LSM4基因与小鼠子宫内膜蜕膜化的关系研究
发布时间：2018-10-17 12:10
【摘要】：目的:探索LSM4基因在子宫内膜组织是否表达及其与子宫内膜蜕膜化的关系,为阐明蜕膜化机制提供新的途径。方法:1.应用原位杂交(ISH)和免疫组织化学(IHC)技术,分别从m RNA和蛋白水平观察LSM4在小鼠子宫内膜的表达及其与孕期子宫蜕膜化的关系;2.应用体外培养子宫内膜基质细胞并诱导蜕膜化实验模型和实时定量PCR技术,检测蜕膜化中LSM4-m RNA表达动态;3.应用RNAi技术对原代基质细胞进行LSM4基因敲降(LSM4-KD)继而诱导蜕膜化,并对LSM4-KD细胞检测蜕膜化标志分子蜕膜催乳素相关蛋白(DPRP)和碱性磷酸酶(AP),考察LSM4基因表达下调是否抑制基质细胞蜕膜化。结果:1.ISH和IHC相互呼应地显示,孕第1天LSM4基因的m RNA和蛋白均表达于子宫内膜腔上皮和腺上皮;植入窗口期的第4天,在上皮和紧邻腔面上皮的子宫内膜浅层基质中有一部分基质细胞LSM4基因显著表达;第5天之后LSM4在上皮表达下调,转而主要表达于内膜基质细胞,与子宫内膜的蜕膜化基本同步;内膜基质层内LSM4阳性细胞随蜕膜化进展而增多,并从内膜浅层扩展到全层,而植入胚周围的浅层基质中阳性细胞则继而减少;至蜕膜化高峰的孕第8天,基质细胞内LSM4-m RNA杂交信号显著减弱,但蛋白阳性信号依然很强;LSM4蛋白同时分布于蜕膜细胞的胞核和胞质,在胞质中LSM4蛋白呈网络状/颗粒状聚集并附着于细胞边界。2.在体外诱导蜕膜化进程中,基质细胞LSM4-m RNA相对拷贝数从0h~96h逐日降低(P0.05);在LSM4-KD实验的阴性对照组中,LSM4-m RNA也呈下降趋势且与DPRP的表达趋势相反。3.与对照组相比,感染LSM4-sh RNA的基质细胞LSM4-m RNA表达量降低极其显著(P0.01),确认了LSM4基因的敲降(LSM4-KD)。LSM4-KD细胞的DPRP-m RNA相对表达量在蜕膜化48h时显著低于对照组(p0.05),96h更显著(p0.01)。对同一种细胞LSM4/DPRP的m RNA量进行相关分析,虽然实验组LSM4-KD导致两者的量均极低,但在蜕膜化的同一时间点,LSM4/DPRP表达量之间有一定相关性。24h两者呈高度正相关,48h却呈高度负相关,而96h时又呈高度正相关。提示LSM4与DPRP可能存在某种调节关系。4.LSM4-KD细胞在蜕膜化96h时,AP活性普遍显著减弱,细胞体积也较小。结论:1.首次发现LSM4基因在小鼠孕早期的子宫内膜呈现时空规律性表达,即围绕植入和蜕膜化起始时段的升高波动和从腔上皮到深层基质的组织空间波动,此组织空间波动也是基质细胞个体蜕膜化起始时段的升高波动;2.首次验证LSM4基因的敲降显著抑制了蜕膜化标志基因DPRP和AP的表达,证明LSM4对基质细胞的蜕膜化起关键作用;3.推测LSM4基因很可能是子宫内膜基质细胞蜕膜化的启动因子,也可能还是子宫内膜接受态的构成分子。
[Abstract]:Aim: to explore the expression of LSM4 gene in endometrium and its relationship with decidualization, and to provide a new approach to elucidate the mechanism of decidualization. Methods: 1. The expression of LSM4 in mouse endometrium and the relationship between LSM4 expression and decidualization during pregnancy were observed by in situ hybridization (ISH) and immunohistochemical (IHC) technique from the level of m RNA and protein. 2. The expression of LSM4-m RNA in decidualization was detected by cultured endometrial stromal cells in vitro, induced decidualization model and real-time quantitative PCR. 3. LSM4 gene knockout (LSM4-KD) was used to induce decidualization in primary stromal cells by RNAi. The down-regulation of LSM4 gene expression in decidua-associated protein (DPRP) and alkaline phosphatase (AP),) was investigated in LSM4-KD cells to determine whether the down-regulation of LSM4 gene could inhibit decidualization of stromal cells. Results: 1.ISH and IHC showed that m RNA and protein of LSM4 gene were expressed in endometrial cavity epithelium and glandular epithelium on the first day of pregnancy. LSM4 gene was expressed significantly in some of the stromal cells in the epithelium and the superficial endometrial matrix adjacent to the surface of the lumen. After 5 days, the expression of LSM4 was down-regulated in the epithelium and mainly expressed in the endometrial stromal cells. LSM4 positive cells in the endometrial stromal layer increased with the development of decidualization and expanded from the superficial layer of the endometrium to the whole layer, while the positive cells in the superficial matrix around the implantation embryo decreased. At the 8th day of gestation, the signal of LSM4-m RNA hybridization in stromal cells was significantly decreased, but the positive signal of protein was still strong, and LSM4 protein was distributed in both nucleus and cytoplasm of decidua cells. In the cytoplasm, the LSM4 protein was network-shaped / granular and attached to the boundary of the cell. 2. In the process of induced decidualization in vitro, the relative copy number of LSM4-m RNA in stromal cells decreased from 0h~96h (P0.05); in the negative control group of LSM4-KD experiment, LSM4-m RNA also showed a decreasing trend and contrary to the expression trend of DPRP. 3. Compared with the control group, the expression of LSM4-m RNA in the stromal cells infected with LSM4-sh RNA was significantly decreased (P0.01), which confirmed the LSM4-KD expression of LSM4 gene. The relative expression of DPRP-m RNA in the LSM4-KD cells was significantly lower than that in the control group at 48 h after decidualization (p0.05), and was more significant at 96 h (p0.01). The m RNA level of LSM4/DPRP of the same cell was analyzed by correlation analysis. Although the quantity of LSM4-KD in the experimental group was very low, at the same time point of decidualization, there was a certain correlation between the expression of LSM4/DPRP and the expression of LSM4/DPRP. There was a high positive correlation between them at 24 h and a high negative correlation at 48 h. At 96 h, there was a high positive correlation. The results suggest that there may be some regulatory relationship between LSM4 and DPRP. At 96 h of decidualization, the AP activity of 4.LSM4-KD cells was significantly decreased and the cell volume was smaller. Conclusion: 1. It is the first time to find that the expression of LSM4 gene in mouse endometrium in early pregnancy is spatiotemporal regular, that is, the fluctuation of tissue space from luminal epithelium to deep matrix, which revolves around the initial period of implantation and decidualization. The spatial fluctuation of the tissue is also the rise of the initiation of decidualization of stromal cells; 2. For the first time, the knockout of LSM4 gene significantly inhibited the expression of decidualization marker genes DPRP and AP, which indicated that LSM4 played a key role in decidualization of stromal cells. 3. It is speculated that LSM4 gene may be the promoter of decidualization of endometrial stromal cells or the constituent molecule of endometrial receptive state.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R714.1
