本文選題：LRF + 內(nèi)質(zhì)網(wǎng)應(yīng)激��； 參考：《西北農(nóng)林科技大學(xué)》2016年博士論文

【摘要】：Luman recruiting factor(LRF)又叫做CREB3 regulate factor(CREBRF),它是一個(gè)內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)的亮氨酸拉鏈蛋白轉(zhuǎn)錄因子。LRF是CREB家族成員Luman的負(fù)反饋調(diào)節(jié)因子,它一方面通過募集Luman進(jìn)入核小體而抑制Luman的活化,另一方面與Luman結(jié)合而加速Luman蛋白的降解。在小鼠分娩與產(chǎn)后期,LRF通過抑制糖皮質(zhì)激素壓力信號進(jìn)而抑制HPA軸的活性。LRF基因敲除鼠中糖皮質(zhì)激素受體表達(dá)異常升高,PRL的表達(dá)下降,導(dǎo)致產(chǎn)后母鼠母性行為缺失。LRF在發(fā)情周期和早期胚胎植入過程中的小鼠子宮中呈規(guī)律性表達(dá),在妊娠小鼠第6-7 d子宮的初級蛻膜區(qū)及次級蛻膜區(qū)的表達(dá)量很高。提示LRF在小鼠子宮中的表達(dá)可能受甾類激素的調(diào)節(jié),而且,LRF可能在胚胎植入或蛻膜化過程中有一定的作用。本試驗(yàn)應(yīng)用qRT-PCR、western blot、免疫組化、慢病毒干擾等技術(shù)檢測了(1)甾類激素對LRF在小鼠子宮中表達(dá)的調(diào)控作用;(2)LRF干擾后對小鼠胚胎植入以及蛻膜化的影響;此外,研究了LRF干擾后對基質(zhì)細(xì)胞體外誘導(dǎo)蛻膜化的影響。1.擴(kuò)增了LRF基因的CDS區(qū)的全長序列,選取三個(gè)LRF的干擾靶點(diǎn),并用T4連接酶將上述堿基序列重組入慢病毒載體。轉(zhuǎn)化入E.coli DH5α感受態(tài)細(xì)菌后,挑取單克隆進(jìn)行PCR鑒定與測序鑒定。將鑒定正確的重組質(zhì)粒與包裝質(zhì)粒用轉(zhuǎn)染試劑TuboFect共轉(zhuǎn)入HEK 293T細(xì)胞進(jìn)行病毒包裝。將得到的慢病毒感染HEK 293T細(xì)胞進(jìn)行慢病毒滴度測定。運(yùn)用qRT-PCR和Western blot技術(shù)檢測LRF過表達(dá)和干擾慢病毒在NIH 3T3細(xì)胞中的過表達(dá)與干擾效果。包裝的LRF過表達(dá)慢病毒能顯著升高外源的LRF蛋白的表達(dá)。包裝的shLRF干擾慢病毒中pCD513B-U6-shLRF-2205片段可以顯著干擾LRF蛋白的表達(dá)。LRF干擾與過表達(dá)慢病毒的運(yùn)用,對進(jìn)一步研究LRF在小鼠中的生理學(xué)功能具有重要意義。2.運(yùn)用western blot、免疫組化等技術(shù)檢測了在甾類激素E2和P4的作用下,LRF蛋白在卵巢摘除小鼠子宮和基質(zhì)細(xì)胞中的表達(dá)。結(jié)果顯示,P4能顯著上調(diào)LRF蛋白在卵巢摘除的小鼠子宮和原代基質(zhì)細(xì)胞中的表達(dá),并且以時(shí)間依賴的方式上升。而且,孕酮受體拮抗劑米非司酮(RU486)能顯著的下調(diào)LRF的表達(dá)。盡管E2處理卵巢摘除小鼠與原代基質(zhì)細(xì)胞后LRF蛋白的表達(dá)沒有明顯的變化,但是,與孕酮處理組相比,E2與P4聯(lián)合處理組中LRF蛋白的表達(dá)顯著下調(diào)。而且,與E2組、對照組相比,在雌激素受體拮抗劑氟維司(ICI 182780)群處理組,LRF蛋白的表達(dá)顯著上調(diào)。這些結(jié)果表明,LRF在小鼠子宮與基質(zhì)細(xì)胞中的表達(dá)經(jīng)由P4-PGR通路調(diào)節(jié),而且雌激素能夠部分地抑制P4上調(diào)的LRF表達(dá)。3.運(yùn)用shLRF干擾慢病毒感染懷孕第3天的小鼠子宮,可以顯著減少妊娠第7-8 d胚胎植入位點(diǎn)的大小與重量。在體外誘導(dǎo)蛻膜化的基質(zhì)細(xì)胞模型中,LRF蛋白和mRNA表達(dá)隨著人工誘導(dǎo)蛻膜化進(jìn)程而顯著升高。shLRF干擾慢病毒感染基質(zhì)細(xì)胞并誘導(dǎo)蛻膜化,蛻膜化標(biāo)記因子dPRP、IGFBP-1和PGR的表達(dá)下調(diào),蛻膜化進(jìn)程也受到抑制。運(yùn)用流式細(xì)胞術(shù)檢測體外蛻膜化基質(zhì)細(xì)胞的細(xì)胞周期與細(xì)胞凋亡發(fā)現(xiàn),shLRF干擾慢病毒感染基質(zhì)細(xì)胞后對細(xì)胞凋亡沒有明顯的影響。但是,蛻膜化基質(zhì)細(xì)胞的細(xì)胞周期在LRF干擾以后被阻滯在了S期。qRT-PCR檢測與S期細(xì)胞周期相關(guān)的或者與蛻膜化相關(guān)的細(xì)胞周期調(diào)節(jié)因子發(fā)現(xiàn),shLRF慢病毒干擾蛻膜細(xì)胞后,cyclin A與cyclin B1的mRNA表達(dá)顯著降低,而cyclin D3與cyclin E的mRNA表達(dá)沒有變化。因此,本試驗(yàn)說明了LRF參與小鼠子宮基質(zhì)蛻膜化的調(diào)節(jié),并通過調(diào)節(jié)細(xì)胞周期因子cyclin A與cyclin B1進(jìn)而調(diào)節(jié)蛻膜化進(jìn)程。本研究證實(shí)了內(nèi)質(zhì)網(wǎng)應(yīng)激的相關(guān)蛋白-LRF在小鼠子宮中的表達(dá)受甾類激素的調(diào)控;而且,LRF通過調(diào)節(jié)細(xì)胞周期相關(guān)基因cyclin A和cyclin B1的表達(dá)參與基質(zhì)細(xì)胞蛻膜化的調(diào)節(jié)。揭示了LRF參與雌性小鼠生殖系統(tǒng)的生理調(diào)節(jié),并為進(jìn)一步研究LRF在哺乳動物生殖過程中的作用提供了一個(gè)起點(diǎn),也為對內(nèi)質(zhì)網(wǎng)應(yīng)激與哺乳動物生殖之間的關(guān)系理解添加了一個(gè)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.

【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】：R714.2

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