本文選題：Hippo信號通路 + 卵巢生殖上皮細(xì)胞��； 參考：《南昌大學(xué)》2016年博士論文

【摘要】：立項背景如今,隨著老齡化社會的出現(xiàn),衰老問題逐漸被人們所重視。在女性,卵巢衰老的速度要明顯快于機體其它器官。卵巢衰老影響生殖健康,引發(fā)絕經(jīng)期的到來、癌癥和多種慢性疾病的發(fā)生。然而,隨著生存環(huán)境的日益惡化、生活壓力不斷加大及藥物治療副作用等因素對女性卵巢功能的損害,使卵源性不孕癥及病理性卵巢功能衰退已成為困擾現(xiàn)代女性的重大社會衛(wèi)生問題。特別是隨著二胎政策的實施,高齡女性對于生活質(zhì)量以及生育壽命的需求日益強烈。故闡明卵巢功能的生理及病理性減退機制、建立有效的干預(yù)手段,已成為醫(yī)學(xué)界刻不容緩的研究課題!近年來卵巢生殖干細(xì)胞(ovarian germline stem cells,OGSCs)的發(fā)現(xiàn)能為有效地增加原始卵泡池、改善卵巢功能、延緩卵巢衰老帶來前所未有的希望!Hippo信號通路是調(diào)控成體干細(xì)胞增殖分化及功能的重要信號通路,我們前期研究發(fā)現(xiàn)Hippo信號通路的主要效應(yīng)因子能觸發(fā)原始卵泡的啟動。但Hippo信號通路是否參與OGSCs功能的調(diào)控以及和生理性及病理性卵巢功能衰老的關(guān)系還有待進(jìn)一步證實。因此本研究中,以KM系小鼠及人卵巢皮層組織為實驗對象:(1)分析Hippo信號通路的主要效應(yīng)因子在卵巢表面上皮(ovarian surface epithelium,OSE)細(xì)胞中的表達(dá)及動態(tài)變化,這種變化與卵巢功能生理性衰老和病理性衰老的關(guān)系;(2)離體觀察Hippo信號通路的主要效應(yīng)因子YAP1在OGSCs增殖分化中的作用;(3)在體分析YAP1經(jīng)卵巢內(nèi)微注射對卵巢功能生理性衰老和病理性衰老的拯救作用。通過實驗為深入探討成年哺乳動物OGSCs的自我更新、分化及衰老等的生物學(xué)過程提供重要材料,為不孕癥的治療、重塑和保護(hù)卵巢功能提供技術(shù)方法和新的藥物作用靶點。第一部分Hippo信號通路主要分子在生理性衰老和病理性衰老卵巢生殖上皮細(xì)胞中的表達(dá)及動態(tài)變化研究目的分析Hippo信號通路的主要分子在卵巢生殖上皮(OSE)細(xì)胞中的表達(dá)及動態(tài)變化,這種變化與卵巢功能生理性衰老和病理性衰老的關(guān)系。研究方法選用7日齡(7days,7D)、2月齡(2months,2M)、10月齡(10months,10M)和20月齡(20months,20M)小鼠卵巢,以及生育旺盛期(青春期后-35歲)、中年期(36-50歲)和絕經(jīng)期(51-60歲)人卵巢皮層樣本為生理性衰老卵巢實驗標(biāo)本;通過雷公藤灌胃(Tripterygium Glycosides,TPT)及環(huán)磷酰胺/馬利蘭片(Cyclophosphamide/Busulfan,CY-BUS)腹腔注射方式,構(gòu)建病理性衰老卵巢模型。分別經(jīng)HE染色,免疫化學(xué)方法及分子生物學(xué)方法等技術(shù),比較Hippo信號通路的主要分子(LATS2,MST1與YAP1)在OSE細(xì)胞中的表達(dá)及動態(tài)變化,以及這種變化與卵巢功能生理性衰老和病理性衰老的關(guān)系。研究結(jié)果(1)在不同年齡小鼠OSE中存在Hippo信號通路相關(guān)分子與OGSCs標(biāo)記因子(MVH與OCT4)共表達(dá)。其中,抑癌因子LATS2,MST1與MVH/OCT4的表達(dá)隨著年齡的增加而逐漸降低;原癌因子YAP1則在2M小鼠OSE中表達(dá)量最高,7D齡其次,20M最低;p YAP1表達(dá)則為7D最高,且p YAP1/YAP1比值隨年齡增加而逐漸降低。(2)在病理性卵巢衰老模型小鼠OSE中也存在Hippo信號通路相關(guān)分子與OGSCs標(biāo)記因子(MVH與OCT4)共表達(dá)。其中,YAP1,MST1與MVH/OCT4在雷公藤和環(huán)磷酰胺/馬利蘭片兩組藥物造成的衰老模型組中均表現(xiàn)為逐漸降低,而LATS2的表達(dá)則呈現(xiàn)出相反趨勢。p YAP1在兩組病理性卵巢衰老小鼠卵巢中均顯著降低,而p YAP1/YAP1的比值則均顯著升高。(3)在不同年齡階段人OSE中同樣存在Hippo信號通路相關(guān)分子與OGSCs標(biāo)記因子(MVH與OCT4)共表達(dá)。其中,MVH/OCT4隨著年齡的增長而逐漸下降,LATS2表達(dá)為青春期最高,YAP1和p YAP1為中年期最高,而MST1則為老年期最低;p YAP1/YAP1則顯示出從青年期到老年期逐漸降低的趨勢。結(jié)論Hippo信號通路相關(guān)分子LATS2,MST1與YAP1和OGSCs標(biāo)記因子MVH與OCT4共表達(dá)于小鼠和人OSE中,兩者表達(dá)的變化存在明顯的時序相關(guān)性,且這種變化與卵巢功能的生理性衰老和病理性衰老亦存在明顯的時序相關(guān)性。表明,Hippo信號通路可能通過調(diào)控OGSCs參與了卵巢功能的生理性衰老和病理性衰老過程。第二部分Hippo信號通路在卵巢生殖干細(xì)胞增殖和分化中的作用研究目的進(jìn)一步完善卵巢生殖干細(xì)胞(OGSCs)的分離、鑒定與培養(yǎng)方法;探明Hippo信號通路對OGSCs增殖分化的影響。研究方法采用兩步酶分離方法及特異性抗體磁珠篩選技術(shù)(magnetic activated cell sorting,MASC)對OGSCs進(jìn)行分離,通過測定MVH,OCT4,Nango,Fraglis,Stella等多種干細(xì)胞和生殖細(xì)胞特異性標(biāo)記因子對OGSCs進(jìn)行鑒定,選用本室改良的方法對OGSCs進(jìn)行培養(yǎng);將攜帶效應(yīng)因子YAP1的過表達(dá)和相應(yīng)sh RNA的干擾慢病毒載體轉(zhuǎn)染至OGSCs,觀察對OGSCs增殖和分化的影響。研究結(jié)果(1)經(jīng)上述方法分離得到的細(xì)胞能共表達(dá)MVH,OCT4,Nango,Fraglis,Stella等干細(xì)胞和生殖細(xì)胞特異性標(biāo)記因子,故而初步鑒定所得細(xì)胞為OGSCs。(2)LATS2,MST1及YAP1可在OGSCs上表達(dá);OGSCs在轉(zhuǎn)染YAP1過表達(dá)慢病毒載體后,其特異性標(biāo)記因子MVH,OCT4的表達(dá)顯著升高,而在轉(zhuǎn)染YAP1sh RNA的干擾慢病毒載體后,MVH,OCT4的表達(dá)則顯著降低。結(jié)論已初步建立了OGSCs的分離、鑒定與培養(yǎng)方法;Hippo信號通路相關(guān)分子存在于OGSCs,并能調(diào)控OGSCs體外增殖。第三部分Hippo信號通路通過調(diào)控卵巢生殖干細(xì)胞的功能在卵巢功能重塑中的作用初探研究目的在體進(jìn)一步驗證Hippo信號通路通過調(diào)控卵巢生殖干細(xì)胞(OGSCs)增殖和分化在卵巢功能重塑中的作用。