【作者】 张丽娟；

【导师】 赵丕文；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2015， 硕士

【摘要】 研究背景卵巢早衰(Premature Ovarian Failure, POF)是指月经初潮正常或青春期延迟,第二特征发育正常的女性在40岁以前出现持续性闭经和性器官萎缩,并伴有卵泡刺激素和黄体生成素升高,而雌激素降低的综合征,为妇科常见的内分泌疾病。流行病学调查显示POF在40岁之前的发病率为1/100,30岁之前为1/1000,20岁之前为1/10000,且发病率呈逐年上升趋势。POF可导致不孕不育及低雌激素状态,也可导致一些其他疾病,严重影响患者生活,影响女性生理和心理健康。卵巢早衰成为当今医学研究的热点和难点。二仙汤是是经过长期临床实践证明疗效确凿的著名方剂。组方药物为：仙茅、淫羊藿、巴戟天、当归、知母、黄柏。在二仙汤中仙茅、淫羊藿温肾阳；巴戟天补肾壮阳,又能补益精血；当归滋肾补肝,活血养血；知母、黄柏滋肾阴,泻相火,对卵巢早衰的临床症状具有明显的改善作用。也常用于更年期综合征,闭经以及其他慢性病。从而成为治疗妇科病证中运用广泛的方剂之一。实验一 顺铂建立大鼠卵巢早衰的实验研究目的：探讨采用临床常用药物顺铂建立化疗性损伤卵巢早衰动物模型的方法,通过不同剂量的顺铂腹腔注射,造成大鼠卵巢功能的损伤,确定最适宜的顺铂浓度,作为建立顺铂所致大鼠卵巢早衰的模型的剂量,并且用于下一步实验,也为今后实验研究提供帮助。方法：筛选出动情周期正常的60只健康雌性大鼠进入实验,随机分为正常组,低剂量组和高剂量组,每组20只。第1、第8天分别按低剂量组和高剂量组分别4mg/kg、6mg/kg腹腔注射顺铂注射液,正常组于同一时间腹腔注射1ML的生理盐水。造模期间每天定时进行阴道脱落细胞涂片实验,观察动情周期的变化及大鼠各项生命体征并记录体重变化。全部大鼠于第二次注射后7d进行取血,测血清中卵泡刺激素(FSH)、雌二醇(E2)、孕酮(Pro)和黄体生成素(LH)水平含量变化。取卵巢组织行病理切片,进行HE染色观察卵巢形态学。结果：(1)大鼠体重给药前各组无明显变化,无统计学差异。给药后不同剂量组大鼠体重均有不同程度下降,且行动缓慢,喜弓背、蜷缩,食欲下降,体毛晦暗无光泽,与正常组比较在注射顺铂8d后高剂量组体重较低剂量组明显下降,正常组体重平稳增长。(2)给药后,正常组大鼠动情周期仍然规律,低剂量组用药后动情周期紊乱,动情间期延长,动情缩短。但停用药一周后部分大鼠动情周期有所恢复。高剂量组大鼠动情周期延长,甚至长期停留在动情间期,不出现动情期。停药后一周动情周期没有恢复。(3)高剂量组与正常组比较大鼠血的FSH值升高有统计学意义(P<0.05)。低剂量组血清中FSH较正常组没有明显差异(P>0.05)。顺铂对大鼠血清中Pro含量的影响,给药前后各组均无明显差异(P>0.05)。E2水平在给药后两组均有下降,与正常组比较有统计学意义(P<0.05)。(4)正常组大鼠卵巢增大,各级卵泡生长活跃,闭锁卵泡很少,且卵泡颗粒细胞层较多。而低剂量组原始卵泡和初级卵泡减少,闭锁卵泡增加,黄体数量减少。高剂量组可见很少次级卵泡和成熟卵泡,大多数为闭锁卵泡,卵泡颗粒细胞层明显变薄,黄体明显减少。结论：本实验研究给予大鼠不同剂量的顺铂注射后,卵巢功能出现不同程度的损伤,而高剂量组模型更为理想。本实验研究为建立稳定的卵巢早衰动物模型提供一种操作简单,成功率高,结果可靠的实验方法。(2)给药后,正常组大鼠动情周期仍然规律,低剂量组用药后动情周期紊乱,动情间期延长,动情缩短。但停用药一周后部分大鼠动情周期有所恢复。高剂量组大鼠动情周期延长,甚至长期停留在动情间期,不出现动情期。停药后一周动情周期没有恢复。(3)高剂量组与正常组比较大鼠血的FSH值升高有统计学意义(P<0.05)。低剂量组血清中FSH较正常组没有明显差异(P>0.05)。顺铂对大鼠血清中Pro含量的影响,给药前后各组均无明显差异(P>0.05)。E2水平在给药后两组均有下降,与正常组比较有统计学意义(P<0.05)。(4)正常组大鼠卵巢增大,各级卵泡生长活跃,闭锁卵泡很少,且卵泡颗粒细胞层较多。而低剂量组原始卵泡和初级卵泡减少,闭锁卵泡增加,黄体数量减少。高剂量组可见很少次级卵泡和成熟卵泡,大多数为闭锁卵泡,卵泡颗粒细胞层明显变薄,黄体明显减少。结论：本实验研究给予大鼠不同剂量的顺铂注射后,卵巢功能出现不同程度的损伤,而高剂量组模型更为理想。本实验研究为建立稳定的卵巢早衰动物模型提供一种操作简单,成功率高,结果可靠的实验方法。实验二二仙汤对大鼠化疗性损伤卵巢颗粒细胞凋亡的影响目的：本实验将以与卵泡凋亡有关的细胞因子和基因为研究主线,根据实验一原理建立化疗性损伤卵巢早衰动物模型,探讨二仙汤对卵泡生长、发育和凋亡的影响,通过整体动物实验,在细胞凋亡的分子水平阐明二仙汤治疗卵巢早衰的作用机制,为研究抗卵巢早衰新药提供理论和实验根据。方法：健康雌性SD大鼠随机分为4组,分别为正常组、二仙汤组、模型组及阳性对照组。除正常组外,按实验一造模方法每组6 mg/kg-1腹腔注射顺铂注射液进行造模。造模成功后连续4周每天上午下午同一时间给予正常组与模型组分别灌胃1ML的蒸馏水,雌激素组灌胃1ML的补佳乐溶液,二仙汤组灌胃1ML配制的二仙汤。每日进行体重及动情周期观察记录。取血清测血清中FSH、E2、Pro和LH水平含量及AMH和INH-B水平含量。取卵巢组织进行HE染色进行形态学观察及各级卵泡计数；TUNEL法检测卵泡颗粒细胞凋亡情况；免疫组化技术检测BAX、Bcl-2和VEGF和蛋白的表达；原位杂交技术检测IL-2基因的表达。结果：(1)正常组体重增长速度较快且平稳。模型组注射顺铂前,体重增长较平均,注射后体重明显下降,后给予灌胃蒸馏水体重无明显变化。中药组给予灌胃二仙汤治疗后体重略有增加。阳性组灌胃雌激素后,体重明显且大幅增加。(2)与正常组比较造模后各组大鼠FSH升高,HL升高,E2降低有统计学意义(P<0.05)；与模型组比较二仙汤组灌胃给药后E2升高,FSH降低有统计学意义；阳性组与模型组比较FSH和HL改变无统计学差异,E2升高有统计学意义(P<0.05)。(3)镜下观察模型组与正常组比较总卵泡数减少,整体而言各组间有差异。正常组比较模型组很少次级卵泡和成熟卵泡,大多数为闭锁卵泡,卵泡颗粒细胞层明显变薄,黄体明显减少。二仙汤组生长卵泡数量较模型组增多,以次级卵泡为主。(4)从凋亡细胞形态判断,模型组与正常比较凋亡颗粒细胞数目明显增多,且细胞质可见着棕黄色,卵泡发育不良。二仙汤组治疗后与模型组比较阳性表达明显减少,阳性组较模型组无明显差异。(5)模型组颗粒细胞BAX蛋白表达量比正常组显著增多,二仙汤组与模型比较表达减少；造模后模型组颗粒细胞Bc1-2蛋白表达量低于模型组,灌胃二仙汤后二仙汤组Bc1-2蛋白表达增加,而二仙汤组与阳性组无显著差异；模型组血管内皮细胞VEGF蛋白表达量明显下降。二仙汤组较模型组表达增多,阳性组表达略有恢复；正常组表达微少,模型组IL-2蛋白表达量比正常组显著增多,二仙汤组和阳性对照组比模型组表达减少。结论：实验证明二仙汤可降低卵巢早衰大鼠自身免疫,减少卵巢抗原靶细胞损伤,保护卵巢功能。也通过调节血清中各激素含量,改变凋亡细胞相关蛋白的表达量来降低顺铂对卵巢的损害,促进卵泡发育,改善并增强卵巢功能。更多还原

