐步衰竭的过程及探讨该动物模型的成功率,和是否是能模拟生理性的卵巢早衰,为卵巢早衰的病因和治疗提供研究方法和实验依据。
研究目的
1.建立环境化学物质VCD致卵巢早衰小鼠模型。
2.观察VCD作用下小鼠卵巢功能变化的相关指标。
3.观察VCD用药后卵巢功能逐步衰竭的过程。
4.提高人们对环境因素的关注。
研究内容和方法
1.研究对象：
雌性昆明小鼠60只,28天龄,体重90±10 g,购自山东大学动物实验中心,温度控制在22±2℃,相对湿度为50%-60%,自由饮食,每天光照时间为12h。
2.研究方法：
2.1实验方案
将60只小鼠随机分为VCD组、佐剂对照组、正常对照组和去势组四组,VCD组和芝麻油组分别每天腹腔注射同等量的VCD和芝麻油5ml／kg,注药2周后每天观察小鼠阴道涂片,观察小鼠动情周期变化。分别于注药后不同时间取卵巢和子宫组织做组织学检查,同时计数各级卵泡数目,观察小鼠子宫和卵巢的形态及组织学变化。
2.2分组与用药
将实验动物随机分为4组：
A组：即VCD用药组20只,每天腹腔注射VCD160mg／kg,即5ml／kgVCD溶液(VCD溶于芝麻油中),连续注射15天；
B组：佐剂对照组即芝麻油组16只,每天注射同等剂量芝麻油5ml／kg,连续注射15
天；
C组：即正常对照组20只,不做任何处理；
D组：即去势组4只,于注药第1天行双侧卵巢切除术。
2.3实验观察指标
①小鼠一般状态观察：观察处理后各组小鼠的活动、饮食及体重变化等情况。
②小鼠动情周期的观察：每天取小鼠阴道分泌物涂片并HE染色,观察小鼠动情周期变化情况。
③小鼠卵巢和子宫组织学变化：分别取不同时间段(d18,d37,d80和d120天)小鼠卵巢和子宫组织做病理切片并HE染色,切片厚度为5μm,每隔20μm取一张切片,光镜下观察小鼠卵巢和子宫的组织学改变。
④不同时间段卵泡计数：于注药后d18,d37,d80和d120天分别卵泡计数,为避免重复计数卵泡,原始卵泡和初级卵泡以卵母细胞核作为标记物计数,次级卵泡和窦卵泡以卵母细胞核仁为标记物计数。每只取20张切片分别计数各级卵泡数目。
结果
1.动情周期变化：注药d60天VCD组动情周期不规则数占80%,d80天全部紊乱。芝麻油组和正常对照组动情周期变化较稳定,去势组小鼠均处于动情间期,无动情周期变化
2.卵泡计数和卵巢组织学变化：注药d18天VCD组原始卵泡消失,初级卵泡较少见,次级卵泡和窦卵泡未见明显减少,注药d37天VCD组初级卵泡偶见或消失,次级卵泡和窦卵泡总数减少至正常对照组的10.08%(8.33／82.67),其卵泡总数为正常对照组3.96%
(11／278),d80天VCD组各阶段生长卵泡数严重减少(P<0.01)；d120天VCD组5只小鼠卵巢体积均明显减小(5／5),镜下见卵巢萎缩明显、皮质增厚、结构紊乱,各级卵泡消失或偶见,正常对照组和芝麻油组可见各级发育中的卵泡.
3.子宫形态及组织学变化：注药d120天VCD组子宫体积萎缩、内膜变薄、腺腔小,类似于去势组子宫变化。正常对照组和芝麻油组子宫内膜较厚,固有层腺体丰富.
结论
1.用职业性环境化学物质VCD能成功建立卵巢早衰动物模型,实验方法简便,建模成功率高。
2.本研究结果显示VCD能选择性破坏原始卵泡和初级卵泡,最终引起次级卵泡及窦卵泡的减少至消失,提示VCD作用于卵巢的靶细胞可能是原始卵泡和初级卵泡,卵巢组织学观察并未见炎型反应及坏死结构,其致卵巢早衰的机制可能是通过凋亡途径。
3.用职业性环境化学物质VCD所致的卵巢早衰过程类似于女性生理性的卵巢早衰过程,这对于卵巢早衰的临床研究和治疗提供一定的帮助。
4.保护女性远离卵巢毒性物质,对女性生殖健康至关重要。
Background
The etiology and mechanism of Premature ovarian failure(POF) are complicated,and there're no reasonable and effective methods to cure.POF would affect patient's life quality seriously and lead to certain diseases directly.For example,premature ovarian failure may lead to infertility,ahead of menostasia and osteoporosis.So,to further discuss the etiology of POF and to search an effective method is the key point to solve the problem.In view of the low feasibility of study in human body,establishing a POF model similar to human's reproductive endocrinology and histology changes is very important to investigate the mechanism and therapy of POF.
