Rg1延缓D-gal POF小鼠模型生殖系统病理损害、改善生殖功能及其机制的研究

发布时间：2018-03-05 00:23

本文选题：卵巢早衰　切入点：人参皂苷Rg1　出处：《重庆医科大学》2017年博士论文　论文类型：学位论文

【摘要】：目的:人参皂苷Rg1(Ginsenoside Rg1)是从中药人参中提取出来的单体成分,具有抗衰老、抗氧化、抗纤维化的功能,并具有类雌激素样活性。与现在常用治疗卵巢早衰(premature ovarian failure POF)的方法激素补充治疗(menopausal hormone therapy MHT)比较副作用较小,并可能改善POF的生殖功能。本研究采用致衰剂D-半乳糖(D-galactose D-gal)建立POF小鼠模型,在建模过程中,当动情周期紊乱时加用Rg1进行治疗,观察其治疗效果,希望为恢复卵巢功能寻找一种新的治疗方法并探讨其作用机制。方法:1、建立POF小鼠模型:选取具有正常动情周期C57小鼠,分为四组,每组10只,四组分别是:正常对照组、低剂量组(D-gal100mg/kg/d)、中剂量组(D-gal 200mg/kg/d)、高剂量组(D-gal300mg/kg/d)。处理过程中观察动情周期;眼眶取血测血清卵泡刺激素(follicle-stimulating hormone FSH),雌二醇(Estradiol 2 E2)、抗苗勒管激素(Anti-Mullerian hormone AMH);称取小鼠体重,卵巢和子宫的重量,计算子宫卵巢的重量系数,HE染色计数各级卵泡数和黄体数,筛选出最佳建模剂量。2、Rg1对生殖系统的作用:选取有正常动情周期的C57小鼠100只,分为四组,每组25只,四组分别是:生理盐水组,Rg1+D-gal组/延缓衰老组,Rg1组,D-gal组/衰老组。观察动情周期变化;眼眶取血测血清性激素;称取小鼠重量,子宫卵巢重量,计算子宫卵巢重量系数;子宫卵巢HE染色,计数各级卵泡和黄体数,观察子宫内膜厚度;子宫卵巢透射电镜观察超微结构变化;检测子宫和卵巢抗氧化指标总超氧化物歧化酶(superoxide dismutase T-SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase GSH-Px),氧化指标丙二醛(malondialdehyde MDA)和炎性指标白介素-1β(Interleukin-1βIL-1β)、白介素-6(interleukin-6 IL-6)、肿瘤坏死因子-α(tumor necrosis factor-αTNF-α);免疫组化检测卵巢颗粒细胞上卵泡刺激素受体(follicle-stimulating hormone receptor FSHr)的表达,并进行定量分析;WB检测卵巢FSHr、P53、P21、P19、P16蛋白的表达量;RT-PCR检测卵巢衰老因子P53、P21、stk,子宫衰老因子Bax、bcl-2 m RNA的表达;合笼实验,每组选取10只小鼠与雄鼠合笼,计数3个分娩周期幼鼠的数量;合笼实验前后D-gal组小鼠肺、肝脏、脾、肾脏HE染色检查,比较其形态学变化。结果:1、复制POF小鼠模型D-gal剂量的筛选实验中,中剂量和高剂量组FSH明显升高,E2、AMH明显降低,卵泡数和黄体数都明显减少,均达到了POF的标准,故本实验后续研究采用中剂量组。2、Rg1对生殖系统的作用研究结果:从注射D-gal的第三周开始衰老组动情周期紊乱,延缓衰老组在整个处理过程中动情周期无明显变化;衰老组卵巢、子宫的重量系数低于延缓衰老组,延缓衰老组与Rg1和生理盐水组无明显差异;卵巢HE染色,衰老组各级卵泡数和黄体数明显减少,延缓衰老组卵泡和黄体数恢复正常,透射电镜衰老组卵泡颗粒细胞中见大量凋亡小体,间质中见坏死组织,延缓衰老组卵泡颗粒细胞未见明显凋亡小体,间质中未见坏死组织;子宫透射电镜,衰老组子宫肌层见大量纤维化,子宫内膜变薄,延缓衰老组子宫的纤维化程度减轻,内膜见腺体结构;衰老组子宫和卵巢上抗氧化指标T-SOD、GSH-Px明显降低,氧化指标MDA明显升高;炎性指标IL-1β、IL-6、TNF-α升高;颗粒细胞上FSHr蛋白的表达量降低;卵巢上P53、P21、P19、P16蛋白表达量明显升高;卵巢上衰老基因P53、P21、stk,子宫上衰老因子Bax、bcl-2 m RNA的表达量明显升高。然而,与衰老组相比较,延缓衰老组抗氧化指标明显升高,氧化指标,炎性指标明显降低,颗粒细胞上FSHr蛋白的表达量增高,子宫和卵巢上衰老因子蛋白和m RNA的表达明显降低。延缓衰老组与生理盐水组,Rg1组在上述指标比较无明显差异。合笼实验前,衰老组肺、肝脏、肾脏、脾HE染色都有明显的炎性改变,经合笼试验这段时间的修复,衰老组肺、肝脏、肾脏、脾的炎性改变已经明显恢复,但是检测三周期分娩幼鼠的数量,衰老组分娩幼鼠的数量随分娩次数增多而逐渐减少,D-gal对生殖功能的影响并没有随时间延长而好转。但是,延缓衰老组分娩幼鼠的数量并未受影响。结论:Rg1通过抗氧化,抗炎性损伤减轻POF小鼠模型卵巢和子宫的病理损害,并通过上调颗粒细胞上FSHr的表达,减少颗粒细胞凋亡,抑制卵泡闭锁,下调衰老信号通路P53-P21-P19/stk、P16、Bax-bcl-2表达改善POF小鼠模型的生殖功能。
[Abstract]:Objective: ginsenoside Rg1 (Ginsenoside Rg1) is a monomer component extracted from ginseng, has anti-aging, anti-oxidation, anti fibrosis function, and has estrogenic activity. And now commonly used in the treatment of premature ovarian failure (premature ovarian failure POF) method of hormone replacement therapy (menopausal hormone therapy MHT comparison) the side effect is small, and may improve the reproductive function of POF. This study used D- galactose induced aging agent (D-galactose D-gal) POF mice model was established, in the modeling process, when the estrous cycle dysfunction with Rg1 in the treatment, observe the treatment effect, hope for the recovery of ovarian function to find a new treatment method and to explore its mechanism. Methods: 1, the establishment of POF mouse model: selection with normal estrous cycle of C57 mice were divided into four groups, 10 rats in each group, four groups: normal control group, low dose group (D-gal100mg/kg/d), The middle dose group (D-gal 200mg/kg/d), high dose group (D-gal300mg/kg/d). To observe the estrous cycle process; orbital blood serum follicle stimulating hormone (follicle-stimulating hormone FSH), estradiol (Estradiol 2 E2), anti Mullerian hormone (Anti-Mullerian hormone AMH); the weight of ovary and uterus in mice. Weight, weight coefficient of uterus and ovary, HE staining the follicle number and the number of corpus luteum, screened the best modeling dose of.2, the effect of Rg1 on the reproductive system: the normal oestrous cycle of 100 C57 mice were divided into four groups, 25 rats in each group, four groups were: normal saline group, Rg1+D-gal group / aging group, Rg1 group, D-gal group / aging group. Observe the changes of estrous cycle; orbital blood serum levels of sex hormones; that the mice weight, weight of uterus and ovary, uterus and ovary weight coefficient calculation; uterine and ovarian HE staining, counting all levels of follicle and corpus luteum The number of observations, the