【Abstract】 Research backgroundPremature Ovarian Failure (POF) is a syndrome that persistent amenorrhoea and atrophy of the sexual organs with increase in follicle-stimulating hormone and luteinizing hormone and decrease in estrogen before forty years old occur to women whose menstruation menarche is normal or puberty is postponed and secondary sex characteristics are well developed. Epidemiological studies have established that morbidity of POF is1/100 before 40,1/1000 before 30,1/10000 before 20 and it is increasing annually. POF can cause infertility, low estradiol level and other diseases which can affect women’s life and physiological and mental health.So POF has become a hot but difficult medical research.Er-xian decoction can improve clinical symptoms of POF and it is a famous and useful prescription by clinical practice.lt is composed of Xianmao, Yinyanghuo, Bajitian, Danggui, Zhimu and Huangbai. Xianmao and Yinyanghuo can warm the kidney-yang. Bajitian can invigorate kidney and strengthen yang, enrich and benefit essence and blood. Danggui can strengthen liver and kidney, activate and nourish blood. Zhimu and Huangbai can nourish kidney-yin and clear away ministerial fire. Moreover, Er-xian decoction is useful for climactic syndrome, amenorrhoea and other chronic diseases. Therefore Er-xian decoction becomes a wide-used prescription in gynaecopathia treatment.Experiment One Study of establishing the rat animal model of premature ovarian failure caused by cisplatinObjective:To investigate the common method of animal model of chemotherapy-induced premature ovarian failure caused by cisplatin, we intraperitoneally injected different volume of cisplatin to cause the damage of ovarian function of rats, so that we could ensure the most suitable concentration for establishment of rat animal model of premature ovarian failure caused by cisplatin.The result can be used in next experiment and helpful for later study.Methods:60 healthy female rats with normal estrous cycle were randomly divided into normal group, low-dose group and high-dose group.Each group has 20 rats. The low-dose group and high-dose group were intraperitoneally injected with cisplatin of 4 mg/kg-1 and 6 mg/kg-1, while normal group were intraperitoneally injected with 1ML normal saline on the 1st and 8th day. During the days, we observed vaginal exfoliated cells of rats to study the change of estrous cycle and vital signs, recorded the weights.7 days after the 2nd injection, we took rat serum for determination of FSH, E2, Pro and LH and ovary for pathological section to observe ovarian tissue morphology by HE coloration.Results:(1) Before injection, weight of rats is not obviously different. After injection, weight of rats in low-dose group and high-dose group were descreased in different degrees. They drumbled, liked arching back and huddling up, lost appetite and their fur was dull. Compared with low-dose group, weight of rats in high-dose group is lighter after 2nd injection, while weight of rats in normal group grows stably.