【作者】 刘帅；

【导师】 燕秋；

【作者基本信息】 大连医科大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 一、目的和意义胚胎植入是一个复杂的发育过程,是子宫内膜与胚胎在时空上同步协调准备的过程。一方面,胚胎发育成具有侵入能力的胚泡阶段;另一方面,子宫内膜发育到允许胚胎侵入的接受态,只有二者同时发育到此阶段,植入才可发生。寡糖作为体内重要的信息分子,参与了细胞-细胞间的分子识别,也介导了细胞内的信号传导。在子宫内膜由非接受态向接受态变化过程中,许多细胞表面寡糖呈阶段特异性变化。Lewis寡糖属于细胞表面寡糖,在胚胎着床过程中发挥重要生物学功能。研究表明:着床窗口期的子宫内膜上皮sLeX表达增加,胚胎上表达L-选凝素(L-selectin),二者相互识别结合,参与胚胎着床。着床窗口期,LeY在人和鼠的子宫内膜上皮高表达,鼠的子宫内膜上皮细胞经LeY抗体预处理后,胚胎的着床率下降;同时,子宫内膜基质金属蛋白酶的活性、表达和分泌被明显抑制。岩藻糖化Lewis寡糖由岩藻糖基转移酶(Fucosylrans-ferase,FUTs)催化合成。目前已发现11种岩藻糖基转移酶( FUT1-11)。根据它们所催化生成的糖苷键类型的不同,可分为α1,2、α1,3和α1,6三类。其中,FUT7属于α1,3岩藻糖基转移酶,是sLeX [NeuAcα2→3Galβ1→4 (Fuc1→3) GlcNAcβ1→R]合成的关键酶。FUT4也属于α1,3岩藻糖基转移酶,催化L-fucose从GDP-fucose转移到H type 2 [Fucα→1Galβ1→4GlcNAcβ1→R]抗原,合成LeY[Fuc1→2Galβ1→4 (Fuc1→3) GlcNAcβ1→R],为LeY寡糖抗原合成关键酶。对FUT7和FUT4表达的调控可以调节细胞表面sLeX及LeY寡糖抗原的表达水平。本文通过对FUT4和FUT7的调控,进而对细胞表面sLeX、LeY寡糖抗原合成的调节,探讨细胞表面寡糖抗原sLeX、LeY在胚胎着床过程中的功能,进一步探讨其在生殖过程中的作用机制,为阐明寡糖在生殖中的作用提供理论依据。二、方法1.利用脂质体转染法将重组质粒转染至人子宫内膜细胞系(RL95-2、HEC-1A),人胚胎细胞系(JAR)中。2.采用RT-PCR方法检测FUT4,FUT7基因表达。3.采用Western blot、间接免疫荧光方法和流式细胞技术检测FUT4、FUT7、sLeX和LeY的表达。4.采用Annexin V/PI流式细胞技术检测细胞凋亡。5.利用黏附实验观察和分析胚胎细胞与子宫内膜细胞黏附情况。三、结果(一)sLeX/L-selectin介导的双向黏附在胚胎着床中的作用1.通过间接免疫荧光方法检测到JAR/RL95-2细胞同时表达sLeX/L-选择素。2.抗体封闭实验表明,sLeX和L-选凝素抗体分别封闭JAR细胞和RL95-2细胞,可以抑制JAR细胞与RL95-2细胞的黏附率。表明:JAR细胞和RL95-2细胞表面sLeX、L-selectin都参与胚胎着床。3.将FUT7过表达质粒分别转染至JAR细胞与RL95-2细胞,可增加细胞表面sLeX寡糖抗原的合成,促进JAR细胞与RL95-2细胞的黏附率;当JAR细胞和RL95-2细胞同时转染FUT7过表达质粒时,黏附率升高最为明显。表明:JAR细胞和RL95-2细胞表面sLeX介导胚胎着床。4.肝素为L-选凝素的抑制剂,当用肝素分别封闭JAR细胞与RL95-2细胞后,可抑制JAR细胞与RL95-2细胞的黏附率,当JAR细胞与RL95-2细胞同时经肝素封闭后,黏附率抑制作用最为明显。表明:JAR细胞和RL95-2细胞表面L-selectin介导胚胎着床。5.RL95-2细胞的凋亡较为转染组比较有所增加,转染空载体组与对照组无显著差异。(二)LeY寡糖抗原与子宫内膜接受潜能的关系1.JAR细胞与RL95-2细胞的黏附率高于其与HEC-1A细胞的黏附率。2.应用Western blot和间接免疫荧光方法检测RL95-2细胞LeY表达高于HEC-1A细胞。3.应用RT-PCT、Western blot和间接免疫荧光方法检测RL95-2细胞FUT4表达高于HEC-1A细胞。4.FUT4-siRNA抑制RL95-2细胞中FUT 4和LeY表达,可抑制胚胎细胞与子宫内膜细胞的黏附率。5.FUT4-过表达质粒促进HEC-1A细胞中FUT4和LeY表达,促进胚胎细胞与子宫内膜细胞的黏附率。6.FUT4-siRNA可以抑制RL95-2细胞中EGFR/MAPK信号通路的激活,FUT4-过表达可以激活HEC-1A细胞中EGFR/MAPK信号通路。四、结论(一)sLeX/L-selectin介导的双向黏附在胚胎着床中的作用1.JAR细胞表面表达L-选凝素,RL95-2细胞表面表达sLeX寡糖抗原,L-选凝素与sLeX寡糖抗原相识别,介导胚胎着床。2.JAR细胞表面表达的sLeX寡糖抗原与RL95-2细胞表面表达的L-选凝素相识别,介导胚胎着床。提示在胚胎细胞与子宫内膜细胞表面的sLeX和L-selectin介导的双向黏附系统在胚胎着床过程中发挥作用。3.sLeX介导的细胞黏附引起RL95-2细胞凋亡。说明sLeX和L-selectin介导的细胞黏附可以进一步引起子宫内膜细胞的凋亡,促进胚胎植入。(二)LeY寡糖抗原与子宫内膜接受潜能的关系1.RL95-2细胞中FUT4和LeY寡糖抗原表达高于HEC-1A细胞。提示:FUT4和LeY在高接受态的子宫内膜中表达高于其在低接受态子宫内膜。FUT4和LeY寡糖抗原可以作为子宫内膜接受态的标志。2.上调或抑制细胞中FUT4表达,可以调节细胞表面LeY寡糖抗原的合成,进而影响胚胎与子宫内膜的黏附率。说明LeY寡糖抗原在胚胎植入的过程中发挥作用。3.FUT4通过调节细胞表面LeY寡糖抗原的合成,进而调控细胞中EGFR/MAPK信号通路的激活,并参与胚胎着床。更多还原

【Abstract】 I. Objective and purposeImplantation is a complex biological process. It requires the synchrony between the appropriately developed embryo and the receptive uterine endometrium. The apptoptiate developed of uterine and embryo is the basis for successful implantation. Oligosaccharides, as constituents of cell surface glycoconjugates, have been shown to have many biological activities during implantation process, which participate in the recongization of cell-cell and signal transduction. A variety of oligosaccharide structures have been identified at embryonic and uterine cell surfaces and change dynamically during progression of the implantation. Some reports show that strong L-selectin staining was present in the human embryo, whereas the expression of selectin oligosaccharide-based ligands was up-regulated during the implantation window, the binding of L-selectin and sialy Lewis X (sLeX) mediated the adhesion of embryo and uterine. Lewis Y (LeY) were highly expressed on both murine embryo and uterine endometrium during implantation period, and the functional