研究方法選用Hippo信號通路中主要效應(yīng)因子YAP1為靶基因,通過卵巢內(nèi)微注射將攜帶效應(yīng)因子YAP1的過表達(dá)和相應(yīng)sh RNA的干擾慢病毒載體分別轉(zhuǎn)染至不孕模型和生育旺盛期小鼠卵巢,觀察對卵巢生殖功能和內(nèi)分泌功能的影響。研究結(jié)果(1)不孕模型小鼠在轉(zhuǎn)染過表達(dá)YAP1后的30-75天期間,可以檢測到卵巢內(nèi)有卵泡再生,原始卵泡啟動激活,子代出生率增加,同時伴有血清中E2和FSH水平上升,卵巢OSE細(xì)胞中YAP1與p YAP1表達(dá)和MVH與OCT4表達(dá)均明顯增高。(2)生育旺盛期小鼠在轉(zhuǎn)染YAP1的干擾慢病毒載體后的30-75天期間,檢測到卵巢內(nèi)卵泡數(shù)量下降,子代出生率減少,同時伴有血清中E2和FSH水平下降,卵巢OSE細(xì)胞中YAP1與p YAP1表達(dá)和MVH與OCT4表達(dá)均明顯降低。結(jié)論調(diào)控Hippo信號通路效應(yīng)分子YAP1可在卵巢功能重塑中發(fā)揮重要作用。
[Abstract]:With the emergence of the aging society, aging problems are becoming more and more important. In women, the aging of the ovary is faster than the other organs of the body. The ovarian senescence affects reproductive health, the arrival of the menopause, the occurrence of cancer and a variety of chronic diseases. However, with the worsening of the living environment, the pressure of life is stressed. The damage of the female ovarian function, the oogenic infertility and the pathological ovarian function decline have become the major social health problems that perplex modern women. Especially with the implementation of the second child policy, the demand for the quality of life and the life expectancy of the older women is increasingly strong. The physiological and pathological degeneration mechanism of ovarian function and the establishment of effective intervention methods have become an urgent research topic in the medical field. In recent years, the discovery of ovarian germline stem cells (OGSCs) can increase the original follicle pool, improve the egg nest function and delay the ovarian senility with unprecedented hope! Hip Po signaling pathway is an important signaling pathway to regulate the proliferation, differentiation and function of adult stem cells. Our previous study found that the main effector factor of the Hippo signaling pathway can trigger the priming of the original follicle. But whether the Hippo signaling pathway is involved in the regulation of OGSCs function and the relationship between the physiological and pathological ovarian functional senescence remains to be advanced. In this study, in this study, KM mice and human ovarian cortical tissues were used as experimental subjects: (1) analysis of the expression and dynamic changes of the main effect factors of the Hippo signaling pathway in the epithelial ovarian epithelial (ovarian surface epithelium, OSE) cells, the relationship between this change and the physiological senescence of the ovary and the pathological senescence of the ovary; (2) in vitro The role of the main effect factor YAP1 of Hippo signaling pathway in OGSCs proliferation and differentiation; (3) in vivo analysis of the effect of YAP1 on ovarian functional physiological senescence and pathological senescence in ovary. The experiment provides important material for the biological processes of self-renewal, differentiation and senescence of adult mammalian OGSCs. Materials to provide technical methods and new drug targets for the treatment of infertility, reshaping and protecting ovarian function. The expression and dynamic changes of the main molecules