The females are born with a finite pool of primordial follicles that cannot be regenerated, the vast majority are lost through a degradative process called atresia,less than 1 percent primordial follicles will mature and ovulate. As a result,exposure to chemicals that cause depletion of the primordial follicles can jeopardize the reproductive capacity of females and may cause early menopause.There're reports that environmental chemicals can destroy the oocyte of follicles and affect the ovary function.Because people are exposed to ever-increasing combinations of environmental chemicals, occupational exposure may increase the risk of infertility and POF. So,there is an increased awareness of environmental chemicals'ovotoxity.
4-vinylcyclohexene diepoxide(VCD) is one of the main metabolic products of 4-vinylcyclohexene(VCH), VCH is a product of rubber, flame retardants, pesticide, plastics and antioxidant production.there're reports that VCH and VCD can selectively destroy the primordial and primary follicles of the mice and Macaque. it was determined that VCD is the ovotoxic form of the parent compound VCH, Consequently, POF model study have utilized VCD. By observing vaginal smear,ovarian histology changes in different time points to evaluate the model.The aim is to observe the ovarian failure gradually,investigate the achievement ratio of the model and to judge the ability of simulating physiology POF,serving as a method and foundation for the study of POF etiology and therapy.
Objective
1. To establish the mice model of POF by using enviromental chemical VCD.
2. To observe and check the relevant markers of ovarian function by VCD.
3. To observe the process of ovarian failure gradually.
4. To show more attention to environment agent.
Materials and Methods
1. Experimental subjects:
The experimental subjects were 60 female kunming mice,28 days old,weighted from 80-100 grams,purchased from the animal experiement center of Shandong university, The room temperature was maintained at 20-24℃and the relative humidity is 50%-60%, Animals were given food and water ad libitum, and maintained on a 12-h light/dark cycle.
2. Experimental methods
2.1 Experimental schedule
60 Kunming mice were divided into four groups at random,including VCD group, adjuvant control group,normal group and castrated group, VCD group and adjuvant control group were treated daily with VCD or vehicle at the same dose 5ml/kg i.p separately.vaginal smear is done 2 weeks after dosing to observe the estrous cycle changes.Meanwhile, Ovaries and uterus were removed and processed for histological evaluation at different time points after the onset of dosing and follicle counts are also performed to observe the morphology and histology changes of the uterus and ovaries.
2.2 Grouping and therapy:
A (VCD group):20 mice, treated daily with VCD 160mg/kg (5ml/kg),15 days, i.p separately.
B (adjuvant control group,sesame oil group):16 mice, treated daily with sesame oil 5ml/kg, 15 days, i.p separately.
C (normal group):20 mice,not be treated.
D (castrated group):4 mice,ovaries were removed on the first day of injecting.
2.3 The outcome measurement
2.3.1 Common conditions observing the mice's activity,diet and weight changes after treating.
2.3.2 Estrous cycles:vaginal discharging smear and HE dyeing to observe the estrous cycle changes.
2.3.3 Ovaries and uterus'histologic changes:Ovaries and uterus were removed and processed for histological evaluation at different time points(on day18,37,80 and 120) after the onset of dosing, the slice thickness is 5 um, we got one slice every 20 um.
2.3.4 Follicle counts at different time points:Follicles were counted on day 18,37,80,120 after the onset of dosing.To avoid double counting,primordial and primary follicles were counted when we saw the oocyte neclei,and secondary and antral follicles'oocyte nucleolus. We counted 20 sections per ovary.
Results
1. Estrus cycles changes:Eighty percent of VCD group mice displayed disruptions in estrous cyclicity on day 60,and all treated mice were in disturbance on day 80.there's a stable estrous cycles of sesame oil and normal group mice and all in diestrus of castrated group.
2. Follicle counts and histology changes of the ovary:On day 18 following onset of treatment, primordial follicles of VCD group disappeared,primary follicles is decreased while larger follicles were unaffected compared with controls; On day 37, primary follicles disappeared or seen by chance and secondary and antral follicles were reduced to 10.08%(8.33/82.67) of normal group,the total number of follicles is 3.96%(11/278) of normal group;On day 80,there was a substantial reduction in the number of secondary and antral follicles (P<0.01);On day 120,the growing follicles disappeared or seen by chance,the ovaries and uterus showed apparently atrophy(5/5),cotex thickened and structure disturbance. There're kinds of follicles in different stages in normal and sesame oil group.
