内质网应激调节蛋白LRF在小鼠子宫内膜蜕膜化中的作用

发布时间：2018-04-28 23:06

本文选题：LRF + 内质网应激　；参考：《西北农林科技大学》2016年博士论文

【摘要】：Luman recruiting factor(LRF)又叫做CREB3 regulate factor(CREBRF),它是一个内质网应激相关的亮氨酸拉链蛋白转录因子。LRF是CREB家族成员Luman的负反馈调节因子,它一方面通过募集Luman进入核小体而抑制Luman的活化,另一方面与Luman结合而加速Luman蛋白的降解。在小鼠分娩与产后期,LRF通过抑制糖皮质激素压力信号进而抑制HPA轴的活性。LRF基因敲除鼠中糖皮质激素受体表达异常升高,PRL的表达下降,导致产后母鼠母性行为缺失。LRF在发情周期和早期胚胎植入过程中的小鼠子宫中呈规律性表达,在妊娠小鼠第6-7 d子宫的初级蜕膜区及次级蜕膜区的表达量很高。提示LRF在小鼠子宫中的表达可能受甾类激素的调节,而且,LRF可能在胚胎植入或蜕膜化过程中有一定的作用。本试验应用qRT-PCR、western blot、免疫组化、慢病毒干扰等技术检测了(1)甾类激素对LRF在小鼠子宫中表达的调控作用;(2)LRF干扰后对小鼠胚胎植入以及蜕膜化的影响;此外,研究了LRF干扰后对基质细胞体外诱导蜕膜化的影响。1.扩增了LRF基因的CDS区的全长序列,选取三个LRF的干扰靶点,并用T4连接酶将上述碱基序列重组入慢病毒载体。转化入E.coli DH5α感受态细菌后,挑取单克隆进行PCR鉴定与测序鉴定。将鉴定正确的重组质粒与包装质粒用转染试剂TuboFect共转入HEK 293T细胞进行病毒包装。将得到的慢病毒感染HEK 293T细胞进行慢病毒滴度测定。运用qRT-PCR和Western blot技术检测LRF过表达和干扰慢病毒在NIH 3T3细胞中的过表达与干扰效果。包装的LRF过表达慢病毒能显著升高外源的LRF蛋白的表达。包装的shLRF干扰慢病毒中pCD513B-U6-shLRF-2205片段可以显著干扰LRF蛋白的表达。LRF干扰与过表达慢病毒的运用,对进一步研究LRF在小鼠中的生理学功能具有重要意义。2.运用western blot、免疫组化等技术检测了在甾类激素E2和P4的作用下,LRF蛋白在卵巢摘除小鼠子宫和基质细胞中的表达。结果显示,P4能显著上调LRF蛋白在卵巢摘除的小鼠子宫和原代基质细胞中的表达,并且以时间依赖的方式上升。而且,孕酮受体拮抗剂米非司酮(RU486)能显著的下调LRF的表达。尽管E2处理卵巢摘除小鼠与原代基质细胞后LRF蛋白的表达没有明显的变化,但是,与孕酮处理组相比,E2与P4联合处理组中LRF蛋白的表达显著下调。而且,与E2组、对照组相比,在雌激素受体拮抗剂氟维司(ICI 182780)群处理组,LRF蛋白的表达显著上调。这些结果表明,LRF在小鼠子宫与基质细胞中的表达经由P4-PGR通路调节,而且雌激素能够部分地抑制P4上调的LRF表达。3.运用shLRF干扰慢病毒感染怀孕第3天的小鼠子宫,可以显著减少妊娠第7-8 d胚胎植入位点的大小与重量。在体外诱导蜕膜化的基质细胞模型中,LRF蛋白和mRNA表达随着人工诱导蜕膜化进程而显著升高。shLRF干扰慢病毒感染基质细胞并诱导蜕膜化,蜕膜化标记因子dPRP、IGFBP-1和PGR的表达下调,蜕膜化进程也受到抑制。运用流式细胞术检测体外蜕膜化基质细胞的细胞周期与细胞凋亡发现,shLRF干扰慢病毒感染基质细胞后对细胞凋亡没有明显的影响。但是,蜕膜化基质细胞的细胞周期在LRF干扰以后被阻滞在了S期。qRT-PCR检测与S期细胞周期相关的或者与蜕膜化相关的细胞周期调节因子发现,shLRF慢病毒干扰蜕膜细胞后,cyclin A与cyclin B1的mRNA表达显著降低,而cyclin D3与cyclin E的mRNA表达没有变化。因此,本试验说明了LRF参与小鼠子宫基质蜕膜化的调节,并通过调节细胞周期因子cyclin A与cyclin B1进而调节蜕膜化进程。本研究证实了内质网应激的相关蛋白-LRF在小鼠子宫中的表达受甾类激素的调控;而且,LRF通过调节细胞周期相关基因cyclin A和cyclin B1的表达参与基质细胞蜕膜化的调节。揭示了LRF参与雌性小鼠生殖系统的生理调节,并为进一步研究LRF在哺乳动物生殖过程中的作用提供了一个起点,也为对内质网应激与哺乳动物生殖之间的关系理解添加了一个新的方向。
[Abstract]:Luman recruiting factor (LRF) is also called CREB3 regulate factor (CREBRF), which is an endoplasmic reticulum stress related leucine zipper transcription factor.LRF is a negative feedback regulator of Luman on the CREB family members. Protein degradation. In the delivery and late stage of mice, the expression of glucocorticoid receptor in the HPA axis activated.LRF knockout mice increased by inhibiting the pressure signal of glucocorticoid and then inhibiting the expression of glucocorticoid receptor in the.LRF knockout mice, and the expression of PRL decreased, resulting in the maternal mouse maternal behavior deletion.LRF in the mouse uterus during the estrous cycle and early embryo implantation. A regular expression was expressed in the primary decidua and secondary decidua areas of the 6-7 D uterus of pregnant mice. It was suggested that the expression of LRF in the mouse uterus may be regulated by steroid hormones, and LRF may have a certain role in the process of embryo implantation or deciduization. This test was conducted with qRT-PCR, Western blot, immunohistochemistry, and lentivirus. Interference and other techniques were used to detect (1) the regulation of steroid hormones on the expression of LRF in the uterus of mice; (2) the effect of LRF interference on the implantation and decidua of mice; furthermore, the effect of LRF interference on the induced decidua of matrix cells in vitro was studied..1. amplified the full length of the LRF gene in the LRF gene, and selected three LRF interference targets. T4 ligase was used to restructure the base sequence into lentivirus vector. After converting into E.coli DH5 alpha receptive bacteria, PCR identification and sequencing identification were carried out. The correct recombinant plasmids and packaging plasmids were transferred to HEK 293T cells by transfection of the transfected reagent TuboFect to HEK 293T cells for viral inclusion. The obtained lentivirus infected HEK 293T cells. QRT-PCR and Western blot