小鼠子宫内膜LIF基因表达与孕激素的关系研究

白血病抑制因子（LIF）是一种多功能活性的糖蛋白，LIF基因在大多数妊娠第4天的小鼠子宫内膜进行着强烈的表达，然而LIF基因表达调控的机理目前尚不清楚，本实验对130只妊娠4－5d的小鼠LIF基因表达和血清中孕激素水平分别进行了检测，发现12只小鼠（占总数的9．23％）无LIF基因表达，无该基因表达小鼠血清中孕激素水平显著低于其它表达的小鼠。实验结果表明：孕激素可能是LIF基因表达的复杂的调控体系中的一个重要的调控因素。     

对小鼠子宫内膜及胚胎LIF基因表达的研究

利用ＲＴ－ＰＣＲ方法,对妊娠的小鼠和非妊娠小鼠子宫内膜、妊娠的小鼠胚胎的ＬＩＦ基因表达进行了研究,孕鼠子宫内膜中存在ＬＩＦ基因的表达,３例非妊娠小鼠子宫内膜ＬＩＦ基因的表达阳性,２只小嫌胚胎中有ＬＩＦ基因的表达。     

S100A10基因在小鼠子宫中的表达及性激素对其表达的调节

S100A10是S100钙结合蛋白家族成员之一,是一种钙依赖性的信号通路的介导分子,近年来有研究表明,它在胚胎植入位点有明显的高表达.文中采用半定量RT-PCR和原位杂交方法,研究了S100A10基因在小鼠中的组织特异性表达,在妊娠初始期胚胎植入位点、妊娠期子宫和正常动情周期子宫中的表达及其受性类固醇激素的调节.观察发现:S100A10基因在小鼠多种组织中都有表达,在卵巢、子宫、睾丸及附睾等一些生殖器官中表达水平尤为高;在妊娠第4天,子宫中S100A10基因表达明显上调,且在子宫内膜植入位点处有非常显著的高表达,其mRNA分布于对系膜侧的腔上皮细胞和功能层的基质细胞,且基质细胞中的信号强于腔上皮细胞,而非植入位点呈现极其微弱的表达;从妊娠第5天直到分娩第1天子宫内都维持恒定的S100A10表达;正常动情周期中,S100A10基因在动情前期和动情期子宫中表达明显上调;卵巢切除模型显示雌激素显著上调S100A10基因的表达,孕激素下调其表达.以上结果提示S100A10可能具有抑制胚胎植入位点腔上皮细胞的过度凋亡和促进基质细胞的增殖与蜕膜化的作用以及参与子宫应激反应过程.     

S100A10基因在小鼠子宫中的表达及性激素对其表达的调节

S100A10是S100钙结合蛋白家族成员之一，是一种钙依赖性的信号通路的介导分子，近年来有研究表明，它在胚胎植入位点有明显的高表达.文中采用半定量RTPCR和原位杂交方法，研究了S100A10基因在小鼠中的组织特异性表达，在妊娠初始期胚胎植入位点、妊娠期子宫和正常动情周期子宫中的表达及其受性类固醇激素的调节.观察发现：S100A10基因在小鼠多种组织中都有表达，在卵巢、子宫、睾丸及附睾等一些生殖器官中表达水平尤为高；在妊娠第4天，子宫中S100A10基因表达明显上调，且在子宫内膜植入位点处有非常显著的高表达，其mRNA分布于对系膜侧的腔上皮细胞和功能层的基质细胞，且基质细胞中的信号强于腔上皮细胞，而非植入位点呈现极其微弱的表达；从妊娠第5天直到分娩第1天子宫内都维持恒定的S100A10表达；正常动情周期中，S100A10基因在动情前期和动情期子宫中表达明显上调；卵巢切除模型显示雌激素显著上调S100A10基因的表达，孕激素下调其表达.以上结果提示S100A10可能具有抑制胚胎植入位点腔上皮细胞的过度凋亡和促进基质细胞的增殖与蜕膜化的作用以及参与子宫应激反应过程.     