3. The mophology and histology changes of the uterus:On day 120,VCD group showed uterus atrophy,endomembrane thinned and gland dicreased,similar to the castrated group.while in normal and sesame oil group,there'er thicker endomembrane and plentiful gland in lamina propria.
Conclusion
1. Anminal model of POF can be successfully established by using environmental chemical VCD, the experimental method is simple and achievement ratio is high.
2. This study showed that VCD. can selectively destroy the primordial and primary follicles,and finally induced the secondary and antral follicles decreased or disappeared, suggested that the target of VCD may be primodial and primary follicles,the histologic observing showed no inflammation or necrosis,suggesting the mechanism of POF model is through apoptosis.
3. The POF model by using VCD is similar to female physiological POF process,which is useful to clinical study and therapy of POF.
4. To protect female humans from ovotoxity chemicals is of vital importance to female's reproductive health.
引文
[1]Hoyer PB,Sipes IG. Assessment of follicle destruction in chemical-induced ovarian toxicity[J]. Annu Rev Pharmacol Toxicol,1996,36:307-331.
[2]Mattison DR.Oocyte destruction by polycyclic aromatic hydrocarbons[J]. Am J Indust Med,1983,4:191-202.
[3]Yu X., Kamijima M, Ichihara G, et al.2-Bromopropane causes ovarian dysfunction by damaging primordial follicles and their oocytes in female rats[J]. Toxicol Appl Pharmacol, 1999,159(3):185-193.
[4]Mayer LP,Devine PJ,Dyer CA,et al.The follicle-deplete mouse ovary produces androgen [J].Biol Reprod,2004,71(1):130-138.
[5]Appt SE,Kaplan JR,Clarkson TB,et al.Destruction of primordial ovarian follicles in adult cynomolgus macaques after exposure to 4-vinylcyclohexene diepoxide:a nonhuman primate model of the menopausal transition[J].Fertil Steril,2006,86:1210-1216.
[6]Doerr JK,Hollis EA and Sipes IG..Species difference in the ovarian toxicity of 1,3-butadiene epoxides in B6C3F1 mice and Sprague-Dawley rats[J]. Toxicology,1996, 113:128-136.
[7]Anasti JN.Premature ovarian failure:an update.Fertil Steril,1998,70(1):1-15.
[8]朱珠,刘嘉茵。环境内分泌干扰物对女性生殖健康影响的研究进展。国外医学·妇产科学分册,2004,06,15：31(3)：179-182.
[9]林建华,严隽鸿,林其德,等。 自身免疫性卵巢衰退小鼠动物模型的建立。上海医学,1999,22(12)：747-8.
[10]蔡立荣,李大金,孙晓溪,等。实验性自身免疫性卵巢炎卵巢功能早衰的机理研究。免疫学,1999,1(1)：15-8.
[11]Bagavant H,Sharp C,Kurth B, et al. Induction and immunohistology of autoimmune ovarian disease in cynomolgus macaques(Macaca fascicularis)[J].Am J Pathology,2002, 160(1):141-9.
[12]Andreu-Vievra C,Chen R,Matzuk MM. Conditional deletion of the retinoblastoma (Rb) gene in ovarian granulosa cells leads to premature ovarian failure[J]. Mol Endocrinol,2008, 22(9):2141-61.
[13]Halperin J,Devi SY,Elizur S,et al. Prolactin signaling through the short form of its receptor represses forkhead transcription factor FOXO3 and its target gene galt causing a severe ovarian defect[J].Mol Endocrinol,2008,22(2):513-22.
[14]Shiina H,Matsumoto T,Sato T,et al. Premature ovarian failure in androgen receptor-deficient mice[J].Proc Natl Acad Sci,2006,103(1):224-9.
[15]Bandyopadhyay S,Chakrabarti J, Baneree J, et, al. Prenatal exposure to high galactose adversely affects initial gonadal pool of germ cells in rats.hum reprod 2003,18(2),276-82.
[16]李洁,杨菁,陈嫒 等 利用不同浓度D(+)半乳糖建立卵巢早衰动物模型。武汉大学学报·医学版2006；27(5)：613-615,619.
[17]李彩霞,王凤英,李玉艳等,小鼠卵巢早衰动物模型的构建,第三军医大学学报2008；30(6)：506-509.
[18]Pocar P, Perazzoli F, Luciano AM, et al. In vitro reproductive toxicity of polychlorinated biphenyls:effects on oocyte maturation and developmental competence in cattle [J]. Mol Reprod Dev,2001,5.8(4):411-416.
[19]Ema M, Kurosaka R, Amano H, et al. Comparative developmental toxicity of butyltin trichloride, dibutyltin dichloride and tributyltin chloride in rats[J]. J Appl Toxicol,1995, 15(4):297-302.
