卵巢激素对小鼠围着床期子宫内膜Le^y寡糖表达的调控

研究表明Le^y寡糖介导了胚胎与子宫内膜之间的识别与粘附,在胚胎植入中起重要作用。其α1,2、α1,3岩藻糖基的合成分别与α1,2岩藻糖基转移酶（FUT1）、α1,3岩藻糖基转移酶（FUT4）的催化作用密切相关,应用Western印迹、免疫组化和半定量RT-PCR方法,观察小鼠妊娠早期、去卵巢后雌孕激素处理的子宫内膜Le^y寡糖抗原以及其合成相关的FUT1、FUT4基因的表达,分析卵巢激素对Le^y寡糖表达的调控,结果显示：妊娠早期,FUT1、FUT4基因的转录水平随孕激素水平上程式而呈下降的趋势,这与Le^y寡糖抗原表达一致。进一步观察发现,去卵巢后经孕激素处理,FUT1、FUT4基因及Le^y寡糖抗原表达均较对照组降低,雌激素处理组表达则明显升高;雌孕激素联合作用介于雌激素组和孕激素组之间,结果表明,孕激素能下调FUT1、FUT4基因的表达。雌激素对其有上调作用,两种激素之间表现为相互拮抗,提示雌孕激素可能通过FUT1、FUT4基因转录水平调控Le^y寡糖抗原在小鼠子宫内膜上皮的表达。     

Le^Y寡糖单克隆抗体对小鼠胚泡基质金属蛋白酶表达和分泌的影响

以LeY 寡糖特异性单克隆抗体AH6为工具中和胚泡表面LeY 寡糖后 ,通过RT PCR、明胶酶谱法、免疫印迹法等方法 ,在体外研究了着床前小鼠胚泡表面LeY 寡糖抗原与其基质金属蛋白酶 (MMP)、金属蛋白酶组织抑制因子 (TIMP1)的表达和分泌之间的关系。结果显示 :胚泡表面LeY 寡糖抗原被中和后仅 1.5h ,胚泡MMP2和MMP9基因转录表达明显下降、而TIMP1基因转录表达则略有升高 ;随后抗体中和引起胚泡MMP2、MMP9的分泌减少 ,而TIMP1的分泌则未见明显变化。结果表明胚泡表面的LeY 寡糖抗原对着床前胚泡的MMP的合成和分泌具有调节作用 ,而且这种作用可能主要是通过调节相应的MMP2和MMP9基因的表达而引起的     

FUT4过表达促进胚胎细胞与子宫内膜细胞的黏附

胚胎与子宫内膜上皮细胞之间的黏附是胚胎成功植入的关键. 岩藻糖基转移酶Ⅳ （FUT4）对胚胎细胞与子宫内膜细胞黏附的影响未见报道.本研究以人子宫内膜细胞 (HEC-1A)和胚胎细胞(JAR)为体外着床模型,观察上调HEC-1A细胞中FUT4表达对JAR细 胞与HEC-1A细胞黏附的影响.RT-PCR和免疫细胞化学检测结果显示,FUT4过表达增加 HEC-1A细胞中FUT4基因及蛋白的表达;免疫细胞化学及Western印迹结果表明,上调HEC-1A细胞中FUT4增加细胞表面LeY的合成;细胞黏附实验结果显示,与未转染组相比较,FUT4过表达增加了JAR细胞与HEC-1A细胞的黏附率.本研究证明,FUT4过表达可以增加细胞表面LeY寡糖抗原的合成,从而促进胚胎细胞与子宫内膜细胞的黏附.     