blockage of LeY with specific anti-LeY antibody in either the embryo side or uterine endometrium side could reduce embryo implantation both in vitro and in vivo.Lewis oligosaccharide belongs to A, B, H Lewis blood group family. Fuco- syltransferases (FUTs) catalyze the synthesis of Lewis oligosaccharides. Until now, 11 fucosyltransferases have been identifided, which composed ofα1,2-,α1,3-,α1,6- fucosyltransferases. FUT7 belongs toα1,3 fucosyltransferases, which is the key enzyme of sLeX [NeuAcα2→3Galβ1→4 (Fuc1→3) GlcNAcβ1→R] synthesis. FUT4 also belongs to 1,3-fucosyltransferases. Based on H type 2 [Fucα→1Galβ1→4GlcNAcβ1→R] epitope, LeY is synthesized by the addition of a Fuc residue to N-acetyl- glucosamine (N-GlcNAc) with theα1, 3-linkage, FUT4 is the key enzyme for the synthesis of LeY. The regulation of FUT7 and FUT4 may control the expression of the sLeX and LeY.In this study, we utilized the FUT7 and FUT4 overexpression plasmid or FUT4- siRNA plasmid to interfere the expression of sLeX and LeY oligosaccharide on cell surface, and evaluate the roles of oligosaccharides on cell surface during implantation. The study will promte the further elucidation of the mechanism of oligosaccharides in embryo implantaion.II. Methods1.The recombinant vector was transfected into cells.2.The expression of FUT4 and FUT7 were detected by the methods of RT-PCR.3.The expression of FUT4 and FUT7, sLeX and LeY were detected by the methods of Western blot, indirect immunofluorescence staining and flow cytometry assay.4.Effect of FUT7 on cell apoptosis was analyzed by Annexin V/PI flow cytome- tric assay.5.Detect the percent adhesion of JAR cell adhesion to RL95-2 or HEC-1A cell monolayer.III. Results(I)Dual adhesion mediated by sLeX/L-selectin in vitro implantation model.1.The expression of sLeX and L-selectin in JAR and RL95-2 cells were detected by indirect immunofluorescence staining. The results showed that the staining of sLeX was present in JAR cellsand RL95-2 cells. L-selectin staining was also observed in JAR cells and RL95-2 cells. It suggests that sLeX and L-selectin were co-existed in either JAR cells or RL95-2 cells.2.To check if the expression of sLeX and L-selectin on both JAR cells and RL95- 2 cells was functionally involved in sLeX/L-selectin adhesion system, the effect of anti- body blocking was undergone using adhesion inhibition assay. The adhesion of JAR cells to RL95-2 cells were inhibited by the specific antibodies blockage. The percent adhesion was significantly decreased when JAR cells was pre-incubated with anti-sLeX antibody or anti-L-selectin antibody. The preincubation of anti-sLeX antibody or anti- L-selectin antibody in RL95-2 cells also caused the similar inhibitory effect.3.FUT7 expression plasmid was transfected into JAR or RL95-2 cells. The results showed that the gene and protein level of FUT7 were elevated by RT-PCR and flow cytometry assay in both JAR and RL95-2 cells transfected with FUT7 expression plasmid compared with that of the control and mock vector transfection. Moreover, the synthesis of sLeX was also increased in JAR or RL95-2 cells after FUT7 expression plasmid transfection by flow cytometry assay. The percent adhesion was analyzed. When FUT7 expression plasmid was co-transfected into both of the cells, the percent adhesion was higher.4.Heparin, the inhibitor of L-selectin, was utilized. Statistical analysis showed that when JAR cells, RL95-2 cells, or both of the cells were incubated with heparin, the percent adhesions were also significantly decreased compared with that of the control. The inhibition was the most obvious when both cells were treated with heparin, comp- ared with the separate addition of heparin to either cell culture.5.The apoptosis was determined by flow cytometry assay after RL95-2 cells were transfected with FUT7 expression plasmid. The data indicated that after co-cultured with JAR cells, the extent of apoptosis in transfected RL95-2 cells was increased, compared with that of the control and mock vector transfected RL95-2 cells.