本文選題：Galectin-3 + 滋養(yǎng)細(xì)胞　； 參考：《復(fù)旦大學(xué)》2012年博士論文

【摘要】：胚胎著床涉及胚胎與母體子宮間極其復(fù)雜而精細(xì)的多因素相互作用。這個(gè)過(guò)程中,子宮內(nèi)膜僅在一個(gè)較短的時(shí)期允許成熟胚胎植入,這個(gè)時(shí)期稱為著床窗,這個(gè)時(shí)期子宮內(nèi)膜具有容受性,目前,子宮內(nèi)膜容受性形成的機(jī)理尚不清楚楚。在胚胎植入過(guò)程中,胚胎滋養(yǎng)細(xì)胞表現(xiàn)出與腫瘤細(xì)胞相似的侵襲能力,在具有容受性的子宮內(nèi)膜表面進(jìn)行粘附、入侵和植入。滋養(yǎng)細(xì)胞侵入子宮內(nèi)膜的全程伴隨著子宮內(nèi)膜上皮細(xì)胞的凋亡以及基質(zhì)細(xì)胞的蛻膜化。 Galectin-3(Gal-3)是本課題組前期篩選到的子宮內(nèi)膜容受性相關(guān)分子之一。我們前期的研究發(fā)現(xiàn)其在子宮內(nèi)膜著床窗口期高表達(dá),且在子宮內(nèi)膜異位癥患者在位內(nèi)膜的Galectin-3表達(dá)降低,可能與子宮內(nèi)膜容受性建立不全有關(guān)。目前對(duì)Galectin-3的研究主要集中在腫瘤方面,其在腫瘤細(xì)胞的生長(zhǎng)、粘附、侵襲、凋亡等方面發(fā)揮著重要的生物學(xué)作用,這些生物學(xué)行為可能在胚胎植入過(guò)程中同樣起著巨大的作用。我們前期研究顯示,Galectin-3能夠調(diào)節(jié)子宮內(nèi)膜細(xì)胞的粘附和增殖。鑒于Galectin-3功能的多樣性,我們對(duì)Galectin-3在母胎界面的其它生物學(xué)行為產(chǎn)生了興趣。既然Galectin-3通過(guò)調(diào)節(jié)腫瘤細(xì)胞的凋亡參與腫瘤細(xì)胞的侵襲,Galectin-3是否也在胚胎植入的過(guò)程中發(fā)揮了凋亡調(diào)節(jié)的作用呢? 本研究在前期工作的基礎(chǔ)上,進(jìn)一步明確Galectin-3在容受性子宮內(nèi)膜形成中的作用。運(yùn)用子宮內(nèi)膜局部沉默Galectin-3的方法研究其在著床中的作用,并用經(jīng)典的體外培養(yǎng)胚胎著床模型——Bewo細(xì)胞株和RL95-2細(xì)胞株間接共培養(yǎng)模型,詳細(xì)地探討了胞內(nèi)及胞外Galectin-3對(duì)子宮內(nèi)膜細(xì)胞的凋亡調(diào)節(jié)作用。論文分為以下三個(gè)部分： 第一部分Galectin-3對(duì)小鼠胚胎著床的影響 目的明確Galectin-3對(duì)小鼠胚胎著床的影響。 方法收集早孕小鼠子宮內(nèi)膜,采用real-time PCR,免疫組織化學(xué),Western blot的方法檢測(cè)早孕小鼠子宮內(nèi)膜中Galectin-3mRNA和蛋白表達(dá)變化情況。帶免疫熒光oligo活體注射小鼠子宮角,熒光顯微鏡下觀察活體轉(zhuǎn)染情況并用Western blot方法驗(yàn)證轉(zhuǎn)染效果。孕鼠于孕9天被處死,研究轉(zhuǎn)染Galectin-3siRNA對(duì)小鼠胚胎著床的影響。 結(jié)果Galectin-3mRNA和蛋白在早孕小鼠子宮內(nèi)膜的表達(dá)高于未孕小鼠,Galectin-3mRNA和蛋白水平分別在孕4天和孕2天達(dá)到高峰。免疫組化結(jié)果顯示：Galectin-3蛋白于孕6～8天腔上皮達(dá)高峰,于孕2～4天腺上皮達(dá)高峰。免疫熒光顯示：活體轉(zhuǎn)染的siRNA進(jìn)入腔上皮細(xì)胞內(nèi)。孕4天蛋白檢測(cè)表明Galectin-3蛋白被Galectin-3siRNA有效下調(diào)。孕9天處死小鼠,與對(duì)照組(Negaive siRNA)相比,Galectin-3siRNA注射側(cè)子宮角著床胚胎數(shù)明顯下調(diào)。 結(jié)論Galectin-3存早孕小鼠子宮內(nèi)膜周期性表達(dá),并干著床期高表達(dá),說(shuō)明Galectin-3可能參與子宮內(nèi)膜容受性的形成。局部下調(diào)Galectin-3(?)減少著床的胚胎數(shù),說(shuō)明Galectin-3影響胚胎著床,可能是通過(guò)參與容受性子宮內(nèi)膜的形成起作用,其表達(dá)下調(diào)可能導(dǎo)致子宮內(nèi)膜容受性缺陷從而導(dǎo)致著床失敗。 第二部分子宮內(nèi)膜上皮細(xì)胞胞內(nèi)Galectin-3的抗凋亡作用 目的探討子宮內(nèi)膜上皮細(xì)胞胞內(nèi)Galectin-3的抗凋亡作用。 方法用星胞霉素處理RL95-2細(xì)胞,MTT法檢測(cè)其對(duì)細(xì)胞活力的影響,吖啶橙染色免疫熒光檢測(cè)和AnnexinV/PI流式細(xì)胞儀檢測(cè)的方法確定其對(duì)RL95-2細(xì)胞凋亡的影響,運(yùn)用real-time PCR和Western blot的方法檢測(cè)Galectin-3表達(dá)變化情況。Galectin-3siRNA轉(zhuǎn)染RL95-2細(xì)胞沉默Galectin-3表達(dá),分析Galectin-3沉默后,星胞霉素對(duì)RL95-2細(xì)胞的凋亡促進(jìn)作用。Real-time PCR和Western blot檢測(cè)不同濃度雌、孕激素對(duì)RL95-2細(xì)胞Galectin-3表達(dá)調(diào)節(jié)情況。沉默Galectin-3表達(dá)后加入雌、孕激素和星胞霉素檢測(cè)雌、孕激素是否通過(guò)調(diào)控Galectin-3表達(dá)來(lái)調(diào)節(jié)RL95-2細(xì)胞的凋亡。 結(jié)果MTT結(jié)果顯示：星胞霉素處理后,RL95-2細(xì)胞活力降低,凋亡率上升,Galectin-3mRNA和蛋白反應(yīng)性上升。Galectin-3siRNA能有效下調(diào)RL95-2細(xì)胞Galectin-3表達(dá)。