of Hippo signaling pathway in the physiological senescence and pathological senescence of ovarian reproductive epithelial cells. Objective to analyze the main molecules of the Hippo signaling pathway in the ovarian reproductive epithelium. OSE) expression and dynamic changes in cells, the relationship between the changes in physiological senescence and pathological senescence of the ovarian function. The methods of study were 7 days of age (7days, 7D), 2 month old (2months, 2M), 10 month old (10months, 10M) and 20 month old (20months, 20M) mouse ovaries, and the birth peak (36-50 years of adolescence), middle age (36-50 years) and menopause (5 The human ovarian cortex samples of 1-60 years old were the experimental specimens of physiological senescent ovary. By intraperitoneal injection of Tripterygium Glycosides (TPT) and cyclophosphamide / Maryland tablets (Cyclophosphamide/Busulfan, CY-BUS) intraperitoneally, the ovarian model of pathological senescence was constructed. HE staining, immuno chemical methods and molecular biological methods were used respectively. The expression and dynamic changes of the main molecules of Hippo signaling pathway (LATS2, MST1 and YAP1) in OSE cells, and the relationship between this change and the physiological senescence and pathological senescence of the ovarian function were compared. The results of the study (1) there was a co expression of Hippo signal pathway related molecules and OGSCs marker factors (MVH and OCT4) in OSE of mice of different ages. In addition, the expression of tumor suppressor factor LATS2, MST1 and MVH/OCT4 gradually decreased with the increase of age, and the expression of primary cancer factor YAP1 in 2M mice OSE was the highest, 7D was second, 20M was the lowest, P YAP1 expression was the highest, and P depreciation ratio gradually decreased with age. (2) there was also a letter in the pathological ovarian aging model mice. Among the two groups, YAP1, MST1 and MVH/OCT4 were gradually reduced in the aging model group caused by two groups of Tripterygium Wilfordii and cyclophosphamide / Maryland tablets, while the expression of LATS2 showed the opposite trend of.P YAP1 in the two groups of pathological ovarian senescent mice. The ratio of P YAP1/YAP1 increased significantly. (3) there was also a co expression of Hippo signaling pathway related molecules and OGSCs marker factors (MVH and OCT4) in human OSE at different ages. MVH/OCT4 decreased gradually with age, and LATS2 was the highest in puberty, YAP1 and P YAP1 was the highest in middle age. P YAP1/YAP1 showed a tendency to decrease gradually from youth to old age. Conclusion Hippo signaling pathway related molecules LATS2, MST1 and YAP1 and OGSCs marker factor MVH and OCT4 are co expressed in mice and OSE, and there is a significant temporal correlation between the two expression changes, and this change is related to the physiological senescence and pathology of ovarian function. Hippo signaling pathway may participate in the physiological senescence and pathological senescence of ovarian function by regulating OGSCs signaling pathway. The role of the second part of Hippo signaling pathway in the