LSM4基因與小鼠子宮內(nèi)膜蛻膜化的關(guān)系研究
發(fā)布時(shí)間：2018-10-17 12:10
【摘要】：目的:探索LSM4基因在子宮內(nèi)膜組織是否表達(dá)及其與子宮內(nèi)膜蛻膜化的關(guān)系,為闡明蛻膜化機(jī)制提供新的途徑。方法:1.應(yīng)用原位雜交(ISH)和免疫組織化學(xué)(IHC)技術(shù),分別從m RNA和蛋白水平觀察LSM4在小鼠子宮內(nèi)膜的表達(dá)及其與孕期子宮蛻膜化的關(guān)系;2.應(yīng)用體外培養(yǎng)子宮內(nèi)膜基質(zhì)細(xì)胞并誘導(dǎo)蛻膜化實(shí)驗(yàn)?zāi)Ｐ秃蛯?shí)時(shí)定量PCR技術(shù),檢測(cè)蛻膜化中LSM4-m RNA表達(dá)動(dòng)態(tài);3.應(yīng)用RNAi技術(shù)對(duì)原代基質(zhì)細(xì)胞進(jìn)行LSM4基因敲降(LSM4-KD)繼而誘導(dǎo)蛻膜化,并對(duì)LSM4-KD細(xì)胞檢測(cè)蛻膜化標(biāo)志分子蛻膜催乳素相關(guān)蛋白(DPRP)和堿性磷酸酶(AP),考察LSM4基因表達(dá)下調(diào)是否抑制基質(zhì)細(xì)胞蛻膜化。結(jié)果:1.ISH和IHC相互呼應(yīng)地顯示,孕第1天LSM4基因的m RNA和蛋白均表達(dá)于子宮內(nèi)膜腔上皮和腺上皮;植入窗口期的第4天,在上皮和緊鄰腔面上皮的子宮內(nèi)膜淺層基質(zhì)中有一部分基質(zhì)細(xì)胞LSM4基因顯著表達(dá);第5天之后LSM4在上皮表達(dá)下調(diào),轉(zhuǎn)而主要表達(dá)于內(nèi)膜基質(zhì)細(xì)胞,與子宮內(nèi)膜的蛻膜化基本同步;內(nèi)膜基質(zhì)層內(nèi)LSM4陽(yáng)性細(xì)胞隨蛻膜化進(jìn)展而增多,并從內(nèi)膜淺層擴(kuò)展到全層,而植入胚周圍的淺層基質(zhì)中陽(yáng)性細(xì)胞則繼而減少;至蛻膜化高峰的孕第8天,基質(zhì)細(xì)胞內(nèi)LSM4-m RNA雜交信號(hào)顯著減弱,但蛋白陽(yáng)性信號(hào)依然很強(qiáng);LSM4蛋白同時(shí)分布于蛻膜細(xì)胞的胞核和胞質(zhì),在胞質(zhì)中LSM4蛋白呈網(wǎng)絡(luò)狀/顆粒狀聚集并附著于細(xì)胞邊界。2.在體外誘導(dǎo)蛻膜化進(jìn)程中,基質(zhì)細(xì)胞LSM4-m RNA相對(duì)拷貝數(shù)從0h~96h逐日降低(P0.05);在LSM4-KD實(shí)驗(yàn)的陰性對(duì)照組中,LSM4-m RNA也呈下降趨勢(shì)且與DPRP的表達(dá)趨勢(shì)相反。3.與對(duì)照組相比,感染LSM4-sh RNA的基質(zhì)細(xì)胞LSM4-m RNA表達(dá)量降低極其顯著(P0.01),確認(rèn)了LSM4基因的敲降(LSM4-KD)。LSM4-KD細(xì)胞的DPRP-m RNA相對(duì)表達(dá)量在蛻膜化48h時(shí)顯著低于對(duì)照組(p0.05),96h更顯著(p0.01)。對(duì)同一種細(xì)胞LSM4/DPRP的m RNA量進(jìn)行相關(guān)分析,雖然實(shí)驗(yàn)組LSM4-KD導(dǎo)致兩者的量均極低,但在蛻膜化的同一時(shí)間點(diǎn),LSM4/DPRP表達(dá)量之間有一定相關(guān)性。24h兩者呈高度正相關(guān),48h卻呈高度負(fù)相關(guān),而96h時(shí)又呈高度正相關(guān)。提示LSM4與DPRP可能存在某種調(diào)節(jié)關(guān)系。4.LSM4-KD細(xì)胞在蛻膜化96h時(shí),AP活性普遍顯著減弱,細(xì)胞體積也較小。結(jié)論:1.首次發(fā)現(xiàn)LSM4基因在小鼠孕早期的子宮內(nèi)膜呈現(xiàn)時(shí)空規(guī)律性表達(dá),即圍繞植入和蛻膜化起始時(shí)段的升高波動(dòng)和從腔上皮到深層基質(zhì)的組織空間波動(dòng),此組織空間波動(dòng)也是基質(zhì)細(xì)胞個(gè)體蛻膜化起始時(shí)段的升高波動(dòng);2.首次驗(yàn)證LSM4基因的敲降顯著抑制了蛻膜化標(biāo)志基因DPRP和AP的表達(dá),證明LSM4對(duì)基質(zhì)細(xì)胞的蛻膜化起關(guān)鍵作用;3.推測(cè)LSM4基因很可能是子宮內(nèi)膜基質(zhì)細(xì)胞蛻膜化的啟動(dòng)因子,也可能還是子宮內(nèi)膜接受態(tài)的構(gòu)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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R714.1
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