[20]Bolon B,Bucci TJ,Warbritton AR,et al.Differential follicle counts as a screen for chemically induced ovarian toxicity in mice:results from continuous breeding bioassays [J].Fundam Appl Toxicol,1997,39(1):1-10.
[21]Ito A,Mafune N and Kimura T. Collaborative work on evaluation of ovarian toxicity.4) Two-or four-week repeated dose study of 4-vinylcyclohexene diepoxide in female rats[J].J Toxicol Sci.2009,34,SP53-58.
[22]Hu X, Christian PJ, Thompson KE,et al. Apoptosis induced in rats by 4-vinylcyclohexene diepoxide is associated with activation of the caspase cascades[J]. Biol Reprod,2001,65(1):87-93.
[23]Hu X, Christian PJ, Sipes IG, et al. Expression and redistribution of cellular bad, bax and bcl-xl protein is associated with VCD-induced ovotoxicity in rats[J]. Biol Reprod,2001,65 (5):1489-1495.
[24]Hu X, Flaws JA, Sipes IG, et al. Activation of mitogen-activated protein kinases and AP-1 transcription factor in ovotoxicity induced by 4-vinylcyclohexene diepoxide in rats[J]. Biol Reprod 2002; 67:718-724.
[25]Haas JR, Christian PJ, Hoyer PB. Effects of impending ovarian failure induced by 4-vinylcyclohexene diepoxide on fertility in C57BL/6 female mice.Comp Med,2007,57 (5):443-9.
[26]Muhammad FS,Goode AK,Kock ND,et al. Effects of 4-vinylcyclohexene diepoxide on peripubertal and adult Sprague-Dawley rats:ovarian,clinical,and pathologic outcomes[J]. Comp Med[J],2009,59(1):46-59.
[27]Lohff JC, Christian PJ, Marion SL,et al. Effect of duration of dosing on onset of ovarian failure in a chemical-induced mouse model of perimenopause.Menopause,2006,13(3):482-8.
[28]Luborsky J L,Meyer P,Sowers M F,et al.Premature menopause in a multi-ethnic population study of the menopause transition[J].Hum Reprod,2003,18(1):199-206.
[1]Oktay K,Newton H,Gosden RG.Transplantation of cryopreserved human ovarian tissue results in follicle growth initiation in SCID mice[J].Fertil Steril,2000,73(3):599-603.
[2]郭毅,李慧。冻存复苏人卵巢皮质在裸鼠体内的发育及卵细胞的提取[J]。中国现代医学杂志,2005,15(17)：2561-2563.
[3]Kim SS, Kang HG, Kim NH,et al. Assessment of the integrity of human oocytes retrieve from cryopreserved ovarian tissue after xenotransplantation. Hum Reprod,2005,20(9),2502-8.
[4]Wang X,Bilolo KK,Qi S,et al.Restoration of fertility in oophorectomized rats after tube-ovarian transplantation[J]. Microsurgery,2002,22(1):30-33.
[5]Arav A,Revel A,Nathan Y, et al. Oocyte recovery, embryo development and ovarian function after cryopreservation and transplantation of whole sheep ovary. Hum Reprod, 2005,20(12):3554-9.
[6]Israely T, Nevo N, Harmelin A, et al. Reducing ischaemic damage in rodent ovarian xenografts transplanted into granulation tissue. Hum Reprod.2006,21(6):1368-79.
[7]Juarez-Oropeza MA, Alvarez-Fernandez G, Lopez V,et al. Steroid metabolism in the cortex and the medulla of the early fetal bovine ovary. J Exp Zool.1993,266(2):102-7.
[8]Abir R, Orvieto R, Raanani H,et al.Parameters affecting Successful transplantation of frozen-thawed human fetal ovaries into immunodefieient mice[J]. Fertil Steril,2003,80(2): 421-428.
[9]Qu J, Godin PA, Nisolle M,et al. Distribution and epidermal growth factor receptor expression of primordial follicles in human ovarian tissue before and after cryopreservation. Hum Reprod.2000,15(2):302-10.
[10]Lan C, Xiao W, Xiao-Hui D, et al. Tissue culture before ransplantation of frozen-thawed human fetal ovarian tissue into immunodeficient mice. Fertil Steril.2008 Dec 22. [Epub ahead of print]
[11]Eimani H, Siadat SF, Eftekhari-Yazdi P, et al. Comparative study between intact and non-intact intramuscular auto-grafted mouse ovaries. Reprod Biomed Online,2009,18(1): 53-60.
[12]Lee RK,Ho,HY,and Yu,SL.et al.Blastocyst development after cryopreservation and subcutaneous transplantation of mouse ovarian tissue. J Assist Reprod Genet,2005,22,95-101.
