本文選題：TSA + MTA　； 參考：《第三軍醫(yī)大學(xué)》2014年碩士論文

【摘要】：關(guān)于人類(lèi)分娩啟動(dòng)機(jī)制的學(xué)說(shuō)很多，但妊娠過(guò)程中子宮如何由靜息轉(zhuǎn)變?yōu)槭湛s狀態(tài)導(dǎo)致分娩開(kāi)始的具體機(jī)制仍未完全闡明。子宮平滑肌細(xì)胞所處的狀態(tài)受到多種激素的調(diào)節(jié)，，其中孕激素在維持其保持靜息及舒張狀態(tài)中起重要作用，它通過(guò)抑制子宮平滑肌細(xì)胞鈣離子轉(zhuǎn)運(yùn)蛋白、抑制催產(chǎn)素分泌、促進(jìn)β-腎上腺素能受體的表達(dá)等機(jī)制來(lái)確保平滑肌細(xì)胞處于松弛狀態(tài)。 大多數(shù)非靈長(zhǎng)類(lèi)動(dòng)物分娩的啟動(dòng)伴有血漿孕激素的下降，而人類(lèi)妊娠有別于其他動(dòng)物：妊娠后期母體中血漿孕激素并未下降，而是與血漿雌激素共同升高。這說(shuō)明人類(lèi)分娩的發(fā)動(dòng)并不需要孕激素(至少是血漿中雌激素)下降的參與。但是研究卻發(fā)現(xiàn)孕激素在人類(lèi)分娩中起重要作用，因?yàn)樵屑に厥荏w拮抗劑無(wú)論在妊娠的任何階段運(yùn)用后均可導(dǎo)致宮縮開(kāi)始,分娩啟動(dòng)。這一現(xiàn)象說(shuō)明：人類(lèi)分娩機(jī)制雖然血清中孕激素水平并未下降，但是“功能性的孕激素”水平卻是降低的——“功能性的孕激素撤退”可能是分娩啟動(dòng)機(jī)制的主要原因。 目前認(rèn)為引起“功能性的孕激素撤退”機(jī)制的原因主要有：①人類(lèi)孕激素受體(progesterone receptor,PR)亞型的變化；②子宮平滑肌局部環(huán)境使孕激素代謝為無(wú)活性的成分；③輔活化因子水平的變化使PR的轉(zhuǎn)錄活性變化；④NFкB的反抑制作用。其中PR受體亞型的變化導(dǎo)致妊娠子宮平滑肌細(xì)胞對(duì)孕激素敏感性改變而啟動(dòng)產(chǎn)程機(jī)制擁有諸多實(shí)驗(yàn)證據(jù)予以支持。 PR基因長(zhǎng)度約為90kb，內(nèi)含8個(gè)內(nèi)顯子序列，位于11q22-23。PR有PRA、PRB兩種亞型，PRA基因N端比PRB基因N端少164個(gè)氨基酸，因此它們的結(jié)構(gòu)和功能都存在很大差異， PRB具有轉(zhuǎn)錄活性，而PRA則發(fā)揮抑制PRB的作用；此外，PRB可以通過(guò)結(jié)合孕激素后起到維持子宮平滑肌舒張狀態(tài)的作用，而PRA則拮抗該作用。大量研究結(jié)果顯示：在人類(lèi)分娩啟動(dòng)前后，子宮平滑肌細(xì)胞PRA表達(dá)量相對(duì)PRB升高，導(dǎo)致二者比列發(fā)生改變，進(jìn)而平滑肌細(xì)胞對(duì)孕激素的敏感性發(fā)生改變，提示PRA與PRB的比例發(fā)生變化可能是分娩啟動(dòng)機(jī)制中的關(guān)鍵因素。而對(duì)不同組織中的研究均發(fā)現(xiàn)表觀(guān)遺傳對(duì)PRs的基因表達(dá)均有顯著影響�？梢院侠硗茰y(cè)，PR基因表觀(guān)遺傳學(xué)修飾可通過(guò)改變PRA、PRB表達(dá)及二者相對(duì)比例變化進(jìn)而在“功能性的孕激素撤退”機(jī)制中發(fā)揮重要作用。 本項(xiàng)目以妊娠子宮平滑肌組織為研究對(duì)象，采用免疫組化、western blot、實(shí)時(shí)定量PCR等方法檢測(cè)分娩發(fā)動(dòng)前后PRA、PRB的表達(dá)變化；通過(guò)ChIP檢測(cè)分娩發(fā)動(dòng)前后子宮平滑肌PR啟動(dòng)子區(qū)H3ac、H4ac、H3K4me3水平。組蛋白乙�；敢种苿┣敲顾谹(trichostatinA, TSA)、H3K4甲基化酶抑制劑5-脫氧-5-甲硫腺苷(5’-deoxy-5’-methylthioadenosine, MTA)處理子宮平滑肌細(xì)胞系前后檢測(cè)PRA及PRB啟動(dòng)子區(qū)乙�；�、甲基化水平變化，初步探討：①產(chǎn)程啟動(dòng)前后，PRs啟動(dòng)子區(qū)乙酰化程度是否發(fā)生變化；②子宮平滑肌細(xì)胞PRs啟動(dòng)子區(qū)乙酰化修飾是否影響PRA、PRB基因的表達(dá)；③PRA、PRB啟動(dòng)子區(qū)乙酰化修飾是否與“功能性孕激素撤退”分娩啟動(dòng)機(jī)制相關(guān)。