(2) After injection, estrous cycle of rats in normal group was normal, while estrous cycle of rats in low-dose group was disordered like longer diestrus and shorter oestrus. But estrous cycle of some rats recovered a week after 2nd injection. However, estrous cycle of rats in high-dose group was longer and even stayed at diestrus without oestrus. Estrous cycle of rats in high-dose group didn’t recover.(3) Compared with normal group, level of FSH in rats in high-dose group elevated (P<0.05), level of FSH in rats in low-dose group elevated not obviously(P>0.05). Level of Pro is not different in each group before and after injection (P>0.05). Compared with normal group, level of E2 in rats in high-dose group and low-dose group decreased (P<0.05).(4) Ovaries of rats in normal group were bigger, follicles of different developing stages were developing well, atretic follicles were few and granulosa cells were full. Primordial follicles and primary follicles in low-dose group decreased, atretic follicles increased, and corpus luteums decreased. Secondary follicles and mature follicles were few and there were many atretic follicles. Granulosa cells were fewer and corpus luteums decreased obviously.Conclusion:After injection of different doses of cisplatin, ovarian function of rats was damaged in different degrees and the effection in high-dose group was more. Our experiment showed an easier and more reliable way to establish animal model of premature ovarian failure.Experiment Two Effect of Er-xian decoction in follicular granulosa cell apoptosis in chemotherapy-induced ovarian failure of ratsObjective:This experiment was on the base on follicle apoptosis related cytokines and genes for the study of the main line, according to Experiment One to establish the animal model of premature ovarian failure, to investigate the Er-xian decoction on the effects of follicle growth, development and apoptosis. Through the whole animal experiment, mechanism of action of treatment of Er-xian decoction on ovarian premature failure was elucidated on the the molecular level of cell apoptosis, theory and experimental basis for the study of new drugs for ovarian premature failure was provided.Methods:Healthy female SD rats were randomly divided into normal group, Erxian Decoction group, model group and positive group. Besides normal group, other groups were intraperitoneally injected with cisplatin of 6 mg/kg. After that, normal group and model group were intragastric infusion with 1ML distilled water, positive group was with 1ML progynova and Erxian Decoction group was with 1ML Erxian Decoction for four weeks at the same time every morning and afternoom. We observed general condition and estrous cycle of rats; took rats serum for