卵巢激素对人子宫内膜FUT9基因表达的调控

新近发现α1,3岩藻糖基转移酶 (FUT9)参与LeX 寡糖的合成。通过逆转录 聚合酶链式反应、免疫印迹分析和免疫组化等方法 ,研究了在不同激素状态下人子宫内膜FUT9基因的表达 ,以及LeX 寡糖表达的调控机制。结果显示 :FUT9基因在人子宫内膜中表达 ,且其mRNA在分泌期的表达水平高于增生期 ,口服米非司酮(mifepristone)后 ,增生期内膜的表达量增加 ;LeX 寡糖主要分布在人子宫内膜腔上皮和腺上皮细胞表面 ,在不同生理时期其变化趋势与FUT9基因相似 ,并且口服米非司酮后 ,分泌期内膜的表达量减少 ;在内膜中检测到 3种(33kD、35kD和 85kD)LeX 寡糖蛋白 ,口服米非司酮后 35kD的LeX 寡糖蛋白消失。结果表明 :FUT9基因在人子宫内膜中有明确表达 ,且其表达受卵巢激素的调节 ,孕激素对其有上调作用 ;卵巢激素在转录水平上实现对LeX 寡糖表达的调节。     

胚泡表面Le^Y寡糖对小鼠胚泡表皮生长因子及其受体表达和分泌的影响

在胚胎与子宫内膜间识别、粘附及胚胎植入过程中 ,阶段特异表达的寡糖抗原LeY 起重要作用。经与特异性单克隆抗体AH6预保温 ,中和胚泡表面LeY 寡糖后 ,通过RT PCR和免疫印迹方法 ,观察了LeY 寡糖对着床前小鼠胚泡表皮生长因子及其受体的表达和分泌的影响。结果显示 ,在体外培养中 ,经AH6封闭胚泡表面的LeY寡糖后 1.5h ,胚泡EGF的转录及分泌明显受到抑制 (P <0 .0 1) ,并且这种抑制作用持续到 6h以上 ;而EGF R表达及分泌仅有轻微降低的趋势。结果提示 ,EGF表达和分泌的降低可能是胚泡表面LeY 寡糖调节胚泡自身发育及随后的着床过程的多种途径之一     

鳞癌细胞增殖能力与岩藻糖基转移酶表达水平及糖蛋白岩藻糖基化修饰有关

岩藻糖基转移酶（fucosyltransferases,FUTs）是一类催化糖蛋白和糖脂发生岩藻糖基化（修饰）酶,主要包括FUT1~FUT9。已有研究证明,很多癌组织中都有不同FUT基因表达升高的现象。本研究证明,表皮鳞癌细胞的增殖能力与几种FUT基因表达水平有关。本文比较研究了人表皮鳞癌A431和SCC12细胞的增殖速度和几种FUT的表达状况,以揭示鳞癌细胞增殖能力与几种FUT基因表达水平的关系。细胞倍增时间结合MTT法揭示,鳞癌A431细胞的倍增时间约为26 h,而鳞癌SCC12细胞的倍增时间约为33 h（P < 0.05）,提示A431细胞增殖速度比SCC12细胞明显加快。与增殖速度一致的是,Western 印迹显示,A431细胞中与DNA合成相关的增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）蛋白表达水平比SCC12细胞高。实时定量PCR（qPCR）检测FUT1-9基因 mRNA转录本,揭示A431细胞中几种FUT基因的mRNA水平均显著高于SCC12细胞。凝集素免疫印迹法和Western 印迹法进一步证明,A431细胞中总蛋白的岩藻糖基化水平比SCC12细胞中的明显升高。敲低FUT4基因表达后,A431细胞中LeY寡糖的表达水平下调,细胞增殖被明显抑制。这些结果证明,较强的表皮鳞癌细胞增殖能力可能与几种FUT基因的高表达,以及糖蛋白的岩藻糖基化（修饰）相关。岩藻糖基转移酶表达水平与临床表皮鳞癌的恶性增生的相关性有待进一步证明。     

