PR启动子区H3ac、H4ac及H3K4me3对分娩启动作用的研究

发布时间：2018-05-07 09:23

本文选题：TSA + MTA　；参考：《第三军医大学》2014年硕士论文

【摘要】：关于人类分娩启动机制的学说很多，但妊娠过程中子宫如何由静息转变为收缩状态导致分娩开始的具体机制仍未完全阐明。子宫平滑肌细胞所处的状态受到多种激素的调节，，其中孕激素在维持其保持静息及舒张状态中起重要作用，它通过抑制子宫平滑肌细胞钙离子转运蛋白、抑制催产素分泌、促进β-肾上腺素能受体的表达等机制来确保平滑肌细胞处于松弛状态。大多数非灵长类动物分娩的启动伴有血浆孕激素的下降，而人类妊娠有别于其他动物：妊娠后期母体中血浆孕激素并未下降，而是与血浆雌激素共同升高。这说明人类分娩的发动并不需要孕激素(至少是血浆中雌激素)下降的参与。但是研究却发现孕激素在人类分娩中起重要作用，因为孕激素受体拮抗剂无论在妊娠的任何阶段运用后均可导致宫缩开始,分娩启动。这一现象说明：人类分娩机制虽然血清中孕激素水平并未下降，但是“功能性的孕激素”水平却是降低的——“功能性的孕激素撤退”可能是分娩启动机制的主要原因。目前认为引起“功能性的孕激素撤退”机制的原因主要有：①人类孕激素受体(progesterone receptor,PR)亚型的变化；②子宫平滑肌局部环境使孕激素代谢为无活性的成分；③辅活化因子水平的变化使PR的转录活性变化；④NFкB的反抑制作用。其中PR受体亚型的变化导致妊娠子宫平滑肌细胞对孕激素敏感性改变而启动产程机制拥有诸多实验证据予以支持。 PR基因长度约为90kb，内含8个内显子序列，位于11q22-23。PR有PRA、PRB两种亚型，PRA基因N端比PRB基因N端少164个氨基酸，因此它们的结构和功能都存在很大差异， PRB具有转录活性，而PRA则发挥抑制PRB的作用；此外，PRB可以通过结合孕激素后起到维持子宫平滑肌舒张状态的作用，而PRA则拮抗该作用。大量研究结果显示：在人类分娩启动前后，子宫平滑肌细胞PRA表达量相对PRB升高，导致二者比列发生改变，进而平滑肌细胞对孕激素的敏感性发生改变，提示PRA与PRB的比例发生变化可能是分娩启动机制中的关键因素。而对不同组织中的研究均发现表观遗传对PRs的基因表达均有显著影响。可以合理推测，PR基因表观遗传学修饰可通过改变PRA、PRB表达及二者相对比例变化进而在“功能性的孕激素撤退”机制中发挥重要作用。本项目以妊娠子宫平滑肌组织为研究对象，采用免疫组化、western blot、实时定量PCR等方法检测分娩发动前后PRA、PRB的表达变化；通过ChIP检测分娩发动前后子宫平滑肌PR启动子区H3ac、H4ac、H3K4me3水平。组蛋白乙酰化酶抑制剂曲骨霉素A(trichostatinA, TSA)、H3K4甲基化酶抑制剂5-脱氧-5-甲硫腺苷(5’-deoxy-5’-methylthioadenosine, MTA)处理子宫平滑肌细胞系前后检测PRA及PRB启动子区乙酰化、甲基化水平变化，初步探讨：①产程启动前后，PRs启动子区乙酰化程度是否发生变化；②子宫平滑肌细胞PRs启动子区乙酰化修饰是否影响PRA、PRB基因的表达；③PRA、PRB启动子区乙酰化修饰是否与“功能性孕激素撤退”分娩启动机制相关。为探讨控制分娩启动的有效环节，防治早产及过期产提供新理论依据。试验结果表明，PR及其亚型在启动组及未启动组的子宫平滑肌细胞核中都有明显的表达。启动组PRA的表达及PRA/PRB比值比未启动组显著升高，但PRB未见明显升高，说明PRA的表达及PRA/PRB比值升高可能与分娩启动机制相关。通过ChIP检测PRA、PRB启动子区的H3、H4乙酰化及H3K4三甲基化水平，结果显示：相比较于产程未启动组产程启动组妊娠子宫平滑肌H3、H4乙酰化水平无论是在PRA还是PRB的启动子区的均未见明显改变，而H3K4三甲基化水平在两个启动子区均显著升高。据此我们认为，产程启动组子宫平滑肌PRA表达增高主要是由其H3K4三甲基化作用而引起的。随后，我们分离、纯化培养未启动子宫平滑肌细胞，并加用TSA、MTA进行干预，发现TSA可显著提高PRA mRNA的表达，MTA可显著抑制其表达，而ChIP检测提示TSA、MTA干预后PRA启动子区H3、H4乙酰化及H3K4三甲基化水平均发生显著改变，并能够进一步影响PRA/PRB比值，提示子宫平滑肌细胞H3、H4乙酰化、及H3K4甲基化均可通过调节PRA的表达改变PRA/PRB比值。细胞水平与组织水平实验得出的结果略有不同，尤其是在乙酰化的影响结果上，组织水平显示乙酰化在启动组和未启动组之间没有明显差异，但细胞水平却显示乙酰化确实可以使PRA、PR的表达量升高，这样的结果可能是因为组织中的表观遗传是一个非常复杂的体系，而体外单纯的药物干预只能单因素的干扰乙酰化或者甲基化水平而没有能够完全模拟体内复杂的调节环境，但是在体外细胞水平的研究，仍然为我们今后的实验方向奠定了基础。
[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.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.3

【共引文献】