techniques were used to detect the overexpression and interference effect of LRF over expression and interference in NIH 3T3 cells. The LRF overexpression of the packaged lentivirus can significantly increase the expression of the exogenous LRF protein. The pCD513B-U6-shLRF-2205 fragment in the packaged shLRF interfering lentivirus can significantly interfere with LRF Protein expression.LRF interference and the use of overexpression of lentivirus, it is of great significance to further study the physiological function of LRF in mice..2. using Western blot, immunohistochemistry and other techniques to detect the expression of LRF protein in ovariectomized mouse uterus and stromal cells under the action of steroid hormone E2 and P4. The results show that P4 can be displayed. The expression of LRF protein in the ovariectomized mouse uterus and primary stromal cells increased in a time dependent manner. Moreover, the progesterone receptor antagonist, mifepristone (RU486), could significantly downregulate the expression of LRF. Although the expression of the LRF protein in the ovariectomized mice and the primary matrix of the E2 was not significantly changed, but the expression of the LRF protein was not significantly changed. Compared with the progesterone treatment group, the expression of LRF protein in the combined treatment group of E2 and P4 was significantly downregulated. Moreover, the expression of LRF protein was significantly up-regulated in the group E2 and the control group, the group treated with the estrogen receptor antagonist, flurosi (ICI 182780) group. These results showed that the expression of LRF in the mouse uterus and stromal cells was regulated by the P4-PGR pathway. Estrogen can partly inhibit the LRF expression of P4 up regulated by.3. using shLRF to interfere with the mouse uterus of third days of pregnancy by interfering with lentivirus infection. The size and weight of the 7-8 D embryo implantation site in pregnancy can be significantly reduced. In the matrix cell model of deciduate induced in vitro, the expression of LRF protein and mRNA is significant with the process of induced decidua. The increase of.ShLRF interfered with the lentivirus infection matrix cells and induced deciduate, the expression of decidua marker factor dPRP, IGFBP-1 and PGR decreased, and the process of decidua was also inhibited. The cell cycle and apoptosis of deciduate stromal cells in vitro were detected by flow cytometry, and shLRF interfered with the apoptosis of the lentivirus. However, the cell cycle of the deciduate stromal cells was blocked after LRF interference in S phase.QRT-PCR detection and the cell cycle of S phase or the cell cycle regulator associated with decidua discovery that shLRF lentivirus interfered decidua cells and the expression of cyclin A and cyclin B1 mRNA expression decreased significantly, while cyclin D3 and cyclin D3 The expression of mRNA in cyclin E has not changed. Therefore, this study shows that LRF participates in the regulation of the matrix decidua of the mouse uterus and regulates the deciduating process by regulating the cell cycle factor cyclin A and cyclin B1. This study confirms that the expression of the protein -LRF in the endoplasmic reticulum stress in the mouse uterus is regulated by steroid hormones, and LR, LR, and LR. F participates in the regulation of matrix cell decidua by regulating the expression of cell cycle related genes, cyclin A and cyclin B1, and reveals the physiological regulation of LRF in the reproductive system of female mice, and provides a starting point for further study of the role of LRF in mammalian reproduction, as well as between endoplasmic reticulum stress and mammalian reproduction. The relationship understanding adds a new direction.

【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】：R714.2

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