ConA的抗着床效应

本文用凝集素为探针,探索糖复合物在胚泡着床中的作用,报道了与甘露糖苷有专一结合的伴刀豆凝集素(ConA)有明显的抗小鼠胚泡着床作用。妊娠4d的小鼠,每只子宫角中注入Con A 25μg,22只子宫角中只有4只子宫角有胚泡着床,着床率为18.2％,与生理盐水对照组的着床率87.5％相比有明显差异。将相同剂量的Con A先与0.4mol/L α-甲基-D-甘露糖苷温育1—2h后再注入子宫,20只子宫角中有15只子宫角有胚泡着床,着床率提高到75％。用辣根过氧化物酶直接标记法证明,着床前子宫内膜细胞表面有Con A受体存在,并随着妊娠天数而增加,尤其是间质细胞,发情期时时为阴性反应,到着床期蜕膜细胞膜表面呈现出大量Con A受体。提示精复合物在着床中的重要作用。与甘露糖苷同样专一结合的,但寡糖结构专一性与Con A不同的豌豆凝集素注入子宫则无抗着床效应,着床率为85.7％。由此可以推测,N-连接的包含二个未被取代的或只在C-2位被取代的α-甘露糖苷的寡糖参于胚泡与子宫内膜相互作用的着床过程。     

人FUT4基因近端启动子结构的初步分析

LeY是一种双岩藻糖化寡糖,在大多数上皮来源的肿瘤细胞（包括乳腺癌、卵巢癌等）中高表达.岩藻糖基转移酶Ⅳ（fucosyltransferase Ⅳ, FUT4）是合成LeY的关键酶. 前期工作发现,FUT4通过增加LeY糖的合成来促进细胞的增殖. 但有关FUT4的转录调控机制尚不清楚. 本文通过对人FUT4基因近端启动子进行生物信息学分析,并构建不同长度启动子序列荧光虫荧光素酶报告基因表达载体,分析其转录活性. 使用First EF程序分析并获得FUT4近端启动子序列,采用PCR 法扩增FUT4基因近端不同长度的启动子序列,定向克隆,获得不同长度的启动子重组质粒. 重组质粒经双酶切及测序鉴定正确. 荧光素酶活性分析不同长度的FUT4 基因启动子片段的转录活性.结果显示,pGL6-FUT4-1.2 kb在MCF-7和MDA- MB-231细胞中转录活性明显升高（P<0.05）.说明FUT4基因启动子区域定位于转 录起始位点上游的-800～-1 600 bp的区域内.     

岩藻糖链与肝癌细胞发生和转移的相关性研究

研究观察了大鼠诱发肝癌过程中,与UEA、LCA凝集素相结合的含岩藻糖糖蛋白尤其是80 ku蛋白的动态变化．在肝癌病人标本中,也观察到了高转移性肝癌细胞比低转移性肝癌细胞表达更多的UEA、LCA相结合的岩藻糖蛋白．岩藻糖寡糖可以构成一些非常重要的黏附分子的结构,如Lewis抗原．继而进一步观察了不同转移潜能的肝癌细胞中Lewis抗原的表达差异,发现高转移性肝癌细胞 (HMCC97H) 比低转移性肝癌细胞(HMCC97L)表达更高的Lewis x 和 b．在肝癌转移动物模型中,转移灶组织中的Lewis抗原合成关键酶α1,3/1,2以及 α1,6 岩藻糖转移酶活性远比对照组高．当肝癌细胞在维甲酸作用以后,细胞表面的Lewis x 或 b 的水平显著下降,α1,3/1,2岩藻糖转移酶活性也显著下降．同时我们观察到Lewis x可以存在于表皮细胞生长因子受体(EGFR)分子上,在维甲酸作用以后,EGFR上的Lewis x抗原和磷酸化水平都显著性下降．上述结果提示岩藻糖化的糖链如Lewis x在肝癌细胞的发生和转移过程中起重要的作用．     

着床期小鼠子宫内膜Le^y糖蛋白的动态变化

为了解子宫内膜Ｌｅ＾ｙ糖蛋白在胚泡着床期间的变化,以及与胚泡表面阶段特异性Ｌｅ＾ｙ抗原出现的关系,应用对Ｌｅ＾ｙ寡糖特的ＡＨ－６单抗为探针,通过ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎｂｌｏｔ免疫酶标染色,观察了小鼠子宫内膜Ｌｅ＾ｙ糖蛋白在着床期的动态变化和分布特点,结果表明：（１）未孕及着床前后的小鼠子宫内膜均含Ｌｅ＾ｙ糖蛋白Ｍｒ５０～２００ｋＤ,未孕内膜的含量明显高于着床期间的样品;（２）在着床日（Ｄ     

