N-乙酰氨基葡萄糖转移酶Ⅲ通过对α5β1整合素N-糖基化修饰调控滋养层细胞的侵袭

发布时间：2018-03-10 19:19

本文选题：N-乙酰氨基葡萄糖转移酶Ⅲ　切入点：N-糖基化　出处：《重庆医科大学》2015年博士论文　论文类型：学位论文

【摘要】：背景：妊娠早期,滋养层细胞黏附并侵入母体子宫内膜是胚胎着床和胎盘形成的关键环节。N-乙酰氨基葡萄糖转移酶Ⅲ(N-acetylglucosaminyltransferase Ⅲ, GnT-Ⅲ)是N-糖基化修饰的一个关键酶,GnT-Ⅲ及其催化产物平分型糖支链结构(bisecting N-acetylglucosamine, bisecting GlcNAc)参与调控肿瘤的转移、侵袭,但GnT-Ⅲ及其催化产物平分型糖支链结构在人妊娠各时期的胎盘发育、滋养层细胞侵袭过程中的功能及作用机制尚不清楚。此外,已有文献报道a5β1整合素上糖基化修饰的改变可引起肿瘤细胞及滋养层细胞侵袭行为的改变。目的：本研究旨在通过比较人正常妊娠不同时期胎盘组织中GnT-Ⅲ及其催化产物平分型GlcNAc结构的表达和分布差异,以及用小干扰RNA法(small interfering RNA, SiRNA)在人绒毛膜癌细胞株JEG-3中建立MGAT3基因(编码GnT-Ⅲ)沉默模型,来探索GnT-Ⅲ和平分型GlcNAc结构对功能蛋白α5β1的N-糖基化修饰和滋养层细胞侵袭的调控,为临床不孕症和自然流产等疾病诊治探寻新的分子靶点。方法：1.我们分别采用免疫组织化学、实时荧光定量PCR (qRT-PCR)、蛋白质印迹、免疫荧光法检测GnT-Ⅲ,用生物素化的蚕豆红细胞凝集素(biotinylated phaseolus vulgaris erythroagglutinin, PHA-E)印迹、PHA-E凝集素组织化学及PHA-E凝集素荧光法检测GnT-Ⅲ的催化产物平分型GlcNAc结构在人正常妊娠各阶段的胎盘组织和不同的人滋养层细胞株中的表达和分布；2.用SiRNA法在人绒毛膜癌细胞株JEG-3中建立MGAT3基因沉默模型,运用CCK-8细胞增殖实验、transwell细胞迁移与侵袭实验、细胞外基质黏附实验技术分析沉默MGAT3基因对细胞增殖、迁移、侵袭及与细胞外基质黏附等生物学效应的影响,用qRT-PCR、蛋白质印迹、免疫共沉淀技术分析沉默MGAT3基因对α5β1整合素的表达和糖基化修饰的影响,并用蛋白质印迹法初步分析相关信号通路的变化。结果：1.在人正常妊娠绒毛组织中,GnT-Ⅲ主要表达于细胞滋养层细胞(cytotrophoblasts, CTBs)和合体滋养层细胞(syncytiotrophoblasts, STBs)的细胞质,且在妊娠前三个月的表达强于妊娠中三个月(P0.05)和妊娠后三个月(P0.05)；GnT-Ⅲ的催化产物平分型GlcNAc结构在妊娠前三个月绒毛中的含量显著高于妊娠后三个月(P0.05)。2. GnT-Ⅲ在绒毛外滋养细胞(extravillous cytotrophoblasts, EVTs)中高表达,且在有EVTs侵入的蜕膜组织也呈高表达,而在无EVTs侵入的蜕膜组织中表达很弱甚至无表达,并且GnT-Ⅲ在有EVTs细胞侵入的蜕膜组织中妊娠前三个月的表达高于妊娠后三个月。3. PHA-E凝集素组织化学分析结果显示平分型GlcNAc结构主要分布在滋养层细胞的表面,且在妊娠前三个月绒毛中大量分布,在足月胎盘绒毛中分布较少,且妊娠前三个月高于妊娠后三个月(P0.05)。4.用SiRNA法在人绒毛膜癌细胞株JEG-3中沉默MGAT3基因(编码GnT-Ⅲ), JEG-3细胞在MGAT3基因沉默后其增殖能力未受到明显影响,而细胞迁移、侵袭能力以及与细胞外基质黏附能力明显下降;MGAT3基因沉默后α5β1整合素的mRNA转录水平和蛋白表达水平没有明显改变,但α5β1整合素上的平分型糖支链含量明显减少。5. Western blot分析结果显示沉默MGAT3基因后ERK和FAK的磷酸化水平下调。结论：该研究结果提示,GnT-Ⅲ在调控滋养层细胞正常迁移、侵润过程中扮演重要角色,α5β1整合素的糖基化修饰可能参与了这一调节过程,深入的机制仍需进一步研究和探索。
[Abstract]:Background: early pregnancy, adhesion of trophoblast cells invade the maternal endometrium and embryo implantation and placental formation is the key link of.N- N-acetylglucosaminyltransferase III (N-acetylglucosaminyltransferase III, GnT- III) is a key enzyme of N- glycosylation, GnT- III and its catalytic product of split type sugar chain structure (bisecting N-acetylglucosamine, bisecting GlcNAc) involved in the regulation of tumor invasion and metastasis, but GnT- III and its catalysate split type sugar branched structure developed in human placenta at different period of pregnancy, trophoblast invasion function and mechanism of action in the process is not clear. In addition, it has been reported that A5 beta 1 integrin on glycosylation changes can cause aggressive behavior tumor cells and trophoblast cells. Objective: This study aims to compare the human placental tissue in different periods of GnT- III and its catalytic normal pregnancy The expression and distribution of difference products bisecting GlcNAc structure, and the use of small interfering RNA (small interfering RNA, SiRNA) to establish MGAT3 gene in human choriocarcinoma cell line JEG-3 (encoding GnT- III) silence model, to explore the regulation of GnT- type III peace GlcNAc structure on the function of protein alpha 5 beta 1 invasion N- sugar group modification and trophoblast cells, explore new molecular targets for clinical diagnosis and treatment of infertility and spontaneous abortion and other diseases. Methods: 1. we used immunohistochemistry, real-time fluorescence quantitative PCR (qRT-PCR), Western blotting, immunofluorescence detection of GnT- III, with bean red blood cell agglutinin biotinylated (biotinylated Phaseolus vulgaris erythroagglutinin, PHA-E blot, GnT- PHA-E III) detection of lectin histochemistry and PHA-E lectin fluorescence catalysate bisecting GlcNAc structure in human placenta tissue of normal pregnancy stages And human trophoblast cell lines with different expression and distribution in 2.; with the method of SiRNA in human choriocarcinoma cell line JEG-3 to establish model of MGAT3 gene silencing, the proliferation of CCK-8 cells, Transwell cell migration and invasion assay, analysis of silencing MGAT3 gene transfer on cell proliferation, the experimental technology of extracellular matrix adhesion, invasion and cell the biological matrix adhesion effect by qRT-PCR, Western blotting, immunoprecipitation expression and glycosylation analysis of MGAT3 gene silencing on the alpha 5 beta 1 integrin modifications, and changes in signal pathways related to a preliminary analysis by Western blotting. Results: 1. in normal pregnancy tissue, GnT- III mainly expressed in trophoblast cells (cytotrophoblasts, CTBs) and trophoblast cells (syncytiotrophoblasts, STBs) in the cytoplasm, and strong expression in the first three months of pregnancy Three months of pregnancy (P0.05) and after three months of pregnancy (P0.05); catalysate bisecting GlcNAc GnT- III in the first three months of pregnancy villi were significantly higher than those after three months of pregnancy (P0.05).2. GnT- III in extravillous trophoblast cells (extravillous, cytotrophoblasts, EVTs) in high expression and also showed high expression and EVTs in the invasion of decidual tissue, while no EVTs invade the decidua expressed little or no expression of GnT- and EVTs in cell invasion in decidua of pregnancy expression three months before than after three months of pregnancy.3. PHA-E lectin histochemical analysis results showed that the surface split type GlcNAc structure is mainly distributed in the trophoblast cells, and a large number of distribution in the first three months of pregnancy villi, less distribution in term placenta in pregnancy and three months before more than three months after pregnancy (P0.05).4. by SiRNA method in human chorionic villi Membrane cancer cell line JEG-3 in silencing the MGAT3 gene (encoding GnT- III), in JEG-3 cells after MGAT3 gene silencing and its proliferation ability was not affected, and the cell migration, invasion and adhesion to extracellular matrix decreased significantly; no significant change in the expression level of MGAT3 gene silencing alpha 5 beta 1 integrin mRNA transcription level but the split type sugar and protein, branched chain alpha 5 beta 1 integrin on the content of.5. significantly decreased Western blot analysis results showed that the down-regulation of ERK and FAK phosphorylation after MGAT3 gene silencing. Conclusion: the results of this study suggest that GnT- of the normal migration in the regulation of trophoblast cells, play an important role in the process of invasion, glycosylation modification of alpha 5 beta 1 integrin may participate in the regulating process, in-depth mechanism still needs further study and exploration.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R714

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