HERP在小鼠卵泡颗粒细胞凋亡中的作用及其机制研究
发布时间：2018-12-11 06:33
【摘要】：哺乳动物的卵巢是一个十分复杂的动态发育的器官,包含不同发育阶段的卵泡,如原始卵泡、初级卵泡、次级卵泡和有腔卵泡。但是,在卵巢的发育过程中,只有为数不多的卵泡能够发育成熟并排卵,绝大多数发生闭锁。在卵泡发育过程中,FSH和LH等促性腺激素发挥了重要作用。FSH和LH能够促进卵泡的发育和优势卵泡的选择,诱导优势卵泡的排卵和黄体的形成。随着研究的深入,除了促性腺激素外,自分泌和旁分泌因子,如生长因子和细胞因子,对于卵泡的发育也起到非常重要的作用。越来越多的研究结果表明内质网应激相关的分子伴侣对于卵泡的发育、闭锁以及黄体化过程起到至关重要的作用。但是目前对于内质网应激相关的分子伴侣调节卵泡发育的具体分子机制的研究还非常的少。HERP(Homocysteine-responsive ER-resident protein)是内质网膜蛋白,内质网应激能够诱导其高表达。HERP通过内质网相关的降解途径(ER-associated protein degradation,ERAD)参与内质网腔内的未折叠或者错误折叠蛋白的降解。HERP能够维持内质网中Ca2+的平衡和线粒体的功能。HERP在多种组织和器官中表达,例如心脏、肝脏、骨骼肌、肾脏和胰腺。研究表明HERP可能参与糖尿病、神经退行性疾病和肌肉衰减症等疾病的发生和发展的过程。本研究通过免疫组化、RNA干扰、western blot、荧光定量PCR、ELISA、流式细胞术和免疫荧光等技术对小鼠卵巢中HERP的分布以及在颗粒细胞中的功能和作用进行了研究。主要的试验结果如下:⑴检测了HERP在仔鼠、未成熟小鼠和正常发情周期小鼠卵巢中的表达。HERP在卵母细胞和排卵前颗粒细胞中表达较高,在卵泡膜细胞和黄体细胞中表达较低;并且在促性腺激素处理和发情周期过程中其表达具有一定的规律性,在发情过程中其表达较高。⑵设计了3条针对小鼠Herp基因的RNAi序列,并成功构建了shRNA慢病毒干扰载体。经过病毒包装得到的干扰慢病毒通过倍比稀释的方法测定其病毒滴度达5-10×107IU/mL。为了鉴定其干扰效果,RAW 264.7细胞经Herp干扰慢病毒转导后,筛选出具有有效干扰效果的干扰片段,测定其干扰效率在80%左右。筛选出稳定抑制Herp基因表达的RAW 264.7细胞,在内质网应激诱导剂的作用下,抑制Herp基因表达能够显著下调内质网应激相关基因的表达,但是在炎症反应相关基因的表达上具有不同的调节作用。⑶体外培养小鼠卵巢颗粒细胞并且转导Herp干扰慢病毒后,测定其干扰效率在70%左右。对颗粒细胞分泌类固醇激素、细胞周期和凋亡进行检测,发现和正常颗粒细胞相比,抑制Herp表达能够通过增加芳香化酶基因Cyp19a1和抑制代谢相关基因Cyplbl的表达从而促进雌激素的分泌;还能够通过抑制细胞周期相关基因cyclin A1、cyclin B1和cyclin D2的表达阻滞细胞周期,在正常体外培养条件下,细胞凋亡率没有显著变化。⑷在赤霉烯酮诱导体外培养的颗粒细胞凋亡过程中,颗粒细胞的凋亡对于赤霉烯酮的处理具有剂量和时间的依赖性,随着浓度的增加和时间的延长颗粒细胞的凋亡率逐渐增加。而且,自噬晚期抑制剂CQ能够显著增加赤霉烯酮诱导的颗粒细胞的凋亡。在赤霉烯酮诱导体外培养的颗粒细胞凋亡过程中,赤霉烯酮能够促进自噬的标志性蛋白LC3-Ⅱ的表达,促进内质网应激相关蛋白GRP78、CHOP和HERP的表达以及抑制MAPK和mTOR信号中pERK和pS6K的表达,内质网应激抑制剂4-PBA能够显著抑制赤霉烯酮诱导的内质网应激相关蛋白的表达以及CQ能够显著增加赤霉烯酮诱导的LC3-Ⅱ的表达,推测赤霉烯酮可能通过增强内质网应激信号和抑制MAPK和mTOR信号诱导细胞自噬的发生。⑸抑制HERP能够显著降低赤霉烯酮诱导体外培养的颗粒细胞的凋亡。在赤霉烯酮处理的颗粒细胞中,干扰HERP增加自噬的标志性蛋白LC3-Ⅱ和BCL-2,而降低活化Caspase-3和BAX的表达。表明抑制HERP表达能够通过增强细胞自噬和抑制凋亡信号,降低赤霉烯酮诱导体外培养颗粒细胞的凋亡。综上所述,本研究从体内和体外两个方面研究了HERP在卵泡颗粒细胞中的作用及其分子调控机制。为HERP功能研究提供了新的理论依据,充实了卵泡发育的研究内容。
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S814
，
本文编号：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.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S814
，
本文编号：2372061
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