Expression of cell surface Lewis X and Y antigens and FUT4 mRNA is increased in Jurkat cells undergoing apoptosis

Cell surface molecules undergo specific changes during apoptosis, including the expression of phosphatidylserine (PS) and some proteins and alterations in sugar chains. Among the various sugar chains on the cell surface, Lewis X (Le(X)) and Lewis Y (Le(Y)) antigens are key determinants for a variety of biological processes. We studied the changes in Le(X) and Le(Y) expression in Jurkat cells, a human T cell line, during apoptosis. Flow cytometry showed that Le(X) and Le(Y) antigen expression was enhanced on the cell surface during apoptosis induced by anti-Fas antibody. To clarify the mechanism of enhanced Le(X) and Le(Y) expression, we assessed the expression levels of fucosyltransferase (FUT1, 2, 3-5-6, 4, and 9) mRNAs that are predominantly expressed in Jurkat cells and which are considered to form Le(X) and Le(Y). The expression of FUT4 mRNA was up-regulated after exposing cells to anti-Fas antibody. Moreover, the increase in Le(X) and Le(Y) antigen levels was significantly suppressed by caspase 3 or 8 inhibitors. These results indicated that the induction of FUT (mainly FUT4), the gene expression of which is mediated by signals downstream of caspase 3, increases Le(X) and Le(Y) expression in apoptotic cells.     

FUT7 antisense sequence inhibits the expression of FUT7/sLeX and adhesion between embryonic and uterine cells

Implantation is a complex developmental event that is initiated by recognition and adhesion of the embryo to the endometrial epithelium. sLeX is an oligosaccharide antigen acting as the ligand of L-selectin, and is stage-specifically expressed in the endometrial epithelium. The adhesion system mediated by L-selectin and sLeX oligosaccharide plays an important role in this process. FUT7 is a key enzyme for sLeX synthesis, and the regulation of sLeX through FUT7 may influence maternal-fetal recognition. In this study, we observed the effect of FUT7 antisense oligodeoxynucleotide on the expression of FUT7 and sLeX, as well as adhesion in an in vitro implantation model consisting of the human uterine epithelial cell line RL95-2 and the human embryonic cell line JAR. Results showed that the expression of FUT7 was significantly decreased, compared with controls, after FUT7 antisense oligodeoxynucleotide transfection into RL95-2 cells, as determined by RT-PCR, Western blotting, and indirect immunofluorescence. Synthesis of sLeX was also decreased, consistent with the FUT7 decrease, as shown by indirect immunofluorescence. The adhesion of embryonic cells to uterine epithelial cells was significantly reduced (P < 0.01) compared with the control. These data indicate that the use of a FUT7 antisense oligodeoxynucleotide can cause a significant reduction of both FUT7 and sLeX antigen, and thereby inhibit the adhesion of embryo cells to endometrium. This approach may provide a new way to regulate reproduction.     

Molecular cloning, genomic mapping, and expression of two secretor blood group alpha (1,2)fucosyltransferase genes differentially regulated in mouse uterine epithelium and gastrointestinal tract