下調(diào)Galectin-3表達(dá)后,星胞霉素對(duì)RL95-2的凋亡促進(jìn)作用加強(qiáng)。雌、孕激素能有效上調(diào)RL95-2細(xì)胞Galectin-3mRNA和蛋白表達(dá)。加入雌、孕激素后, Galectin-3被沉默的RL95-2細(xì)胞凋亡率高于加入Negative siRNA的對(duì)照組。 結(jié)論子宮內(nèi)膜上皮細(xì)胞胞內(nèi)Galectin-3具有抗凋亡作用。雌、孕激素能調(diào)控子宮內(nèi)膜上皮細(xì)胞中Galectin-3表達(dá),雌、孕激素調(diào)控子宮內(nèi)膜上皮細(xì)胞的凋亡,Galectin-3可能是這個(gè)凋亡途徑的下游分子之一。 第三部分激素對(duì)滋養(yǎng)細(xì)胞Galectin-3的調(diào)節(jié)以及胞外Galectin-3對(duì)子宮內(nèi)膜細(xì)胞的凋亡促進(jìn)作用 目的探討激素對(duì)滋養(yǎng)細(xì)胞Galectin-3的表達(dá)及分泌調(diào)節(jié)以及Galectin-3在母胎界面對(duì)子宮內(nèi)膜上皮細(xì)胞的凋亡調(diào)節(jié)作用。 方法體外培養(yǎng)滋養(yǎng)細(xì)胞和子宮內(nèi)膜上皮細(xì)胞：Bewo和RL95-2細(xì)胞株,不同濃度雌激素、孕激素和人促絨毛性腺激素處理Bewo細(xì)胞。Real-time PCR、 Western blot和ELISA的方法檢測(cè)三種激素對(duì)Bewo細(xì)胞表達(dá)和分泌Galectin-3的影響。不同濃度重組蛋白Galectin-3作用于RL95-2細(xì)胞,BrdU法和AnnexinV/PI流式細(xì)胞儀的方法檢測(cè)其對(duì)RL95-2細(xì)胞增殖和凋亡調(diào)節(jié)作用。重組蛋白Galectin-3加入RL95-2細(xì)胞,流式細(xì)胞儀檢測(cè)其對(duì)RL95-2細(xì)胞integrinβ1/β3的表達(dá)影響。共培養(yǎng)Bewo和RL95-2細(xì)胞,加入Galectin-3中和性抗體,進(jìn)一步明確Bewo細(xì)胞分泌的Galectin-3蛋白對(duì)RL95-2細(xì)胞的凋亡促進(jìn)作用。 結(jié)果各個(gè)濃度雌、孕激素和人促絨毛性腺激素均能有效上調(diào)Bewo細(xì)胞Galectin-3mRNA和蛋白表達(dá),并促進(jìn)Bewo細(xì)胞分泌Galectin-3蛋白。Galectin-3重組蛋白促進(jìn)RL95-2細(xì)胞的凋亡,并上調(diào)細(xì)胞表面integrin β1的表達(dá),而對(duì)integrin β3無(wú)影響。共培養(yǎng)Bewo細(xì)胞和RL95-2細(xì)胞發(fā)現(xiàn)RL95-2細(xì)胞的凋亡率明顯上升,而Galectin-3中和性抗體能扭轉(zhuǎn)這種情況。 結(jié)論雌、孕激素和促絨毛膜性腺激素調(diào)控滋養(yǎng)細(xì)胞表達(dá)和分泌Galectin-3。滋養(yǎng)細(xì)胞分泌的Galectin-3促進(jìn)子宮內(nèi)膜上皮細(xì)胞凋亡,而這種促凋亡作用可能是通過(guò)上調(diào)integrin β1表達(dá)達(dá)到的。
[Abstract]:Embryo implantation involves the extremely complex and fine multifactor interaction between the embryo and the mother's womb. In this process, the endometrium only allows the implantation of mature embryos in a short period of time. This period is called the implantation window. The endometrium is receptive at this time. At present, the mechanism of endometrial receptivity is not yet clear. During embryo implantation, embryonic trophoblastic cells exhibit similar invasiveness with tumor cells, adhered to the surface of the endometrium with receptivity, invasion and implantation. The whole process of trophoblast invasion of endometrium is accompanied by the apoptosis of endometrial epithelial cells and decidua of matrix cells.
Galectin-3 (Gal-3) is one of the endometrial receptive molecules that have been screened at the early stage of our group. Our previous study found that it was highly expressed in the endometrium implantation window, and the expression of Galectin-3 in the eutopic endometrium of endometriosis patients decreased, which may be related to the establishment of endometrial receptivity. At present, Galectin-3 The research mainly focuses on the tumor, which plays