早孕小鼠子宫内膜组织中IASPP表达对胚胎着床的作用

目的:探讨早孕小鼠子宫内膜组织中P53凋亡刺激蛋白抑制因子(IASPP)的表达对胚胎着床的作用.方法:对NIH雌鼠进行阴道涂片以确定其动情周期,采用实时荧光定量PCR、Western blot以及免疫组化等分别在分子和蛋白水平上检测雌鼠子宫内膜上IASPP的表达情况;对实验组小鼠左侧子宫角注射IASPP抑制剂,最后测量...     

孕激素对子宫内膜上皮细胞增殖调控机制研究进展

子宫内膜是卵巢激素的主要靶器官,女性进入青春期后,在下丘脑垂体的调节下,雌激素、孕激素出现周期性分泌,子宫内膜的形态和功能也随之发生改变.黄体期,血清中上升的孕激素可拮抗雌激素的作用,抑制子宫内膜上皮细胞的增殖,使其分化为具有分泌功能的上皮细胞.孕激素抑制子宫内膜上皮细胞增殖并促使其分化的机制主要是:一方面孕激素通过降低子宫内膜上皮细胞雌激素受体(ER)的表达,降低颗粒细胞分泌细胞色素P450芳香化酶的表达和活性,增加17β-HSDII在子宫内膜局部的表达和活性,增强雌激素代谢,从而抑制雌激素对子宫内膜上皮细胞的促增殖作用,间接抑制子宫内膜上皮细胞的增殖;另一方面孕激素通过与子宫内膜上皮细胞的孕激素受体结合后进入核内,调节细胞周期调控蛋白、原癌基因等参与细胞周期调控的分子表达,直接抑制子宫内膜上皮细胞的增殖并促使其分化.     

Survivin在子宫腺肌病中的表达及与VEGF相关性研究

目的探讨凋亡抑制基因Survivin在子宫腺肌病的表达及其与VEGF的相关性。方法应用免疫组织化学S—P法,检测50例子宫腺肌病异位及在位内膜组织中Survivin、VEGF蛋白的表达,并与正常在位子宫内膜组织30例进行对照。结果子宫腺肌病异位内膜组织中Survivin蛋白阳性表达率为80％,明显高于正常内膜及在位内膜组织的表达率16．67％、16％（P〈0．05）;子宫腺肌病异位内膜组织中VEGF蛋白阳性表达率为56％,明显高于正常内膜及在位内膜组织的表达率38％、20％（P〈0．05）;Survivin蛋白表达与VEGF蛋白呈正相关（P〈0．01）。结论Survivin可通过抑制异位内膜细胞凋亡,参与血管形成对子宫腺肌病的发生、发展起作用;Survivin与VEGF在子宫腺肌病的发生、发展中起协同作用。     

SWAP-70在小鼠正常动情周期和围植入期子宫中的表达及其调节

SWAP-70是鸟苷酸交换因子家族成员之一，是PI3K介导的酪氨酸磷酸化受体下游通路的信号分子之一. 以往的研究及我们的前期工作初步表明它在小鼠子宫和恒河猴胚胎植入位点有高表达，提示此分子可能参与了胚胎植入的调节. 为此，本研究在小鼠模型中，采用原位杂交、免疫组化和半定量RT-PCR方法，系统地研究了SWAP-70在妊娠初始期胚胎植入位点、妊娠期子宫和动情周期子宫中的表达及其受雌、孕激素的调节. 结果表明SWAP-70在植入位点有非常显著的高表达，其mRNA和蛋白分布于胚泡以及对系膜侧的腔上皮细胞和临近胚泡的子宫基质细胞，而在非植入位点表达强度很弱；在妊娠第7天和第9天的母胎界面上，滋养层巨细胞、迷走滋养层和海绵滋养层都维持较强的SWAP-70表达；正常动情周期中，SWAP-70在动情期子宫中表达明显上调；卵巢切除模型显示雌激素显著上调SWAP-70的表达，孕激素能够阻断雌激素的作用. 以上结果提示，SWAP-70可能参与胚胎植入过程中多种细胞行为的调节，如胚泡黏附、基质细胞的增殖与蜕膜化以及滋养层细胞的浸润和分化过程，同时在动情周期子宫内膜细胞功能调控中发挥一定作用.     