[13]Jeremias E, Bedaiwy MA, Gurunluoglu R, et al. Heterotopic autotransplan-tation of the ovary with micro vascular anastomosis:a novel surgical technique. Fertil Steril,2002, 77(6):1278-1282.
[14]Imhof M, Bergmeister H, Lipovac M,et al.Orthotopic microvascular reanastomosis of whole cryopreserved ovine ovaries resulting in pregnancy and live birth. Fertil Steril. 2006,85:1208-15.
[15]Courbiere B, Caquant L, Mazoyer C,et al. Difficulties improving ovarian functional recovery by microvascular transplantation and whole ovary vitrification. Fertil Steril,2009 Jun;91(6):2697-706.
[16]Hubner K, Fuhrmann G, Christenson LK, et al.Derivation of oocytes from mouse embryonic stem cells. Science 2003,300(5623):1251-1256.
[17]Nayernia K, Nolte J, Michelmann HW,et al. In vitro-diferentiated embryonic stem cells give rise to male gametes that can generate offspring mice[J].Dev Cell,2006,11(1):125-132.
[18]Dyce PW,Wen L, Li J. In vitro germline potential of stem cells derived from fetal porcine skin[J]. Nat Cell Biol,2006,8(4):384-390.
[19]Danner S,Kajahn J,Geismann C,et al.Derivation of oocyte-like cells from a clonal pancreatic stem cell line[J].Mol Hum Reprod,2007, 13(1):11-20.
[20]Johnson J, Bagley J, Skaznik-Wikiel M, et al.Oocyte generation in adult mammalian ovaries by putative germ cells derived from bone marrow and peripheral blood [J]. Cell, 2005,122:303-315.
[21]Bukovsky A, Caudle MR, Svetlikova M,Upadhyaya NB.2004. Origin of germ cells and formation of new primary follicles in adult human ovaries.Reprod Biol Endocrinol,2004, 2:20.
[22]Bukovsky A, Ayala ME, Dominguez R,et al.2007. Bone marrow derived cells and alternative pathways of oogenesis in adult rodents. Cell Cycle 6:2306-2309.
[23]Lee HJ, Selesniemi K, Niikura Y et al. Bone Marrow Transplantation Generates Immature Oocytes and Rescues Long-Term Fertility in a Preclinical Mouse Model of Chemotherapy-Induced Premature Ovarian Failure[J].J Clin Oncol,2007,25(22): 3198-3204.
[24]Fu X,He Y,Xie C,et al.Bone marrow mesenchymal stem cell transplantation improves ovarian function and structure in rats with chemotherapy-induced ovarian damage. Cytotherapy,2008,10(4):353-63.
[25]陈贵安,蔡学泳。人类卵母细胞冻存的临床应用[J]。中国实用妇科与产科杂志,2007,23(1)：30-32。
[26]Lim J M, Fukui Y, Ono H. Developmental competence of bovine oocytes f rozen at various maturation stages followed by in vitro maturation and fertilization[J].Theriogenology, 1992,37:351-361.
[27]Luvoni GC,Pellizzari P.Embryo development in vitro of cat oocytes cryopreserved at different maturation stages[J]. Theriogenology,2000,53(8):1529-40.
[28]宋文妍,孙莹璞,金海霞,等。不同冷冻方法对小鼠不同成熟时期卵母细胞纺锤体的影响[J]。现代妇产科进展,2007,16(5)：362-365.
[29]Ghadami M, Salama S, Khatoon N, et al. Toward gene therapy of primary ovarian failure:adenovirus expressing human FSH receptor corrects the Finnish C566T mutation[J]. MHR-Basic Science of Reproductive Medicine,2008,14(1):9-15.
[30]Dong J, Albertini DF, Nishimori K,et al.Growth differentiation factor-9 is required during early ovarian folliculogenesis[J].Nature,1996,383:531-535.
[31]Shimizu T, Miyahayashi Y, Yokoo M, et al. Molecular cloning of porcine growth differentiation factor 9(GDF-9) cDNA and its role in early folliculogenesis:direct ovarian injection of GDF-9 gene fragments promotes early folliculogenesis[J]. Reproduction,2004, 128:537-543.
[32]Wu X, Chen L, Brown CA, et al. Interelationship of growth differentiation factor 9 and inhibin in early folliculogenesisand ovarian tumorigenesis in mice. Mol Endocrinol,2004, 18(6):1509-19.
[33]Horejsi J,Novakova D,Martinek J. Circulating ovarian autoantibodies and FSH and LH levels in adolescent girls with primary menstrual cycle disorders[J]. J Pediatr Adolesc Gynecol,1996,9:74-78.
