HERP在小鼠卵泡顆粒細(xì)胞凋亡中的作用及其機(jī)制研究
發(fā)布時(shí)間：2018-12-11 06:33
【摘要】：哺乳動(dòng)物的卵巢是一個(gè)十分復(fù)雜的動(dòng)態(tài)發(fā)育的器官,包含不同發(fā)育階段的卵泡,如原始卵泡、初級卵泡、次級卵泡和有腔卵泡。但是,在卵巢的發(fā)育過程中,只有為數(shù)不多的卵泡能夠發(fā)育成熟并排卵,絕大多數(shù)發(fā)生閉鎖。在卵泡發(fā)育過程中,FSH和LH等促性腺激素發(fā)揮了重要作用。FSH和LH能夠促進(jìn)卵泡的發(fā)育和優(yōu)勢卵泡的選擇,誘導(dǎo)優(yōu)勢卵泡的排卵和黃體的形成。隨著研究的深入,除了促性腺激素外,自分泌和旁分泌因子,如生長因子和細(xì)胞因子,對于卵泡的發(fā)育也起到非常重要的作用。越來越多的研究結(jié)果表明內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)的分子伴侶對于卵泡的發(fā)育、閉鎖以及黃體化過程起到至關(guān)重要的作用。但是目前對于內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)的分子伴侶調(diào)節(jié)卵泡發(fā)育的具體分子機(jī)制的研究還非常的少。HERP(Homocysteine-responsive ER-resident protein)是內(nèi)質(zhì)網(wǎng)膜蛋白,內(nèi)質(zhì)網(wǎng)應(yīng)激能夠誘導(dǎo)其高表達(dá)。HERP通過內(nèi)質(zhì)網(wǎng)相關(guān)的降解途徑(ER-associated protein degradation,ERAD)參與內(nèi)質(zhì)網(wǎng)腔內(nèi)的未折疊或者錯(cuò)誤折疊蛋白的降解。HERP能夠維持內(nèi)質(zhì)網(wǎng)中Ca2+的平衡和線粒體的功能。HERP在多種組織和器官中表達(dá),例如心臟、肝臟、骨骼肌、腎臟和胰腺。研究表明HERP可能參與糖尿病、神經(jīng)退行性疾病和肌肉衰減癥等疾病的發(fā)生和發(fā)展的過程。本研究通過免疫組化、RNA干擾、western blot、熒光定量PCR、ELISA、流式細(xì)胞術(shù)和免疫熒光等技術(shù)對小鼠卵巢中HERP的分布以及在顆粒細(xì)胞中的功能和作用進(jìn)行了研究。主要的試驗(yàn)結(jié)果如下:⑴檢測了HERP在仔鼠、未成熟小鼠和正常發(fā)情周期小鼠卵巢中的表達(dá)。HERP在卵母細(xì)胞和排卵前顆粒細(xì)胞中表達(dá)較高,在卵泡膜細(xì)胞和黃體細(xì)胞中表達(dá)較低;并且在促性腺激素處理和發(fā)情周期過程中其表達(dá)具有一定的規(guī)律性,在發(fā)情過程中其表達(dá)較高。⑵設(shè)計(jì)了3條針對小鼠Herp基因的RNAi序列,并成功構(gòu)建了shRNA慢病毒干擾載體。經(jīng)過病毒包裝得到的干擾慢病毒通過倍比稀釋的方法測定其病毒滴度達(dá)5-10×107IU/mL。為了鑒定其干擾效果,RAW 264.7細(xì)胞經(jīng)Herp干擾慢病毒轉(zhuǎn)導(dǎo)后,篩選出具有有效干擾效果的干擾片段,測定其干擾效率在80%左右。篩選出穩(wěn)定抑制Herp基因表達(dá)的RAW 264.7細(xì)胞,在內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)劑的作用下,抑制Herp基因表達(dá)能夠顯著下調(diào)內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)基因的表達(dá),但是在炎癥反應(yīng)相關(guān)基因的表達(dá)上具有不同的調(diào)節(jié)作用。⑶體外培養(yǎng)小鼠卵巢顆粒細(xì)胞并且轉(zhuǎn)導(dǎo)Herp干擾慢病毒后,測定其干擾效率在70%左右。對顆粒細(xì)胞分泌類固醇激素、細(xì)胞周期和凋亡進(jìn)行檢測,發(fā)現(xiàn)和正常顆粒細(xì)胞相比,抑制Herp表達(dá)能夠通過增加芳香化酶基因Cyp19a1和抑制代謝相關(guān)基因Cyplbl的表達(dá)從而促進(jìn)雌激素的分泌;還能夠通過抑制細(xì)胞周期相關(guān)基因cyclin A1、cyclin B1和cyclin D2的表達(dá)阻滯細(xì)胞周期,在正常體外培養(yǎng)條件下,細(xì)胞凋亡率沒有顯著變化。⑷在赤霉烯酮誘導(dǎo)體外培養(yǎng)的顆粒細(xì)胞凋亡過程中,顆粒細(xì)胞的凋亡對于赤霉烯酮的處理具有劑量和時(shí)間的依賴性,隨著濃度的增加和時(shí)間的延長顆粒細(xì)胞的凋亡率逐漸增加。而且,自噬晚期抑制劑CQ能夠顯著增加赤霉烯酮誘導(dǎo)的顆粒細(xì)胞的凋亡。在赤霉烯酮誘導(dǎo)體外培養(yǎng)的顆粒細(xì)胞凋亡過程中,赤霉烯酮能夠促進(jìn)自噬的標(biāo)志性蛋白LC3-Ⅱ的表達(dá),促進(jìn)內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白GRP78、CHOP和HERP的表達(dá)以及抑制MAPK和mTOR信號中pERK和pS6K的表達(dá),內(nèi)質(zhì)網(wǎng)應(yīng)激抑制劑4-PBA能夠顯著抑制赤霉烯酮誘導(dǎo)的內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白的表達(dá)以及CQ能夠顯著增加赤霉烯酮誘導(dǎo)的LC3-Ⅱ的表達(dá),推測赤霉烯酮可能通過增強(qiáng)內(nèi)質(zhì)網(wǎng)應(yīng)激信號和抑制MAPK和mTOR信號誘導(dǎo)細(xì)胞自噬的發(fā)生。⑸抑制HERP能夠顯著降低赤霉烯酮誘導(dǎo)體外培養(yǎng)的顆粒細(xì)胞的凋亡。在赤霉烯酮處理的顆粒細(xì)胞中,干擾HERP增加自噬的標(biāo)志性蛋白LC3-Ⅱ和BCL-2,而降低活化Caspase-3和BAX的表達(dá)。表明抑制HERP表達(dá)能夠通過增強(qiáng)細(xì)胞自噬和抑制凋亡信號,降低赤霉烯酮誘導(dǎo)體外培養(yǎng)顆粒細(xì)胞的凋亡。綜上所述,本研究從體內(nèi)和體外兩個(gè)方面研究了HERP在卵泡顆粒細(xì)胞中的作用及其分子調(diào)控機(jī)制。為HERP功能研究提供了新的理論依據(jù),充實(shí)了卵泡發(fā)育的研究內(nèi)容。
[Abstract]:The ovary of a mammal is a very complex, dynamic developed organ, including follicles at different stages of development, such as the original follicle, the primary follicle, the secondary follicle, and the follicle. However, in the development of the ovary, only a few of the follicles can develop and ovulate, most of which are locked. In the process of the development of the follicle, the gonadotropins such as FSH and LH play an important role. FSH and LH can promote the development of the follicle and the selection of the dominant follicle, and induce the ovulation of the dominant follicle and the formation of the corpus luteum. In addition to the in-depth of the study, the autocrine and paracrine factors, such as growth factors and cytokines, play a very important role in the development of the follicle. More and more studies have shown that endoplasmic reticulum stress-related molecular chaperones play a critical role in the