為探討控制分娩啟動(dòng)的有效環(huán)節(jié)，防治早產(chǎn)及過(guò)期產(chǎn)提供新理論依據(jù)。 試驗(yàn)結(jié)果表明，PR及其亞型在啟動(dòng)組及未啟動(dòng)組的子宮平滑肌細(xì)胞核中都有明顯的表達(dá)。啟動(dòng)組PRA的表達(dá)及PRA/PRB比值比未啟動(dòng)組顯著升高，但PRB未見(jiàn)明顯升高，說(shuō)明PRA的表達(dá)及PRA/PRB比值升高可能與分娩啟動(dòng)機(jī)制相關(guān)。 通過(guò)ChIP檢測(cè)PRA、PRB啟動(dòng)子區(qū)的H3、H4乙�；癏3K4三甲基化水平，結(jié)果顯示：相比較于產(chǎn)程未啟動(dòng)組產(chǎn)程啟動(dòng)組妊娠子宮平滑肌H3、H4乙�；綗o(wú)論是在PRA還是PRB的啟動(dòng)子區(qū)的均未見(jiàn)明顯改變，而H3K4三甲基化水平在兩個(gè)啟動(dòng)子區(qū)均顯著升高。據(jù)此我們認(rèn)為，產(chǎn)程啟動(dòng)組子宮平滑肌PRA表達(dá)增高主要是由其H3K4三甲基化作用而引起的。隨后，我們分離、純化培養(yǎng)未啟動(dòng)子宮平滑肌細(xì)胞，并加用TSA、MTA進(jìn)行干預(yù)，發(fā)現(xiàn)TSA可顯著提高PRA mRNA的表達(dá)，MTA可顯著抑制其表達(dá)，而ChIP檢測(cè)提示TSA、MTA干預(yù)后PRA啟動(dòng)子區(qū)H3、H4乙�；癏3K4三甲基化水平均發(fā)生顯著改變，并能夠進(jìn)一步影響PRA/PRB比值，提示子宮平滑肌細(xì)胞H3、H4乙�；⒓癏3K4甲基化均可通過(guò)調(diào)節(jié)PRA的表達(dá)改變PRA/PRB比值。細(xì)胞水平與組織水平實(shí)驗(yàn)得出的結(jié)果略有不同，尤其是在乙�；挠绊懡Y(jié)果上，組織水平顯示乙�；趩�(dòng)組和未啟動(dòng)組之間沒(méi)有明顯差異，但細(xì)胞水平卻顯示乙酰化確實(shí)可以使PRA、PR的表達(dá)量升高，這樣的結(jié)果可能是因?yàn)榻M織中的表觀(guān)遺傳是一個(gè)非常復(fù)雜的體系，而體外單純的藥物干預(yù)只能單因素的干擾乙酰化或者甲基化水平而沒(méi)有能夠完全模擬體內(nèi)復(fù)雜的調(diào)節(jié)環(huán)境，但是在體外細(xì)胞水平的研究，仍然為我們今后的實(shí)驗(yàn)方向奠定了基礎(chǔ)。
[Abstract]:There are many theories about the initiation mechanism of human childbirth, but the specific mechanism of how the uterus changes from resting to contraction during pregnancy has not been fully elucidated. The state of the uterine smooth muscle cells is regulated by a variety of hormones, in which progestin plays an important role in maintaining its resting and diastolic state. By inhibiting the calcium transporter of the uterine smooth muscle cells, inhibiting the secretion of oxytocin, promoting the expression of beta adrenergic receptor and so on, the smooth muscle cells are in a relaxed state.