[34]付莉,黄冰玉,赵怡璇,等。口服耐受对实验性卵巢早衰抗透明带抗体的抑制作用研究[J]。现代妇产科进展,2006,15(9)：681-683。
[35]jinyao L,huali J,fuchun Z et al. Treatment of autoimmune ovarian disease by co-administration with mouse zona pellucida protein 3 and DNA vaccine through induction of adaptive regulatory T cells[J]. J Gene Med,2008,10(7):810-820.
[2]Mattison DR.Oocyte destruction by polycyclic aromatic hydrocarbons[J]. Am J Indust Med,1983,4:191-202.
[3]Yu X., Kamijima M, Ichihara G, et al.2-Bromopropane causes ovarian dysfunction by damaging primordial follicles and their oocytes in female rats[J]. Toxicol Appl Pharmacol, 1999,159(3):185-193.
[4]Mayer LP,Devine PJ,Dyer CA,et al.The follicle-deplete mouse ovary produces androgen [J].Biol Reprod,2004,71(1):130-138.
[5]Appt SE,Kaplan JR,Clarkson TB,et al.Destruction of primordial ovarian follicles in adult cynomolgus macaques after exposure to 4-vinylcyclohexene diepoxide:a nonhuman primate model of the menopausal transition[J].Fertil Steril,2006,86:1210-1216.
[6]Doerr JK,Hollis EA and Sipes IG..Species difference in the ovarian toxicity of 1,3-butadiene epoxides in B6C3F1 mice and Sprague-Dawley rats[J]. Toxicology,1996, 113:128-136.
[7]Anasti JN.Premature ovarian failure:an update.Fertil Steril,1998,70(1):1-15.
[8]朱珠,刘嘉茵。环境内分泌干扰物对女性生殖健康影响的研究进展。国外医学·妇产科学分册,2004,06,15：31(3)：179-182.
[9]林建华,严隽鸿,林其德,等。 自身免疫性卵巢衰退小鼠动物模型的建立。上海医学,1999,22(12)：747-8.
[10]蔡立荣,李大金,孙晓溪,等。实验性自身免疫性卵巢炎卵巢功能早衰的机理研究。免疫学,1999,1(1)：15-8.
[11]Bagavant H,Sharp C,Kurth B, et al. Induction and immunohistology of autoimmune ovarian disease in cynomolgus macaques(Macaca fascicularis)[J].Am J Pathology,2002, 160(1):141-9.
[12]Andreu-Vievra C,Chen R,Matzuk MM. Conditional deletion of the retinoblastoma (Rb) gene in ovarian granulosa cells leads to premature ovarian failure[J]. Mol Endocrinol,2008, 22(9):2141-61.
[13]Halperin J,Devi SY,Elizur S,et al. Prolactin signaling through the short form of its receptor represses forkhead transcription factor FOXO3 and its target gene galt causing a severe ovarian defect[J].Mol Endocrinol,2008,22(2):513-22.
[14]Shiina H,Matsumoto T,Sato T,et al. Premature ovarian failure in androgen receptor-deficient mice[J].Proc Natl Acad Sci,2006,103(1):224-9.
[15]Bandyopadhyay S,Chakrabarti J, Baneree J, et, al. Prenatal exposure to high galactose adversely affects initial gonadal pool of germ cells in rats.hum reprod 2003,18(2),276-82.
[16]李洁,杨菁,陈嫒 等 利用不同浓度D(+)半乳糖建立卵巢早衰动物模型。武汉大学学报·医学版2006；27(5)：613-615,619.
[17]李彩霞,王凤英,李玉艳等,小鼠卵巢早衰动物模型的构建,第三军医大学学报2008；30(6)：506-509.
[18]Pocar P, Perazzoli F, Luciano AM, et al. In vitro reproductive toxicity of polychlorinated biphenyls:effects on oocyte maturation and developmental competence in cattle [J]. Mol Reprod Dev,2001,5.8(4):411-416.
[19]Ema M, Kurosaka R, Amano H, et al. Comparative developmental toxicity of butyltin trichloride, dibutyltin dichloride and tributyltin chloride in rats[J]. J Appl Toxicol,1995, 15(4):297-302.
[20]Bolon B,Bucci TJ,Warbritton AR,et al.Differential follicle counts as a screen for chemically induced ovarian toxicity in mice:results from continuous breeding bioassays [J].Fundam Appl Toxicol,1997,39(1):1-10.
[21]Ito A,Mafune N and Kimura T. Collaborative work on evaluation of ovarian toxicity.4) Two-or four-week repeated dose study of 4-vinylcyclohexene diepoxide in female rats[J].J Toxicol Sci.2009,34,SP53-58.