proliferation and differentiation of ovarian reproductive stem cells is to improve the separation of ovarian reproductive stem cells (OGSCs). Identification and culture methods; the effect of Hippo signaling pathway on the proliferation and differentiation of OGSCs. The methods used two step enzyme separation method and specific antibody magnetic bead screening technique (magnetic activated cell sorting, MASC) to separate OGSCs, and determine a variety of stem cell and germ cell specific markers, such as MVH, OCT4, Nango, Fraglis, and reproductive cells. OGSCs was identified by factors and OGSCs was cultured in this room. The overexpression of the effect factor YAP1 and the interfering lentivirus vector of the corresponding sh RNA were transfected to OGSCs to observe the effect of OGSCs on the proliferation and differentiation of OGSCs. The results (1) the cells separated by the above methods could co express MVH, OCT4, Nango, Fraglis, Stella. OGSCs. (2) LATS2, MST1 and YAP1 can be expressed on OGSCs, and OGSCs in YAP1 overexpression of lentivirus vector, the expression of its specific marker factor MVH, the expression of OCT4 is significantly increased, and the expression of MVH, and the expression of MVH Significant reduction. Conclusion OGSCs isolation, identification and culture methods have been preliminarily established; Hippo signaling pathway related molecules exist in OGSCs, and can regulate the proliferation of OGSCs in vitro. The third part of Hippo signaling pathway in ovarian function remodeling by regulating the function of ovarian reproductive stem cells in ovarian function remodeling, the purpose of further verification of Hippo letter in vivo Through regulating the proliferation and differentiation of ovarian reproductive stem cells (OGSCs) in ovarian function remodeling, the method selected the main effect factor YAP1 in the Hippo signaling pathway as the target gene. Through microinjection of the ovary, the overexpression of the effect factor YAP1 and the corresponding sh RNA interfering lentivirus vector were transfected into the infertility model respectively. The effect of the ovaries on the reproductive function and the endocrine function of the ovary was observed. The results of the study (1) during the 30-75 day after transfection of YAP1, the rats in the infertile model could detect the follicle regeneration in the ovary, the activation of the original follicle, the increase of the birth rate of the progeny, the increase of the level of E2 and FSH in the serum, and the OS of the ovary. The expression of YAP1 and P YAP1 in E cells and the expression of MVH and OCT4 were significantly increased. (2) during the 30-75 day after transfecting YAP1 with the lentivirus vector, the number of follicles in the ovary decreased, the birth rate of the progeny decreased, and the level of E2 and FSH decreased in the serum, and the YAP1 and P expressions in the OSE cells of the ovary were expressed in the ovary OSE cells. Conclusion the regulation of Hippo signaling pathway effector molecule YAP1 can play an important role in ovarian remodeling.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R711.75