Fucosylated oligosaccharides have been proposed to be involved in multiple cell-cell interactions, including mouse blastocyst adhesion and intestine-microbe interactions. To begin to define the regulation and function of terminal alpha(1,2)fucosylated carbohydrates in these and other tissues, we isolated and characterized a 85-kilobase (kb) genomic region of mouse chromosome 7, 23.2 centimorgans analogous to human chromosome 19q13.3 that encodes three alpha(1,2)fucosyltransferases. Gene-specific DNA probes from the open reading frames of the mouse fucosyltransferase genes corresponding to human FUT1, FUT2, and SEC1 demonstrate distinct tissue-specific expression patterns by Northern blot analyses. Flow cytometry profiles of cultured cells transfected with DNA segments containing the open reading frames of the mouse genes confirm that each encodes an alpha(1,2)fucosyltransferase. In uterus and colon, a 3.3-kb FUT2 mRNA represents the major fucosyltransferase gene expressed. Steady-state FUT2 mRNA levels are cyclically regulated during the estrus cycle, increasing 10-fold from early diestrus to a relative maximum in proestrus. In contrast, SEC1 and FUT1 do not show prominently regulated expression in uterus. FUT2 expression localizes to luminal uterine epithelium by in situ hybridization, implying that this gene determines expression of cell surface Fucalpha1-->2Galbeta epitopes proposed to mediate blastocyst adhesion.     

Expression of the Lewis group carbohydrate antigens during Xenopus development

We have examined the pattern of expression of the Lewis group carbohydrate antigens during the development of African toad Xenopus laevis. One of these antigens, Lewis x (Le(x), also known as SSEA-1), was previously shown to be involved in cell-cell adhesion in early mouse embryos and teratocarcinoma stem cells. Recently another member of these antigens, sialyl-Le(x), was found to be one of the major ligands for the selectin family of cell-cell adhesion molecules. In order to study the role of carbohydrate-mediated cell adhesion during Xenopus development, we first studied the expression pattern of the Le(x). We found that Le(x)was not expressed in early embryos, started to be expressed at the tail bud stage in anterior regions of the body such as the cement gland or head skin, and was gradually showed more posterial expression at later stages. At tadpole stage, it was also expressed on specific cell bodies in brain, and in axon region in brain and neural retina. Antibodies against Le(x)blocked neurite outgrowth in the explant culture of tadpole brain. One of the candidates for Le(x)carrier protein in the tadpole brain is a 200 kDa glycoprotein detected by Western blotting. In adult tissues, it was expressed in brain, testis, and gut, but not in kidney, lung, spleen, ovary, or muscle. We also examined the expression patterns of other Lewis group antigens. Among them, sialyl-Le(x)was expressed on endothelial cells and on leukocytes, suggesting the possibility that it functions as a ligand for selectin in Xenopus.     

Coordinate expression of X and Y haptens during murine embryogenesis

The X hapten (Gal beta 1----4[Fuc alpha 1----3]GlcNAc) may play an important role in the adhesion of blastomeres during compaction. Therefore, we have investigated more thoroughly developmental changes in the fucosylation of lactoseries carbohydrate chains and the enzymatic basis of these fucosylation changes using well-characterized monoclonal antibodies. The Y hapten (Fuc alpha 1----2Gal beta 1----4[Fuc alpha 1----3]GlcNAc) and polymeric X haptens were detected by fluorescence-activated flow cytometry on murine embryonal carcinoma cells. In paraffin sections of postimplantation mouse embryos, the Y hapten was detected in the embryonic ectoderm and visceral endoderm on Days 5.5-7.5; this pattern of antigen expression is identical to that previously reported for the X hapten (SSEA-1). Thus, the Gal:alpha 1----2 (H) and GlcNAc:alpha 1----3 (X) fucosyltransferases appear to be co-regulated during embryogenesis. Reciprocal changes in X and Y hapten expression were observed, however, during preimplantation development. Unlike the X hapten, the Y hapten is expressed maximally on 16-cell morulae and 32- to 64-cell blastocysts. Eight-cell embryos cultured to the blastocyst stage in vitro did not acquire the Y hapten, however, suggesting a role for the uterine environment in carbohydrate antigen expression. Homogenates of F9 embryonal carcinoma cells were found to possess a potent GlcNAc:alpha 1----3 fucosyltransferase activity, as well as a weaker Gal:alpha 1----2 fucosyltransferase activity, using paragloboside as a substrate. The results suggest that embryonic cell surface carbohydrate phenotypes represent a balance in the competition between glycosyltransferases for available substrates. Rapid changes in carbohydrate expression during development may reflect intermediate states of cellular commitment and determination that are critical for lineage formation and morphogenesis.