bFGF调控CD34对子宫内膜癌细胞生长的影响

目的探讨碱性成纤维细胞生长因子（bFGF）调控CD34对子宫内膜癌细胞生长的影响。方法免疫组化法检测正常子宫内膜和子宫内膜癌组织中bFGF、微血管密度（MVD）和增殖细胞核抗原（PCNA）的表达。用TUNEL原位凋亡法检测细胞凋亡并计算凋亡率（AR）。结果子宫内膜癌组织中bFGF、MVD和PCNA表达均明显高于正常子宫内膜组织,而AR低于正常子宫内膜组织。bFGF、MVD和PCNA均呈正相关。bFGF和MVD与AR均呈负相关。结论bFGF促进肿瘤细胞增殖,抑制肿瘤细胞凋亡。     

Livin和Fhit在子宫内膜癌中的表达及意义

采用免疫组化SP法检测Livin和Fhit在30例正常子宫内膜和70例子宫内膜癌组织中的表达,研究凋亡抑制蛋白Livin和Fhit在子宫内膜癌中的表达和意义。结果显示Livin蛋白在正常子宫内膜组织中表达明显低于子宫内膜癌组织（P〈0.05）;Fhit蛋白在正常子宫内膜组织中表达明显高于子宫内膜癌组织（P〈0.05）;在子宫内膜癌组织中,随组织学分级和病理分期的增加,Livin蛋白表达阳性率逐渐上升,Fhit蛋白表达阳性率逐渐下降,而且Fhit蛋白表达与淋巴结转移相关（P〈0.05）;Livin蛋白和Fhit蛋白表达呈负相关（P〈0.05）。结果提示Livin和Fhit异常表达可能与子宫内膜癌的发生、发展关系密切,联合检测Livin和Fhit有助于子宫内膜癌早期诊断、治疗及预后判断。     

Forced expression of theOct-4 gene influences differentiation of embryonic stem cells

By transfecting an Oct-4 expression plasmid into embryonic stem cells (ES cells), the ES-O cell line was constructed, which sustained the expression of Oct-4 gene when induced by retinoic acid. Forced expression of Oct-4 gene could not sustain the stem property of ES-O cells without the differentiation inhibiting factor LIF, but if LIF exists, forced expression of Oct-4 gene could enhance the ability to sustain the undifferentiation state and inhibit cell differentiation induced by retinoic acid. It was indicated that Oct-4 must cooperate with LIF to sustain the undifferentiation state of ES cells. During the cell differentiation, ES-O cells tend to differentiate into neural cells, suggesting that forced expression of Oct-4 gene may be in relation with the differentiation of neuroderm.     

Blastocyst implantation depends on maternal expression of leukaemia inhibitory factor.

A critical point during mammalian pregnancy is the implantation of the blastocyst when the embryo attaches to the wall of the uterus. The autonomously developing preimplantation embryo then becomes dependent on the maternal environment for its continued development. Little is known about the regulation of implantation, except that a complex interaction between peptide and steroid hormones synchronizes the preparation of the uterus for implantation with the development of the embryo. Whether the implantation event is under maternal or embryonic control is also unclear (reviewed in refs 1, 2). We have previously shown that a cytokine, leukaemia inhibitory factor (LIF), is expressed in the uterine endometrial glands specifically on the fourth day of pregnancy. This burst of expression is under maternal control and always precedes implantation of the blastocyst. Here we report that transient expression of LIF in mice is essential for implantation. Females lacking a functional LIF gene are fertile, but their blastocysts fail to implant and do not develop. The blastocysts, however, are viable and, when transferred to wild-type pseudopregnant recipients, they can implant and develop to term.     