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 ;Hippo signaling:A hub of growth control,tumor suppression and pluripotency maintenance[J];遺傳學(xué)報;2011年10期

2 許傳銘;萬福生;;哺乳動物Hippo信號通路:腫瘤治療的新標(biāo)靶[J];遺傳;2012年03期

3 李敏睿;張盛洪;鐘碧慧;;Hippo信號通路在惡性腫瘤及干細(xì)胞中的作用[J];胃腸病學(xué)和肝病學(xué)雜志;2013年04期

4 朱忠勝;張春林;;腫瘤中Hippo-TAZ信號轉(zhuǎn)導(dǎo)通路研究進(jìn)展[J];國際骨科學(xué)雜志;2013年03期

5 史夢婕;邵松軍;李潔媚;周艷芳;黃培春;;Hippo通路與腫瘤相關(guān)性研究進(jìn)展[J];中國細(xì)胞生物學(xué)學(xué)報;2014年03期

6 李桂蘭;張麗;閆華超;;Hippo信號通路與腫瘤發(fā)生相關(guān)性研究進(jìn)展[J];基礎(chǔ)醫(yī)學(xué)與臨床;2010年09期

7 鮑乾坤;何金龍;朱毅;;Hippo-YAP通路在心血管系統(tǒng)中的作用[J];生理科學(xué)進(jìn)展;2014年03期

8 Jiliang Zhou;;An emerging role for Hippo-YAP signaling in cardiovascular development[J];The Journal of Biomedical Research;2014年04期

9 Masato Enomoto;Tatsushi Igaki;;Deciphering tumor-suppressor signaling in flies:Genetic link between Scribble/Dlg/Lgl and the Hippo pathways[J];遺傳學(xué)報;2011年10期

10 張靖;朱金水;;Hippo-YAP信號通路與胃癌相關(guān)性研究[J];國際消化病雜志;2011年04期

相關(guān)會議論文 前3條

1 Zhan Yang;;MicroRNA-155 and Hippo/STK3 Form a Feedback Loop to Participate in Vascular Smooth Muscle Cell Proliferation[A];中國生物化學(xué)與分子生物學(xué)會第十一次會員代表大會暨2014年全國學(xué)術(shù)會議論文集——專題報告五[C];2014年

2 Hongtan Wu;Luyao Wei;Suyuan Ji;Fuqin Fan;Funiu Qin;Lanfen Chen;Dawang Zhou;;MST1/2-Yap signaling pathway in organ size control and cancer development[A];生命的分子機器及其調(diào)控網(wǎng)絡(luò)——2012年全國生物化學(xué)與分子生物學(xué)學(xué)術(shù)大會摘要集[C];2012年

3 Zhan Yang;Bin Zheng;Yu Zhang;Xin-hua Zhang;Ming He;Dong Ma;Jin-kun Wen;;MicroRNA-155 and Hippo/STK3 Form a Feedback Loop to Participate in Vascular Smooth Muscle Cell Proliferation[A];第十二屆全國脂質(zhì)與脂蛋白學(xué)術(shù)會議論文匯編[C];2014年