(II)The relationship between potension of uterine receptivity and LeY oligo- saccharide.1.The percent adhesion of JAR to RL95-2 cell monolayer was higher than HEC- 1A cell monolayer; and co-culture the JAR cells with RL95-2 cell monolayer or HEC- 1A cell monolayer, respectively, the extension of JAR cells on RL95-2 cell monolayer was more obviously than that on HEC-1A cell monolayer.2.For LeY assay, the expression of LeY on RL95-2 cells was also higher than on HEC-1A cells by Western blot and indirect immunofluorescence staining.3.For FUT4 detect assay, RT-PCR, Western blot and indirect immunofluores- cence staining were utilized. Results revealed that the expression of FUT4 in RL95-2 cells was higher than in HEC-1A cells on gene level and protein level4.To detect the effect of FUT4-siRNA on the RL95-2 cells, different assays were employed. RT-PCR found that the expression of FUT4 gene was dramatically decreased by FUT4-siRNA transfection in comparison to untransfected controls. Western blot, indirect immunofluorescence staining and flow cytometry revealed all that the protein expression level of FUT4 was much lower in FUT4-siRNA transfected RL95-2 cells in comparison to untransfected controls. The percent adhesion of JAR cell to RL95-2 cell monolayer was been inhibited.5.To explore the effect of FUT4 on LeY synthesis, FUT4 expression plasmid was transfected into HEC-1A cells. The results showed that the gene and protein level of FUT4 were elevated in FUT4 expression plasmid transfected cell compare to untrans- fected control by RT-PCR, Western blot, indirect immunofluorescence staining and flow cytometry assay. The synthesis level of LeY is also augment parallel with the increased of FUT4. The percent adhesion of JAR cell to HEC-1A cell monolayer was been evaluated.6.To study the effect of FUT4 expression on EGFR/MAPK signaling pathway, tyrosine phosphorylation of EGFR and ERK1/2 was assessed by Western blotting. After stimulating with EGF (100 ng/ml) for 10 min, sample was harvested. In RL95-2 cells, FUT4 knocking down cells expressed less pEGFR, pTYr and pERK1/2. Meanwhile, overexpression of FUT4 in HEC-1A cells could increase the synthesis of pEGFR, pTYr and pERK1/2.IV. Conclusions(I) Dual adhesion mediated by sLeX/L-selectin in vitro implantation model.1.The binding of L-selectin on JAR cells and sLeX on RL95-2 cells mediates the embryo implantation.2.The binding of sLeX on JAR cells and L-selectin on RL95-2 cells mediates the embryo implantation. Dual adhesion mediated by sLeX/L-selectin participate the embryo implantation.3.Adhesion mediated by sLeX increases apoptosis of RL95-2 cells.(II) The relationship between potension of uterine receptivity and LeY oligo- saccharide.1.The expression FUT4 and LeY on RL95-2 cells is higher than which on HEC- 1A cells. The results indicate that FUT4 and LeY may been seemed as endometrial receptivity markers.2.Regulation of FUT4 can control the expression of LeY oligosaccharide, further increase the percent adhesion of embryo to uterine.3.FUT4 can regulate the LeY epression which further mediates the EGFR/MAPK signaling pathway and participates embryo implantation.更多还原