determination of FSH, E2, Pro, LH, AMH and INH-B; observation of ovarian tissue morphology by HE coloration and count of all levels of follicle; analysis of granulosa cell apoptosis by TUNEL; the expression of immunohistochemical detection of BAX, Bcl-2 and VEGF protein.; the expression of IL-2 by in situ hybridization.Results:(1) Weight of rats in normal group grows fast and stably. Before injection, weight of rats is not obviously different. After injection, weight of rats in Erxian Decoction group, model group and positive group descreased obviously. After intragastric infusion with distilled wate, weight of rats didn’t change obviously. After intragastric infusion with Erxian Decoction, weight of rats gained slightly. After intragastric infusion with progynova, weight of rats gained obviously.(2) Compared with normal group, LH elevated and E2 decreased in other groups(P<0.05). Compared with model group, E2 elevated and FSH decreased in Erxian Decoction group. FSH and LH in model group and positive group were not different obviously, while E2 of model group decreased(P<0.05).(3) The total number of follicles decreased in model group compared to normal group, there are differences between groups on the whole. Compared with normal group, there were rarely secondary follicles or mature follicles, mostly atresia follicles, follicular granulosa cells layer was significantly thin, corpus luteum decreased significantly in model group. Compared with model group, Erxian Decoction group had more follicles, mostly were secondary follicles.(4) Judging from apoptotic cells form, the model group compared with the normal group, the number of apoptotic granulosa cells increased obviously, and the cytoplasm visible tan, follicular dysplasia. After Erxian Decoction treatment, positive expression decreased significantl. There was no obvious difference between positive group and model group.(5) The expression of BAX significantly increased. The expression of Bcl-2 significantly decreased in model group compared to normal group. After treatment of Er-Xian decoction, the expression of BAX decreased, expression of Bcl-2 increased. There was no significant difference between positive group and Er-Xian decoction group. The expression of VEGF protein in model group was obviously lower than the normal group, which in positive group and Er-Xian decoction group was higher. The expression of IL-2 was little, it is more in model group and less in positive group and Er-Xian decoction group compared to model group.Conclusion:The experiment proves that Er-Xian decoction can reduce autoimmune and ovarian antigen target cell damage in ovarian premature failure rats, to protect the ovarian function. By adjusting the hormone content in serum and changing the expression of related proteins in apoptotic cells, Er-Xian decoction can reduce the damage to the ovaries by cisplatin, promote the follicular development and improve and enhance the ovarian function.更多还原