development, atresia and colonization of the follicle. However, there are few studies on the specific molecular mechanism of the regulation of follicular development for molecular chaperones associated with endoplasmic reticulum stress. HERP (Homocysteine-response protein) is the endoplasmic reticulum membrane protein, and the endoplasmic reticulum stress can induce its high expression. HERP is involved in the degradation of unfolded or misfolded proteins in the endoplasmic reticulum lumen through the endoplasmic reticulum-associated degradation pathway (ERAD). HERP can maintain the balance of Ca2 + and the function of mitochondria in the endoplasmic reticulum. The HEPs are expressed in a variety of tissues and organs, such as heart, liver, skeletal muscle, kidney and pancreas. The study shows that HERP may be involved in the pathogenesis and development of diabetes, neurodegenerative diseases and muscle attenuation. The distribution of HERP and its function and function in the ovary of mice were studied by immunohistochemistry, RNA interference, western blot, fluorescence quantitative PCR, ELISA, flow cytometry and immunofluorescence. The main results of the test are as follows: The expression of HERP in the ovary of the mouse, the immature mouse and the normal estrus cycle is detected. The expression of HERP in the granulosa cells of the oocytes and the ovulatory cells is high, and the expression of HERP is lower in the follicular membrane cells and the yellow cells; and the expression of the HERP in the process of the gonadotropin treatment and the estrus cycle has certain regularity, and the expression of the HERP in the estrus process is high. In this paper, three RNAi sequences for mouse Herp gene were designed and shRNA lentiviral interference vector was successfully constructed. The virus titer of 5-10-107IU/ mL was determined by the time-to-fold dilution method of the interfering lentivirus obtained by the virus packaging. In order to identify the interference effect, after the cells of RAW 264.7 cell were transduced with Herp-interference lentivirus, the interference fragment with effective interference effect was screened, and the interference efficiency of RAW 264.7 cells was about 80%. and the expression of the endoplasmic reticulum stress-related gene can be significantly reduced under the action of the endoplasmic reticulum stress inducing agent and the expression of the endoplasmic reticulum stress-related gene can be remarkably reduced under the action of the endoplasmic reticulum stress inducing agent, but the expression of the related gene of the inflammation reaction has different regulating effects. In vitro culture of the mouse ovary granulosa cells and the transduction of Herp to the lentivirus, the interference efficiency was determined to be about 70%. It is found that the inhibition of Herp expression can promote the secretion of estrogen by increasing the expression of the aromatase gene Cyp19a1 and the inhibition of the metabolism-related gene Cyplbl, compared with the normal granulosa cells. It is also possible to block the cell cycle by inhibiting the expression of cyclin A1, cyclin B1 and cyclin D2 in the cell cycle, and the cell apoptosis rate is not significantly changed under normal in vitro culture conditions. In the process of the apoptosis of the granulosa cells cultured in vitro, the apoptosis of the granulosa cells has a dose and time-dependent dependence on the treatment of the alketene, and the apoptosis rate of the granulosa cells gradually increases with the increase of the concentration and the time. Moreover, the autophagy inhibitor CQ can significantly increase the apoptosis of the granulosa cells induced by the alketene. In the process of inducing the apoptosis of the granulosa cells cultured in vitro, the alketene can promote the expression of the autophagy protein LC3-II, promote the expression of