[22]Hu X, Christian PJ, Thompson KE,et al. Apoptosis induced in rats by 4-vinylcyclohexene diepoxide is associated with activation of the caspase cascades[J]. Biol Reprod,2001,65(1):87-93.
[23]Hu X, Christian PJ, Sipes IG, et al. Expression and redistribution of cellular bad, bax and bcl-xl protein is associated with VCD-induced ovotoxicity in rats[J]. Biol Reprod,2001,65 (5):1489-1495.
[24]Hu X, Flaws JA, Sipes IG, et al. Activation of mitogen-activated protein kinases and AP-1 transcription factor in ovotoxicity induced by 4-vinylcyclohexene diepoxide in rats[J]. Biol Reprod 2002; 67:718-724.
[25]Haas JR, Christian PJ, Hoyer PB. Effects of impending ovarian failure induced by 4-vinylcyclohexene diepoxide on fertility in C57BL/6 female mice.Comp Med,2007,57 (5):443-9.
[26]Muhammad FS,Goode AK,Kock ND,et al. Effects of 4-vinylcyclohexene diepoxide on peripubertal and adult Sprague-Dawley rats:ovarian,clinical,and pathologic outcomes[J]. Comp Med[J],2009,59(1):46-59.
[27]Lohff JC, Christian PJ, Marion SL,et al. Effect of duration of dosing on onset of ovarian failure in a chemical-induced mouse model of perimenopause.Menopause,2006,13(3):482-8.
[28]Luborsky J L,Meyer P,Sowers M F,et al.Premature menopause in a multi-ethnic population study of the menopause transition[J].Hum Reprod,2003,18(1):199-206.
[1]Oktay K,Newton H,Gosden RG.Transplantation of cryopreserved human ovarian tissue results in follicle growth initiation in SCID mice[J].Fertil Steril,2000,73(3):599-603.
[2]郭毅,李慧。冻存复苏人卵巢皮质在裸鼠体内的发育及卵细胞的提取[J]。中国现代医学杂志,2005,15(17)：2561-2563.
[3]Kim SS, Kang HG, Kim NH,et al. Assessment of the integrity of human oocytes retrieve from cryopreserved ovarian tissue after xenotransplantation. Hum Reprod,2005,20(9),2502-8.
[4]Wang X,Bilolo KK,Qi S,et al.Restoration of fertility in oophorectomized rats after tube-ovarian transplantation[J]. Microsurgery,2002,22(1):30-33.
[5]Arav A,Revel A,Nathan Y, et al. Oocyte recovery, embryo development and ovarian function after cryopreservation and transplantation of whole sheep ovary. Hum Reprod, 2005,20(12):3554-9.
[6]Israely T, Nevo N, Harmelin A, et al. Reducing ischaemic damage in rodent ovarian xenografts transplanted into granulation tissue. Hum Reprod.2006,21(6):1368-79.
[7]Juarez-Oropeza MA, Alvarez-Fernandez G, Lopez V,et al. Steroid metabolism in the cortex and the medulla of the early fetal bovine ovary. J Exp Zool.1993,266(2):102-7.
[8]Abir R, Orvieto R, Raanani H,et al.Parameters affecting Successful transplantation of frozen-thawed human fetal ovaries into immunodefieient mice[J]. Fertil Steril,2003,80(2): 421-428.
[9]Qu J, Godin PA, Nisolle M,et al. Distribution and epidermal growth factor receptor expression of primordial follicles in human ovarian tissue before and after cryopreservation. Hum Reprod.2000,15(2):302-10.
[10]Lan C, Xiao W, Xiao-Hui D, et al. Tissue culture before ransplantation of frozen-thawed human fetal ovarian tissue into immunodeficient mice. Fertil Steril.2008 Dec 22. [Epub ahead of print]
[11]Eimani H, Siadat SF, Eftekhari-Yazdi P, et al. Comparative study between intact and non-intact intramuscular auto-grafted mouse ovaries. Reprod Biomed Online,2009,18(1): 53-60.
[12]Lee RK,Ho,HY,and Yu,SL.et al.Blastocyst development after cryopreservation and subcutaneous transplantation of mouse ovarian tissue. J Assist Reprod Genet,2005,22,95-101.
[13]Jeremias E, Bedaiwy MA, Gurunluoglu R, et al. Heterotopic autotransplan-tation of the ovary with micro vascular anastomosis:a novel surgical technique. Fertil Steril,2002, 77(6):1278-1282.
[14]Imhof M, Bergmeister H, Lipovac M,et al.Orthotopic microvascular reanastomosis of whole cryopreserved ovine ovaries resulting in pregnancy and live birth. Fertil Steril. 2006,85:1208-15.