相關(guān)重要報紙文章 前2條

1 記者 胡德榮;我學(xué)者發(fā)現(xiàn)原癌蛋白質(zhì)YAP抑制劑[N];健康報;2014年

2 記者 徐瑞哲;人造“克星”抑制胃癌[N];解放日報;2014年

相關(guān)博士學(xué)位論文 前10條

1 安健;Nedd4-like E3泛素連接酶調(diào)控Angiomotin/p130蛋白穩(wěn)定性的分子機制研究[D];復(fù)旦大學(xué);2011年

2 劉歡;Hippo通路效應(yīng)蛋白YAP調(diào)控基因轉(zhuǎn)錄的機制與功能研究[D];浙江大學(xué);2015年

3 劉鵬;HBV preS2 通過miR-338-3p 調(diào)控Hippo通路效應(yīng)分子TAZ促進(jìn)肝細(xì)胞肝癌作用研究[D];山東大學(xué);2015年

4 朱國華;上調(diào)miR-130b通過滅活Hippo信號通路來增強膠質(zhì)瘤干細(xì)胞的表型[D];新疆醫(yī)科大學(xué);2015年

5 韓笑;Hippo信號通路中TEAD蛋白質(zhì)的結(jié)構(gòu)與功能研究[D];吉林大學(xué);2016年

6 郭鵬達(dá);RARγ的缺失通過調(diào)控Hippo-Yap信號通路促進(jìn)結(jié)直腸癌的發(fā)生和轉(zhuǎn)移[D];蘇州大學(xué);2016年

7 周菁;YAP在哮喘發(fā)生過程中的表達(dá)情況及其功能探討[D];南昌大學(xué);2016年

8 鄧軍;CUL4A通過調(diào)控Hippo通路促胃癌細(xì)胞增殖和侵襲的分子機制[D];南昌大學(xué);2016年

9 李佳;Hippo信號通路在調(diào)節(jié)卵巢生殖干細(xì)胞功能和延緩卵巢衰老中的作用[D];南昌大學(xué);2016年

10 劉乃華;磷酸二酯酶5/PKG通過Hippo/TAZ信號調(diào)控前列腺癌腫瘤干細(xì)胞干性及其分子機制研究[D];浙江大學(xué);2016年

相關(guān)碩士學(xué)位論文 前10條

1 王小燕;Alcf八個氨基酸差異選擇性剪接對核定位功能的影響及其抗體制備[D];重慶醫(yī)科大學(xué);2015年

2 陸亞紅;家蠶BmYki不同剪接體的功能研究[D];蘇州大學(xué);2016年

3 宋春暖;家蠶Hippo通路成員的全基因組鑒定與分析[D];西南大學(xué);2016年

4 梁坤;Hippo信號通路在結(jié)直腸癌中的表達(dá)及臨床意義[D];青島大學(xué);2014年

5 魏文超;Hippo信號通路在胃粘膜癌變過程中的作用[D];青島大學(xué);2013年

6 向晟;Hippo通路主要組分與小鼠原始卵泡池大小的時序相關(guān)性研究[D];南昌大學(xué);2014年

7 錢瑩;家蠶Hippo信號通路主要基因的研究[D];蘇州大學(xué);2014年

8 沈龍霞;HCMV經(jīng)Hippo-YAP信號通路對絨毛外細(xì)胞滋養(yǎng)細(xì)胞功能影響的體外研究[D];泰山醫(yī)學(xué)院;2014年

9 陳柯宏;LATS1基因去甲基化對人腎癌細(xì)胞生物學(xué)功能及其Hippo-YAP信號通路的影響[D];重慶醫(yī)科大學(xué);2014年

10 尹聰;Hippo通路與自噬共調(diào)節(jié)IUGR新生仔豬肝臟代謝的機理研究[D];華中農(nóng)業(yè)大學(xué);2013年

，

本文編號：1982607