the endoplasmic reticulum stress-related protein GRP78, CHOP and HERP, and inhibit the expression of pERK and pS6K in the MAPK and mTOR signals, The endoplasmic reticulum stress inhibitor 4-PBA can obviously inhibit the expression of the endoplasmic reticulum stress-related protein and the expression of the CQ in the endoplasmic reticulum stress-induced endoplasmic reticulum stress-induced endoplasmic reticulum stress-related protein, It is suggested that the alketene may induce autophagy in the cells by enhancing the endoplasmic reticulum stress signal and inhibiting the MAPK and mTOR signaling. The inhibition of HERP can significantly reduce the apoptosis of the granulosa cells cultured in vitro. The expression of Caspase-3 and BAX was reduced by interfering with the increase of the autophagy protein LC3-II and BCL-2 in the granulosa cells treated with the alketene. It is shown that the inhibition of HERP expression can decrease the apoptosis of the granulosa cells in vitro by enhancing the autophagy of the cells and inhibiting the apoptosis signals. To sum up, this study has studied the role of HERP in the granulosa cell of the follicle and its molecular control mechanism from two aspects in vivo and in vitro. It provides a new theoretical basis for the study of the HERP function, and enriches the research contents of the development of the follicle.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S814
，
本文編號：2372061
[Abstract]:The ovary of a mammal is a very complex, dynamic developed organ, including follicles at different stages of development, such as the original follicle, the primary follicle, the secondary follicle, and the follicle. However, in the development of the ovary, only a few of the follicles can develop and ovulate, most of which are locked. In the process of the development of the follicle, the gonadotropins such as FSH and LH play an important role. FSH and LH can promote the development of the follicle and the selection of the dominant follicle, and induce the ovulation of the dominant follicle and the formation of the corpus luteum. In addition to the in-depth of the study, the autocrine and paracrine factors, such as growth factors and cytokines, play a very important role in the development of the follicle. More and more studies have shown that endoplasmic reticulum stress-related molecular chaperones play a critical role in the development, atresia and colonization of the follicle. However, there are few studies on the specific molecular mechanism of the regulation of follicular development for molecular chaperones associated with endoplasmic reticulum stress. HERP (Homocysteine-response protein) is the endoplasmic reticulum membrane protein, and the endoplasmic reticulum stress can induce its high expression. HERP is involved in the degradation of unfolded or misfolded proteins in the endoplasmic reticulum lumen through the endoplasmic reticulum-associated degradation pathway (ERAD). HERP can maintain the balance of Ca2 + and the function of mitochondria in the endoplasmic reticulum. The HEPs are expressed in a variety of tissues and organs, such as heart, liver, skeletal muscle, kidney and pancreas. The study shows that HERP may be involved in the pathogenesis and development of diabetes, neurodegenerative diseases and muscle attenuation. The distribution of HERP and its function and function in the ovary of mice were studied by immunohistochemistry, RNA interference, western blot, fluorescence quantitative PCR, ELISA, flow cytometry and immunofluorescence. The main results of the test are as follows: The expression of HERP in the ovary of the mouse, the immature mouse and the normal estrus cycle is detected. The expression of HERP in the granulosa cells of the oocytes and the ovulatory cells is high, and the expression of HERP is lower in the follicular membrane cells and the yellow cells; and the expression