[15]Courbiere B, Caquant L, Mazoyer C,et al. Difficulties improving ovarian functional recovery by microvascular transplantation and whole ovary vitrification. Fertil Steril,2009 Jun;91(6):2697-706.
[16]Hubner K, Fuhrmann G, Christenson LK, et al.Derivation of oocytes from mouse embryonic stem cells. Science 2003,300(5623):1251-1256.
[17]Nayernia K, Nolte J, Michelmann HW,et al. In vitro-diferentiated embryonic stem cells give rise to male gametes that can generate offspring mice[J].Dev Cell,2006,11(1):125-132.
[18]Dyce PW,Wen L, Li J. In vitro germline potential of stem cells derived from fetal porcine skin[J]. Nat Cell Biol,2006,8(4):384-390.
[19]Danner S,Kajahn J,Geismann C,et al.Derivation of oocyte-like cells from a clonal pancreatic stem cell line[J].Mol Hum Reprod,2007, 13(1):11-20.
[20]Johnson J, Bagley J, Skaznik-Wikiel M, et al.Oocyte generation in adult mammalian ovaries by putative germ cells derived from bone marrow and peripheral blood [J]. Cell, 2005,122:303-315.
[21]Bukovsky A, Caudle MR, Svetlikova M,Upadhyaya NB.2004. Origin of germ cells and formation of new primary follicles in adult human ovaries.Reprod Biol Endocrinol,2004, 2:20.
[22]Bukovsky A, Ayala ME, Dominguez R,et al.2007. Bone marrow derived cells and alternative pathways of oogenesis in adult rodents. Cell Cycle 6:2306-2309.
[23]Lee HJ, Selesniemi K, Niikura Y et al. Bone Marrow Transplantation Generates Immature Oocytes and Rescues Long-Term Fertility in a Preclinical Mouse Model of Chemotherapy-Induced Premature Ovarian Failure[J].J Clin Oncol,2007,25(22): 3198-3204.
[24]Fu X,He Y,Xie C,et al.Bone marrow mesenchymal stem cell transplantation improves ovarian function and structure in rats with chemotherapy-induced ovarian damage. Cytotherapy,2008,10(4):353-63.
[25]陈贵安,蔡学泳。人类卵母细胞冻存的临床应用[J]。中国实用妇科与产科杂志,2007,23(1)：30-32。
[26]Lim J M, Fukui Y, Ono H. Developmental competence of bovine oocytes f rozen at various maturation stages followed by in vitro maturation and fertilization[J].Theriogenology, 1992,37:351-361.
[27]Luvoni GC,Pellizzari P.Embryo development in vitro of cat oocytes cryopreserved at different maturation stages[J]. Theriogenology,2000,53(8):1529-40.
[28]宋文妍,孙莹璞,金海霞,等。不同冷冻方法对小鼠不同成熟时期卵母细胞纺锤体的影响[J]。现代妇产科进展,2007,16(5)：362-365.
[29]Ghadami M, Salama S, Khatoon N, et al. Toward gene therapy of primary ovarian failure:adenovirus expressing human FSH receptor corrects the Finnish C566T mutation[J]. MHR-Basic Science of Reproductive Medicine,2008,14(1):9-15.
[30]Dong J, Albertini DF, Nishimori K,et al.Growth differentiation factor-9 is required during early ovarian folliculogenesis[J].Nature,1996,383:531-535.
[31]Shimizu T, Miyahayashi Y, Yokoo M, et al. Molecular cloning of porcine growth differentiation factor 9(GDF-9) cDNA and its role in early folliculogenesis:direct ovarian injection of GDF-9 gene fragments promotes early folliculogenesis[J]. Reproduction,2004, 128:537-543.
[32]Wu X, Chen L, Brown CA, et al. Interelationship of growth differentiation factor 9 and inhibin in early folliculogenesisand ovarian tumorigenesis in mice. Mol Endocrinol,2004, 18(6):1509-19.
[33]Horejsi J,Novakova D,Martinek J. Circulating ovarian autoantibodies and FSH and LH levels in adolescent girls with primary menstrual cycle disorders[J]. J Pediatr Adolesc Gynecol,1996,9:74-78.
[34]付莉,黄冰玉,赵怡璇,等。口服耐受对实验性卵巢早衰抗透明带抗体的抑制作用研究[J]。现代妇产科进展,2006,15(9)：681-683。
[35]jinyao L,huali J,fuchun Z et al. Treatment of autoimmune ovarian disease by co-administration with mouse zona pellucida protein 3 and DNA vaccine through induction of adaptive regulatory T cells[J]. J Gene Med,2008,10(7):810-820.