an important biological role in the growth, adhesion, invasion and apoptosis of the tumor cells. These biological behaviors may also play a great role in the process of embryo implantation. Our previous study showed that Galectin-3 could regulate the adhesion and proliferation of endometrium cells. In view of Ga The diversity of lectin-3 functions, we are interested in other biological behavior of Galectin-3 in the maternal fetal interface. Since Galectin-3 is involved in the invasion of tumor cells by regulating the apoptosis of tumor cells, does Galectin-3 also play the role of apoptosis regulation in the process of embryo implantation?
On the basis of earlier work, this study further clarified the role of Galectin-3 in the formation of receptive endometrium. The role of Galectin-3 in the implantation of endometrium was studied by using the method of local silence of endometrium, and the classical culture model of embryo implantation, Bewo cell line and RL95-2 cell line, was used in detail. The effects of intracellular and extracellular Galectin-3 on the apoptosis of endometrial cells were discussed. The paper is divided into three parts:
Part 1 the effect of Galectin-3 on mouse embryo implantation
Objective to clarify the effect of Galectin-3 on embryo implantation in mice.
Methods the endometrium of early pregnancy mice was collected. The changes of Galectin-3mRNA and protein expression in the endometrium of early pregnant mice were detected by real-time PCR, immunohistochemistry and Western blot. The mouse uterus angle was injected with immunofluorescent oligo in vivo. The transfection of the living body was observed under the fluorescence microscope and the transfection was verified by Western blot method. The pregnant mice were sacrificed on the 9 day of pregnancy. The effect of Galectin-3siRNA transfection on embryo implantation in mice was studied.
Results the expression of Galectin-3mRNA and protein in the endometrium of early pregnant mice was higher than that of the unpregnant mice. The levels of Galectin-3mRNA and protein reached the peak at 4 days of pregnancy and 2 days of pregnancy respectively. The results of immunohistochemistry showed that Galectin-3 protein was at the peak of the upper cavities of the 6~8 day of pregnancy and reached the peak at the 2~4 day of pregnancy. Immunofluorescence showed Si in vivo transfected. RNA was entered into the cavity epithelial cells. The 4 day gestation protein test showed that the Galectin-3 protein was effectively downregulated by Galectin-3siRNA. The mice were killed at 9 days of pregnancy. Compared with the control group (Negaive siRNA), the number of embryos in the corner of the uterus of the Galectin-3siRNA injection was obviously down.