of the HERP in the process of the gonadotropin treatment and the estrus cycle has certain regularity, and the expression of the HERP in the estrus process is high. In this paper, three RNAi sequences for mouse Herp gene were designed and shRNA lentiviral interference vector was successfully constructed. The virus titer of 5-10-107IU/ mL was determined by the time-to-fold dilution method of the interfering lentivirus obtained by the virus packaging. In order to identify the interference effect, after the cells of RAW 264.7 cell were transduced with Herp-interference lentivirus, the interference fragment with effective interference effect was screened, and the interference efficiency of RAW 264.7 cells was about 80%. and the expression of the endoplasmic reticulum stress-related gene can be significantly reduced under the action of the endoplasmic reticulum stress inducing agent and the expression of the endoplasmic reticulum stress-related gene can be remarkably reduced under the action of the endoplasmic reticulum stress inducing agent, but the expression of the related gene of the inflammation reaction has different regulating effects. In vitro culture of the mouse ovary granulosa cells and the transduction of Herp to the lentivirus, the interference efficiency was determined to be about 70%. It is found that the inhibition of Herp expression can promote the secretion of estrogen by increasing the expression of the aromatase gene Cyp19a1 and the inhibition of the metabolism-related gene Cyplbl, compared with the normal granulosa cells. It is also possible to block the cell cycle by inhibiting the expression of cyclin A1, cyclin B1 and cyclin D2 in the cell cycle, and the cell apoptosis rate is not significantly changed under normal in vitro culture conditions. In the process of the apoptosis of the granulosa cells cultured in vitro, the apoptosis of the granulosa cells has a dose and time-dependent dependence on the treatment of the alketene, and the apoptosis rate of the granulosa cells gradually increases with the increase of the concentration and the time. Moreover, the autophagy inhibitor CQ can significantly increase the apoptosis of the granulosa cells induced by the alketene. In the process of inducing the apoptosis of the granulosa cells cultured in vitro, the alketene can promote the expression of the autophagy protein LC3-II, promote the expression of the endoplasmic reticulum stress-related protein GRP78, CHOP and HERP, and inhibit the expression of pERK and pS6K in the MAPK and mTOR signals, The endoplasmic reticulum stress inhibitor 4-PBA can obviously inhibit the expression of the endoplasmic reticulum stress-related protein and the expression of the CQ in the endoplasmic reticulum stress-induced endoplasmic reticulum stress-induced endoplasmic reticulum stress-related protein, It is suggested that the alketene may induce autophagy in the cells by enhancing the endoplasmic reticulum stress signal and inhibiting the MAPK and mTOR signaling. The inhibition of HERP can significantly reduce the apoptosis of the granulosa cells cultured in vitro. The expression of Caspase-3 and BAX was reduced by interfering with the increase of the autophagy protein LC3-II and BCL-2 in the granulosa cells treated with the alketene. It is shown that the inhibition of HERP expression can decrease the apoptosis of the granulosa cells in vitro by enhancing the autophagy of the cells and inhibiting the apoptosis signals. To sum up, this study has studied the role of HERP in the granulosa cell of the follicle and its molecular control mechanism from two aspects in vivo and in vitro. It provides a new theoretical basis for the study of the HERP function, and enriches the research contents of the development of the follicle.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S814
，
本文編號：2372061
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