[Abstract]:Aim: to explore the expression of LSM4 gene in endometrium and its relationship with decidualization, and to provide a new approach to elucidate the mechanism of decidualization. Methods: 1. The expression of LSM4 in mouse endometrium and the relationship between LSM4 expression and decidualization during pregnancy were observed by in situ hybridization (ISH) and immunohistochemical (IHC) technique from the level of m RNA and protein. 2. The expression of LSM4-m RNA in decidualization was detected by cultured endometrial stromal cells in vitro, induced decidualization model and real-time quantitative PCR. 3. LSM4 gene knockout (LSM4-KD) was used to induce decidualization in primary stromal cells by RNAi. The down-regulation of LSM4 gene expression in decidua-associated protein (DPRP) and alkaline phosphatase (AP),) was investigated in LSM4-KD cells to determine whether the down-regulation of LSM4 gene could inhibit decidualization of stromal cells. Results: 1.ISH and IHC showed that m RNA and protein of LSM4 gene were expressed in endometrial cavity epithelium and glandular epithelium on the first day of pregnancy. LSM4 gene was expressed significantly in some of the stromal cells in the epithelium and the superficial endometrial matrix adjacent to the surface of the lumen. After 5 days, the expression of LSM4 was down-regulated in the epithelium and mainly expressed in the endometrial stromal cells. LSM4 positive cells in the endometrial stromal layer increased with the development of decidualization and expanded from the superficial layer of the endometrium to the whole layer, while the positive cells in the superficial matrix around the implantation embryo decreased. At the 8th day of gestation, the signal of LSM4-m RNA hybridization in stromal cells was significantly decreased, but the positive signal of protein was still strong, and LSM4 protein was distributed in both nucleus and cytoplasm of decidua cells. In the cytoplasm, the LSM4 protein was network-shaped / granular and attached to the boundary of the cell. 2. In the process of induced decidualization in vitro, the relative copy number of LSM4-m RNA in stromal cells decreased from 0h~96h (P0.05); in the negative control group of LSM4-KD experiment, LSM4-m RNA also showed a decreasing trend and contrary to the expression trend of DPRP. 3. Compared with the control group, the expression of LSM4-m RNA in the stromal cells infected with LSM4-sh RNA was significantly decreased (P0.01), which confirmed the LSM4-KD expression of LSM4 gene. The relative expression of DPRP-m RNA in the LSM4-KD cells was significantly lower than that in the control group at 48 h after decidualization (p0.05), and was more significant at 96 h (p0.01). The m RNA level of LSM4/DPRP of the same cell was analyzed by correlation analysis. Although the quantity of LSM4-KD in the experimental group was very low, at the same time point of decidualization, there was a certain correlation between the expression of LSM4/DPRP and the expression of LSM4/DPRP. There was a high positive correlation between them at 24 h and a high negative correlation at 48 h. At 96 h, there was a high positive correlation. The results suggest that there may be some regulatory relationship between LSM4 and DPRP. At 96 h of decidualization, the AP activity of 4.LSM4-KD cells was significantly decreased and the cell volume was smaller. Conclusion: 1. It is the first time to find that the expression of LSM4 gene in mouse endometrium in early pregnancy is spatiotemporal regular, that is, the fluctuation of tissue space from luminal epithelium to deep matrix, which revolves around the initial period of implantation and decidualization. The spatial fluctuation of the tissue is also the rise of the initiation of decidualization of stromal cells; 2. For the first time, the knockout of LSM4 gene significantly inhibited the expression of decidualization marker genes DPRP and AP, which indicated that LSM4 played a key role in decidualization of stromal cells. 3. It is speculated that LSM4 gene may be the promoter of decidualization of endometrial stromal cells or the constituent molecule of endometrial receptive state.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R714.1
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