Conclusion Galectin-3 has periodic expression of endometrium in early pregnant mice and high expression of dry implantation stage, indicating that Galectin-3 may participate in the formation of endometrium receptivity. The number of Galectin-3 (?) reduces the number of embryos of the implantation, indicating that Galectin-3 affects the implantation of the embryo, which may be involved in the formation of the receptive endometrium and its expression. Down regulation may lead to endometrial receptivity defects, resulting in implantation failure.
The second part is the anti apoptotic effect of intracellular Galectin-3 in endometrial epithelial cells.
Objective to investigate the anti apoptotic effect of intracellular Galectin-3 in endometrial epithelial cells.
Methods RL95-2 cells were treated with cytomycin, and the effect on cell viability was detected by MTT. The effect of acridine orange staining immunofluorescence and AnnexinV/PI flow cytometry on the apoptosis of RL95-2 cells was determined. Real-time PCR and Western blot were used to detect the change of Galectin-3 expression and.Galectin-3siRNA transfection RL9. 5-2 cells silenced the expression of Galectin-3, and after Galectin-3 silencing, the apoptosis of RL95-2 cells was promoted by the cell mycomycin,.Real-time PCR and Western blot were used to detect the different concentrations of female and progestin to the Galectin-3 expression of RL95-2 cells. After the silence of Galectin-3 expression, estrogen, progesterone and astrocyin were used to detect female, and whether progesterone was used. Over regulation of Galectin-3 expression can regulate the apoptosis of RL95-2 cells.
Results the results of MTT showed that after the treatment of cytosine, the activity of RL95-2 cells decreased, the rate of apoptosis increased, and the increase of Galectin-3mRNA and protein reactive.Galectin-3siRNA could effectively reduce the expression of Galectin-3 in RL95-2 cells. After the downregulation of Galectin-3, the apoptosis of RL95-2 was enhanced by the downregulation of Galectin-3. Cell Galectin-3mRNA and protein expression. After adding estrogen and progesterone, the apoptosis rate of Galectin-3 silenced RL95-2 cells was higher than that of Negative siRNA.
Conclusion Galectin-3 can inhibit apoptosis in endometrium epithelial cells. Estrogen and progestin can regulate the expression of Galectin-3 in endometrial epithelial cells. Estrogen and progesterone regulate the apoptosis of endometrial epithelial cells. Galectin-3 may be one of the downstream molecules of this apoptotic pathway.
The third part is the regulation of hormones on trophoblast Galectin-3 and the effect of extracellular Galectin-3 on the apoptosis of endometrial cells.
Objective to investigate the regulation of hormones on the expression and secretion of Galectin-3 in trophoblastic cells and the regulation of Galectin-3 on the apoptosis of endometrial epithelial cells at the maternal fetal interface.
Methods in vitro culture of trophoblast and endometrium epithelial cells: Bewo and RL95-2 cell lines, different concentrations of estrogen, progestin and human chorionic gonadotropin treated Bewo cells.Real-time PCR, Western blot and ELISA to detect the effect of three hormones on the expression and secretion of Galectin-3 in Bewo cells. The effect of n-3 on the proliferation and apoptosis of RL95-2 cells was detected by RL95-2 cells, BrdU and AnnexinV/PI flow cytometry. The recombinant protein Galectin-3 added to RL95-2 cells, and the expression of integrin beta 1/ beta 3 in RL95-2 cells was detected by flow cytometry. One step was to clarify the effect of Galectin-3 protein secreted by Bewo cells on the apoptosis of RL95-2 cells.
Results all the concentrations of female, progestin and human chorionic gonadotropin can effectively increase the expression of Galectin-3mRNA and protein in Bewo cells, promote the secretion of Galectin-3 protein.Galectin-3 recombinant protein in Bewo cells and promote the apoptosis of RL95-2 cells, and increase the expression of integrin beta 1 on the cell surface, but have no effect on integrin beta 3. Co culture Bewo cells. And RL95-2 cells found that the apoptosis rate of RL95-2 cells increased significantly, while Galectin-3 neutralizing antibody could reverse this situation.
Conclusion estrogen, progesterone and chorionic gonadotropin regulate the expression and secretion of Galectin-3 secreted by Galectin-3. trophoblastic cells to promote the apoptosis of endometrium epithelial cells, which may be achieved by up regulation of integrin beta 1.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R321

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 李婧;談?dòng)?;中西醫(yī)對(duì)子宮內(nèi)膜容受性研究進(jìn)展[J];山東中醫(yī)藥大學(xué)學(xué)報(bào);2014年03期

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本文編號(hào)：2018720