The initiation of labor in most non primates is accompanied by a drop in plasma progestin, while human pregnancy is different from that of other animals: plasma progesterone does not decrease in the late pregnancy, but increases with the plasma estrogen. This suggests that the initiation of human birth does not require the participation of progestin (at least the estrogen in the plasma). It is found that progestin plays an important role in human childbirth, because the progestin receptor antagonist can lead to the onset of uterine contraction and the initiation of childbirth at any stage of pregnancy. This phenomenon indicates that although the level of progestin in the human childbirth mechanism does not decline, the level of "functional progesterone" is reduced. Low "functional withdrawal of progestin" may be the main reason for the initiation of labor.
The main reasons for the mechanism of "functional progestin withdrawal" are as follows: (1) changes in the progesterone receptor (PR) subtype of human progestin receptor; (2) the local environment of the uterine smooth muscle makes progesterone metabolism an inactive component; (3) the alteration of the level of the activator makes the transcriptional activity of PR change; (4) the reverse inhibition of NF B The changes in the PR receptor subtype lead to a variety of experimental evidence to support the changes in progesterone sensitivity in pregnancy uterine smooth muscle cells and the initiation of the mechanism of labor.
The length of the PR gene is about 90KB, containing 8 introns, located in 11q22-23.PR with PRA, PRB two subtypes, and PRA gene N ends with 164 amino acids less than the N terminal of PRB gene, so their structure and function are very different. PRB has a transcriptional activity, while PRA plays the role of inhibiting PRB. Furthermore, it can be recovered by combining progestin to dimension. A large number of studies showed that the expression of PRA in the uterine smooth muscle cells increased relative to PRB before and after the initiation of human childbirth, leading to a change in the number of two groups and the changes in the sensitivity of the smooth muscle cells to progesterone, suggesting a change in the proportion of PRA to PRB. It is the key factor in the initiation mechanism of childbirth. And the epigenetic inheritance has a significant influence on the gene expression of PRs in different tissues. It is reasonable to speculate that the epigenetic modification of PR gene can play a heavy role in the mechanism of "functional progestin withdrawal" by changing the PRA, PRB expression and the relative proportion of the two. It's going to work.
This project took the uterine smooth muscle tissue of pregnancy as the research object, using immunohistochemistry, Western blot, real-time quantitative PCR and other methods to detect the changes of PRA, PRB expression before and after delivery. ChIP was used to detect H3ac, H4ac, H3K4me3 of PR promoter region of uterine smooth muscle before and after delivery. Histone acetylase inhibitor, osseomycin A (trichost), was detected by ChIP. AtinA, TSA), H3K4 methylation inhibitor 5- deoxy -5- methionine adenosine (5 '-deoxy-5' -methylthioadenosine, MTA) before and after the treatment of uterine smooth muscle cell lines to detect the level of PRA and PRB promoter region acetylation, the level of methylation changes. Whether the acetylated modification of the PRs promoter region of the smooth muscle cells affects the expression of PRA and PRB gene; (3) whether the acetylation of the PRB promoter region is related to the initiation mechanism of "functional progestin withdrawal", which provides a new theoretical basis for the prevention and control of premature delivery and overdue production.
The results showed that PR and its subtypes were clearly expressed in the nucleus of the uterine smooth muscle in the starting and unstarted groups. The expression of PRA and the ratio of PRA/PRB in the starting group were significantly higher than those in the uninitiated group, but the PRB was not significantly elevated, indicating that the expression of PRA and the increase of the ratio of PRA/PRB may be related to the mechanism of the initiation of delivery.
The levels of H3, H4 acetylation and H3K4 three methylation in the promoter region of the PRB were detected by ChIP. The results showed that the level of H4 acetylation was not significantly changed in the promoter region of the PRA or PRB compared to the H3 in the pregnancy of the production process group, and the level of H3K4 three methylation was significant in the two promoter regions. On this basis, we think that the increased expression of PRA in the uterine smooth muscle was mainly caused by its H3K4 trimethylation. Then, we isolated, purified and cultured no uterine smooth muscle cells, and added TSA, MTA to improve the expression of PRA mRNA significantly, and MTA could significantly inhibit its expression and ChIP detection. TSA, MTA, PRA promoter region H3, H4 acetylation and H3K4 three methylated water significantly changed, and can further affect the PRA/PRB ratio, suggesting that H3, H4 acetylation, and H3K4 methylation of uterine smooth muscle cells can change PRA/PRB ratio by regulating PRA expression. The results of cell level and tissue level are slightly less. At the same time, especially in the effect of acetylation, the tissue level showed no significant difference between the starting and the non starting groups, but the cell level showed that acetylation did make the expression of PRA and PR elevated, which may be because epigenetics in the tissue is a very complex system, and in vitro simple Drug intervention can only interfere with the level of acetylation or methylation but not fully simulate the complex regulatory environment in the body, but the study of cell level in vitro still lays the foundation for our future experimental direction.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R714.3

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