Galectin-3对子宫内膜容受性建立过程中细胞凋亡的影响

发布时间：2018-06-14 19:35

本文选题：Galectin-3 + 滋养细胞　；参考：《复旦大学》2012年博士论文

【摘要】：胚胎着床涉及胚胎与母体子宫间极其复杂而精细的多因素相互作用。这个过程中,子宫内膜仅在一个较短的时期允许成熟胚胎植入,这个时期称为着床窗,这个时期子宫内膜具有容受性,目前,子宫内膜容受性形成的机理尚不清楚楚。在胚胎植入过程中,胚胎滋养细胞表现出与肿瘤细胞相似的侵袭能力,在具有容受性的子宫内膜表面进行粘附、入侵和植入。滋养细胞侵入子宫内膜的全程伴随着子宫内膜上皮细胞的凋亡以及基质细胞的蜕膜化。 Galectin-3(Gal-3)是本课题组前期筛选到的子宫内膜容受性相关分子之一。我们前期的研究发现其在子宫内膜着床窗口期高表达,且在子宫内膜异位症患者在位内膜的Galectin-3表达降低,可能与子宫内膜容受性建立不全有关。目前对Galectin-3的研究主要集中在肿瘤方面,其在肿瘤细胞的生长、粘附、侵袭、凋亡等方面发挥着重要的生物学作用,这些生物学行为可能在胚胎植入过程中同样起着巨大的作用。我们前期研究显示,Galectin-3能够调节子宫内膜细胞的粘附和增殖。鉴于Galectin-3功能的多样性,我们对Galectin-3在母胎界面的其它生物学行为产生了兴趣。既然Galectin-3通过调节肿瘤细胞的凋亡参与肿瘤细胞的侵袭,Galectin-3是否也在胚胎植入的过程中发挥了凋亡调节的作用呢? 本研究在前期工作的基础上,进一步明确Galectin-3在容受性子宫内膜形成中的作用。运用子宫内膜局部沉默Galectin-3的方法研究其在着床中的作用,并用经典的体外培养胚胎着床模型——Bewo细胞株和RL95-2细胞株间接共培养模型,详细地探讨了胞内及胞外Galectin-3对子宫内膜细胞的凋亡调节作用。论文分为以下三个部分：第一部分Galectin-3对小鼠胚胎着床的影响目的明确Galectin-3对小鼠胚胎着床的影响。方法收集早孕小鼠子宫内膜,采用real-time PCR,免疫组织化学,Western blot的方法检测早孕小鼠子宫内膜中Galectin-3mRNA和蛋白表达变化情况。带免疫荧光oligo活体注射小鼠子宫角,荧光显微镜下观察活体转染情况并用Western blot方法验证转染效果。孕鼠于孕9天被处死,研究转染Galectin-3siRNA对小鼠胚胎着床的影响。结果Galectin-3mRNA和蛋白在早孕小鼠子宫内膜的表达高于未孕小鼠,Galectin-3mRNA和蛋白水平分别在孕4天和孕2天达到高峰。免疫组化结果显示：Galectin-3蛋白于孕6～8天腔上皮达高峰,于孕2～4天腺上皮达高峰。免疫荧光显示：活体转染的siRNA进入腔上皮细胞内。孕4天蛋白检测表明Galectin-3蛋白被Galectin-3siRNA有效下调。孕9天处死小鼠,与对照组(Negaive siRNA)相比,Galectin-3siRNA注射侧子宫角着床胚胎数明显下调。结论Galectin-3存早孕小鼠子宫内膜周期性表达,并干着床期高表达,说明Galectin-3可能参与子宫内膜容受性的形成。局部下调Galectin-3(?)减少着床的胚胎数,说明Galectin-3影响胚胎着床,可能是通过参与容受性子宫内膜的形成起作用,其表达下调可能导致子宫内膜容受性缺陷从而导致着床失败。第二部分子宫内膜上皮细胞胞内Galectin-3的抗凋亡作用目的探讨子宫内膜上皮细胞胞内Galectin-3的抗凋亡作用。方法用星胞霉素处理RL95-2细胞,MTT法检测其对细胞活力的影响,吖啶橙染色免疫荧光检测和AnnexinV/PI流式细胞仪检测的方法确定其对RL95-2细胞凋亡的影响,运用real-time PCR和Western blot的方法检测Galectin-3表达变化情况。Galectin-3siRNA转染RL95-2细胞沉默Galectin-3表达,分析Galectin-3沉默后,星胞霉素对RL95-2细胞的凋亡促进作用。Real-time PCR和Western blot检测不同浓度雌、孕激素对RL95-2细胞Galectin-3表达调节情况。沉默Galectin-3表达后加入雌、孕激素和星胞霉素检测雌、孕激素是否通过调控Galectin-3表达来调节RL95-2细胞的凋亡。结果MTT结果显示：星胞霉素处理后,RL95-2细胞活力降低,凋亡率上升,Galectin-3mRNA和蛋白反应性上升。Galectin-3siRNA能有效下调RL95-2细胞Galectin-3表达。下调Galectin-3表达后,星胞霉素对RL95-2的凋亡促进作用加强。雌、孕激素能有效上调RL95-2细胞Galectin-3mRNA和蛋白表达。加入雌、孕激素后, Galectin-3被沉默的RL95-2细胞凋亡率高于加入Negative siRNA的对照组。结论子宫内膜上皮细胞胞内Galectin-3具有抗凋亡作用。雌、孕激素能调控子宫内膜上皮细胞中Galectin-3表达,雌、孕激素调控子宫内膜上皮细胞的凋亡,Galectin-3可能是这个凋亡途径的下游分子之一。第三部分激素对滋养细胞Galectin-3的调节以及胞外Galectin-3对子宫内膜细胞的凋亡促进作用目的探讨激素对滋养细胞Galectin-3的表达及分泌调节以及Galectin-3在母胎界面对子宫内膜上皮细胞的凋亡调节作用。方法体外培养滋养细胞和子宫内膜上皮细胞：Bewo和RL95-2细胞株,不同浓度雌激素、孕激素和人促绒毛性腺激素处理Bewo细胞。Real-time PCR、 Western blot和ELISA的方法检测三种激素对Bewo细胞表达和分泌Galectin-3的影响。不同浓度重组蛋白Galectin-3作用于RL95-2细胞,BrdU法和AnnexinV/PI流式细胞仪的方法检测其对RL95-2细胞增殖和凋亡调节作用。重组蛋白Galectin-3加入RL95-2细胞,流式细胞仪检测其对RL95-2细胞integrinβ1/β3的表达影响。共培养Bewo和RL95-2细胞,加入Galectin-3中和性抗体,进一步明确Bewo细胞分泌的Galectin-3蛋白对RL95-2细胞的凋亡促进作用。结果各个浓度雌、孕激素和人促绒毛性腺激素均能有效上调Bewo细胞Galectin-3mRNA和蛋白表达,并促进Bewo细胞分泌Galectin-3蛋白。Galectin-3重组蛋白促进RL95-2细胞的凋亡,并上调细胞表面integrin β1的表达,而对integrin β3无影响。共培养Bewo细胞和RL95-2细胞发现RL95-2细胞的凋亡率明显上升,而Galectin-3中和性抗体能扭转这种情况。结论雌、孕激素和促绒毛膜性腺激素调控滋养细胞表达和分泌Galectin-3。滋养细胞分泌的Galectin-3促进子宫内膜上皮细胞凋亡,而这种促凋亡作用可能是通过上调integrin β1表达达到的。
[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.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R321

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