Generation of tetraploid complementation mice from embryonic stem cells cultured with chemical defined medium

Mouse embryonic stem cells(mESCs)derived from inner cell mass(ICM)of pre-implantation embryos,can maintain undifferentiated state when cultured in N2B27 medium supplemented with GSK3inhibitor CHIR99021 and MEK inhibitor PD0325901(‘‘2i’’)and leukemia inhibitor factor(LIF).Compare to conventional culture medium,all components of this medium are defined.With the N2B27 medium,‘‘2i’’and LIF,mESCs can contribute to the germline of the chimeric embryos,however,whether the‘‘all-ES cells’’mice can been generated by tetraploid complementation is unclear yet,while the tetraploid complementation serve as a golden standard to assess the pluripotency of ES cells.Here,our study showed that mESCs derived and cultured with the N2B27 complete medium could generate fertile mice by tetraploid complementation.In addition,the survival rate of tetraploid complementation mice produced by inbred mES cell lines is higher than the conventional culture condition,and increased the percentage of Oct4 positive cells contrast to conventional medium either.Therefore,the N2B27 medium supplemented with‘‘2i’’and LIF is an alternative choice forthe derivation and long-term culture of mouse embryonic stem cells.     

Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells

Embryonic stem (ES) cells, the totipotent outgrowths of blastocysts, can be cultured and manipulated in vitro and then returned to the embryonic environment where they develop normally and can contribute to all cell lineages. Maintenance of the stem-cell phenotype in vitro requires the presence of a feeder layer of fibroblasts or of a soluble factor, differentiation inhibitory activity (DIA) produced by a number of sources; in the absence of DIA the ES cells differentiate into a wide variety of cell types. We recently noted several similarities between partially purified DIA and a haemopoietic regulator, myeloid leukaemia inhibitory factor (LIF), a molecule which induces differentiation in M1 myeloid leukaemic cells and which we have recently purified, cloned and characterized. We demonstrate here that purified, recombinant LIF can substitute for DIA in the maintenance of totipotent ES cell lines that retain the potential to form chimaeric mice.     

Scriptaid affects histone acetylation and the expression of development-related genes at different stages of porcine somatic cell nuclear transfer embryo during early development

Although the somatic cell nuclear transfer(SCNT) technique has been used extensively for cloning and generating transgenic pigs,the cloning efficiency is still very low.It has been proposed that the low efficiency of this technique is the result of incomplete epigenetic reprogramming and abnormal gene expression during early embryonic development.In this study,we investigate the effect of Scriptaid,a low-toxicity histone deacetylase inhibitor,on the developmental competence of porcine SCNT embryos.We found that treating SCNT embryos with 500 nmol/L Scriptaid for 15 h after activation significantly enhanced the blastocyst formation rate(27.7%) compared with the untreated group(control)(12.2%,P<0.05).Using an immunofluorescence technique to measure the average fluorescence intensity,we also found that treating SCNT embryos with Scriptaid increased the level of histone acetylation on histone H3 at lysine 14(acH3K14).Furthermore,treating embryos with Scriptaid increased the expression level of three genes that play important roles during embryonic development(Oct4,Klf4 at the blastocyst stage and Nanog at the 4-cell stage).Moreover,the expression level of the apoptosis-related gene Caspase-3 was significantly lower in the Scriptaid-treated SCNT embryos compared with the control SCNT embryos at the 4-cell and blastocyst stages.In conclusion,these results indicate that Scriptaid treatment improves the development and nuclear reprogramming of porcine SCNT embryos.     

Correction of RNA aberrant splice increases foreign gene expression in transgenic mice

Transgenic mice with mammary gland secreting human granulocyte colony stimulating factor (G-CSF) were produced using mice whey acid protein gene promoter. It was found that there was very low expression level in mammary gland. Human G-CSF cDNA was obtained by RT-PCR from transgenic mice mammary gland. Sequence analysis showed that this G-CSF gene deleted the 4th exon, and compared with human G-CSF genomic DNA, there were donor and acceptor splice sites in the deletion fragment. It was considered that the 3rd and 4th introns also delete in G-CSF fragment. The transgenic construct was corrected by deleting the 3rd and 4th introns to construct the minigene, which was used to produce transgenic mice by microinjection. Northern blot showed that G-CSF expression using the new construct increased 5.4 times as that before in transgenic mice. The results suggested that it was possible that RNA aberrant splice result in low expression in transgenic mice.