本文選題：卵巢早衰　切入點：人參皂苷Rg1　出處：《重慶醫(yī)科大學》2017年博士論文　論文類型：學位論文

【摘要】：目的:人參皂苷Rg1(Ginsenoside Rg1)是從中藥人參中提取出來的單體成分,具有抗衰老、抗氧化、抗纖維化的功能,并具有類雌激素樣活性。與現(xiàn)在常用治療卵巢早衰(premature ovarian failure POF)的方法激素補充治療(menopausal hormone therapy MHT)比較副作用較小,并可能改善POF的生殖功能。本研究采用致衰劑D-半乳糖(D-galactose D-gal)建立POF小鼠模型,在建模過程中,當動情周期紊亂時加用Rg1進行治療,觀察其治療效果,希望為恢復卵巢功能尋找一種新的治療方法并探討其作用機制。方法:1、建立POF小鼠模型:選取具有正常動情周期C57小鼠,分為四組,每組10只,四組分別是:正常對照組、低劑量組(D-gal100mg/kg/d)、中劑量組(D-gal 200mg/kg/d)、高劑量組(D-gal300mg/kg/d)。處理過程中觀察動情周期;眼眶取血測血清卵泡刺激素(follicle-stimulating hormone FSH),雌二醇(Estradiol 2 E2)、抗苗勒管激素(Anti-Mullerian hormone AMH);稱取小鼠體重,卵巢和子宮的重量,計算子宮卵巢的重量系數(shù),HE染色計數(shù)各級卵泡數(shù)和黃體數(shù),篩選出最佳建模劑量。2、Rg1對生殖系統(tǒng)的作用:選取有正常動情周期的C57小鼠100只,分為四組,每組25只,四組分別是:生理鹽水組,Rg1+D-gal組/延緩衰老組,Rg1組,D-gal組/衰老組。觀察動情周期變化;眼眶取血測血清性激素;稱取小鼠重量,子宮卵巢重量,計算子宮卵巢重量系數(shù);子宮卵巢HE染色,計數(shù)各級卵泡和黃體數(shù),觀察子宮內(nèi)膜厚度;子宮卵巢透射電鏡觀察超微結(jié)構(gòu)變化;檢測子宮和卵巢抗氧化指標總超氧化物歧化酶(superoxide dismutase T-SOD)、谷胱甘肽過氧化物酶(glutathione peroxidase GSH-Px),氧化指標丙二醛(malondialdehyde MDA)和炎性指標白介素-1β(Interleukin-1βIL-1β)、白介素-6(interleukin-6 IL-6)、腫瘤壞死因子-α(tumor necrosis factor-αTNF-α);免疫組化檢測卵巢顆粒細胞上卵泡刺激素受體(follicle-stimulating hormone receptor FSHr)的表達,并進行定量分析;WB檢測卵巢FSHr、P53、P21、P19、P16蛋白的表達量;RT-PCR檢測卵巢衰老因子P53、P21、stk,子宮衰老因子Bax、bcl-2 m RNA的表達;合籠實驗,每組選取10只小鼠與雄鼠合籠,計數(shù)3個分娩周期幼鼠的數(shù)量;合籠實驗前后D-gal組小鼠肺、肝臟、脾、腎臟HE染色檢查,比較其形態(tài)學變化。結(jié)果:1、復制POF小鼠模型D-gal劑量的篩選實驗中,中劑量和高劑量組FSH明顯升高,E2、AMH明顯降低,卵泡數(shù)和黃體數(shù)都明顯減少,均達到了POF的標準,故本實驗后續(xù)研究采用中劑量組。2、Rg1對生殖系統(tǒng)的作用研究結(jié)果:從注射D-gal的第三周開始衰老組動情周期紊亂,延緩衰老組在整個處理過程中動情周期無明顯變化;衰老組卵巢、子宮的重量系數(shù)低于延緩衰老組,延緩衰老組與Rg1和生理鹽水組無明顯差異;卵巢HE染色,衰老組各級卵泡數(shù)和黃體數(shù)明顯減少,延緩衰老組卵泡和黃體數(shù)恢復正常,透射電鏡衰老組卵泡顆粒細胞中見大量凋亡小體,間質(zhì)中見壞死組織,延緩衰老組卵泡顆粒細胞未見明顯凋亡小體,間質(zhì)中未見壞死組織;子宮透射電鏡,衰老組子宮肌層見大量纖維化,子宮內(nèi)膜變薄,延緩衰老組子宮的纖維化程度減輕,內(nèi)膜見腺體結(jié)構(gòu);衰老組子宮和卵巢上抗氧化指標T-SOD、GSH-Px明顯降低,氧化指標MDA明顯升高;炎性指標IL-1β、IL-6、TNF-α升高;顆粒細胞上FSHr蛋白的表達量降低;卵巢上P53、P21、P19、P16蛋白表達量明顯升高;卵巢上衰老基因P53、P21、stk,子宮上衰老因子Bax、bcl-2 m RNA的表達量明顯升高。然而,與衰老組相比較,延緩衰老組抗氧化指標明顯升高,氧化指標,炎性指標明顯降低,顆粒細胞上FSHr蛋白的表達量增高,子宮和卵巢上衰老因子蛋白和m RNA的表達明顯降低。延緩衰老組與生理鹽水組,Rg1組在上述指標比較無明顯差異。合籠實驗前,衰老組肺、肝臟、腎臟、脾HE染色都有明顯的炎性改變,經(jīng)合籠試驗這段時間的修復,衰老組肺、肝臟、腎臟、脾的炎性改變已經(jīng)明顯恢復,但是檢測三周期分娩幼鼠的數(shù)量,衰老組分娩幼鼠的數(shù)量隨分娩次數(shù)增多而逐漸減少,D-gal對生殖功能的影響并沒有隨時間延長而好轉(zhuǎn)。但是,延緩衰老組分娩幼鼠的數(shù)量并未受影響。結(jié)論:Rg1通過抗氧化,抗炎性損傷減輕POF小鼠模型卵巢和子宮的病理損害,并通過上調(diào)顆粒細胞上FSHr的表達,減少顆粒細胞凋亡,抑制卵泡閉鎖,下調(diào)衰老信號通路P53-P21-P19/stk、P16、Bax-bcl-2表達改善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.

【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R711.75

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