thickness of endometrium; uterine and ovarian transmission electron microscopy to observe the ultrastructural changes of uterus and ovary; detection of antioxidant index of total superoxide dismutase (superoxide dismutase T-SOD), glutathione peroxidase (glutathione peroxidase GSH-Px), oxidation index (malondialdehyde MDA) and the content of inflammatory markers interleukin -1 beta (Interleukin-1 beta beta IL-1), interleukin -6 (interleukin-6 IL-6), tumor necrosis factor alpha (tumor alpha TNF- alpha necrosis factor-); immunohistochemical detection of ovarian granulosa cells of follicle stimulating hormone receptor (follicle-stimulating hormone receptor FSHr) expression and quantitative analysis; WB P53, P21 detection of ovarian FSHr, P19, P16 expression the detection of P53 protein; ovarian aging factor RT-PCR P21, STK, uterine aging factor Bax, the expression of Bcl-2 m RNA; cage experiment, each group selected 10 mice and male rats were mated, counting 3 delivery The number of cycles in the cage; before and after the experiment group D-gal mice lung, liver, spleen, kidney HE staining and compared the morphological changes. Results: 1, experimental screening copy POF mouse model of D-gal dose, middle dose and high dose group significantly increased FSH, E2, AMH decreased significantly, the number of follicles and corpus luteum number all decreased, reached the standard of POF, this study used in subsequent experiments in middle dose group.2, Rg1 results on the reproductive system of the role of research: from the beginning of the third week old D-gal injection group estrous cycle disorder, aging group in the whole treatment process in the estrous cycle had no obvious change; aging group ovarian weight. The coefficient of uterus aging below group, aging group with Rg1 and saline group had no significant difference; ovarian HE staining, aging group numbers of follicles and luteal aging group significantly reduced the number of follicles and corpus luteum number returned to normal, TEM attenuation Granulosa cells in old group showed a large number of apoptotic bodies, necrotic tissue stroma, aging group granulosa cells had no obvious apoptotic bodies, no necrosis in stroma; uterine TEM, aging group myometrial mass fibrosis, endometrial thinning and fibrosis in delaying aging group uterus reduced endometrial gland structure; aging group of uterus and ovary on antioxidant indexes of T-SOD, GSH-Px decreased significantly, MDA oxidation index increased obviously; inflammatory index of IL-1 beta, IL-6, TNF- were elevated; granulosa cells on the expression of FSHr protein is lower; ovary P53, P21, P19, P16 protein expression was significantly increased; aging gene P53, P21 STK, ovary, uterus on aging factor Bax, expression of Bcl-2 m RNA were obviously increased. However, compared with the aging group, aging group significantly increased the antioxidant index, oxidation index, inflammatory index decreased significantly, fine particles The amount of FSHr protein expression in cells increased, uterus and ovary aging factor expression protein and m RNA decreased significantly. Aging group and the saline group, Rg1 group in the above indexes had no significant difference. Cage aging group before the experiment, lung, liver, kidney, spleen and HE staining have obvious inflammatory the change of the repair cage test this time, aging group lung, liver, kidney, spleen inflammatory change has obvious recovery, but the number three in the delivery cycle detection, the number of aging group were decreased as the delivery delivery number increase, the effect of D-gal on reproductive function and not with time better. However, the number of aging group in delivery was not affected by Rg1. Conclusion: the antioxidant, reduce the pathological damage of POF mice model of ovarian and uterine inflammatory injury, and increase the expression of FSHr on granulosa cells, reduced granulosa cells Apoptosis, inhibition of follicle atresia, and down regulation of senescence signal pathway P53-P21-P19/stk, P16, Bax-bcl-2 expression improve the reproductive function of POF mice model.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R711.75

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