小鼠胚泡黏附时子宫内膜nm23-M1/NDPK A的表达

nm23家族除与肿瘤转移抑制有关,它还参与调节正常细胞的发育、增殖、分化及凋亡等过程。运用RT-PCR、Western blot 和免疫组织化学技术,分析小鼠胚泡黏附时子宫内膜着床点和着床旁组织nm23-M1/NDPK A 的表达,以未交配鼠作对照,为进一步阐明胚泡着床的机制提供有意义的实验依据。RT-PCR 结果显示,小鼠胚泡黏附时子宫内膜nm23-M1/NDPK A mRNA 表达明显高于对照组,并且着床点明显高于着床旁,Western blot 和免疫组织化学分析nm23-M1/NDPK A 蛋白表达,也得到一致的结果。提示nm23-M1/NDPK A 参与胚泡着床这一重要生命活动过程。     

双向凝胶电泳-飞行时间质谱技术鉴定小鼠胚泡黏附时子宫内膜nm23-M2／NDPK B的表达

运用双向聚丙烯酰胺凝胶电泳(2DPAGE)分析未交配小鼠子宫内膜和妊娠第五天(D5)小鼠子宫内膜胚泡黏附时植入位点及其旁组织蛋白质组。差异蛋白质组学显示,等电点(isoelectric point,pI)约7．1、分子量(molecular weight,Mw)约18kDa的蛋白质点在D5小鼠子宫内膜特别是植入位点表达上调。对此蛋白质点用基质辅助激光解析电离飞行时间质谱(matrix-assisted laser desorption／ionization time of flying mass spectrometry,MALDI—TOF—MS)测定其胶内酶解后的肽质量指纹谱(Peptide Mass Fingerprint,PMF),经Mascot：Peptide Mass Fingerprint中SWISS-PROT数据库查询后,鉴定该蛋白质为鼠源性nm23-M2／NDPKB。RT—PCR和免疫组织化学结果也显示D5小鼠子宫内膜nm23-M2／NDPK B mRNA和蛋白表达明显增加。提示nm23-M2／NDPKB参与胚泡着床这一重要生命活动过程。     

双向凝胶电泳-飞行时间质谱技术鉴定小鼠胚泡黏附时子宫内膜nm23-M2/NDPK B的表达

运用双向聚丙烯酰胺凝胶电泳(2DPAGE)分析未交配小鼠子宫内膜和妊娠第五天(D_5)小鼠子宫内膜胚泡黏附时植入位点及其旁组织蛋白质组。差异蛋白质组学显示,等电点(isoelectricpoint,pI)约7.1、分子量(molecularweight,Mw)约18kDa的蛋白质点在D_5小鼠子宫内膜特别是植入位点表达上调。对此蛋白质点用基质辅助激光解析电离飞行时间质谱(matrix—assistedlaserdesorpion/ionizationtimeofflyingmassspectrometry,MALDI-TOF-MS)测定其胶内酶解后的肽质量指纹谱(PeptideMassFingerprint,PMF),经Mascot:PeptideMassFingerprint中SWISS-PROT数据库查询后,鉴定该蛋白质为鼠源性nm23-M2/NDPKB。RT-PCR和免疫组织化学结果也显示D_5小鼠子宫内膜nm23-M2/NDPKBmRNA和蛋白表达明显增加。提示nm23-M2/NDPKB参与胚泡着床这一重要生命活动过程。     

小鼠胚泡着床期子宫内膜LongSAGE基因文库构建

按照LongSAGE文库构建原理,先后经子宫内膜总RNA提取、cDNA合成、130bp双标签的产生、PCR扩增、34bp双标签形成、连接成串联体、与载体连接、转化大肠杆菌等相关步骤构建小鼠胚泡着床期子宫内膜LongSAGE基因文库。经证实所构建文库的阳性克隆率达100%,克隆中插入的串联体长度介于400￣500bp之间,成功构建了小鼠胚泡着床期子宫内膜LongSAGE基因文库。     

p16INK4a在早孕小鼠子宫内膜的表达及其对胚泡着床的影响

本研究旨在检测肿瘤抑制基因p16INK4a(inhibitor of cyclin-dependent kinase 4a)在早孕小鼠子宫内膜中的表达规律,探讨p16INK4a在小鼠胚胎着床过程中的作用.采用荧光定量PCR(FQ-PCR)和免疫组织化学方法分别检测未孕小鼠及孕小鼠第2、3、4、5、7天子宫内膜p16INK4a mRNA和蛋白的表达;子宫角注射p16INK4a抗体观察胚泡着床数.FQ-PCR结果显示孕小鼠子宫内膜组织p16INK4amRNA的表达高于未孕小鼠,且随着妊娠天数的增加呈现表达逐渐增强的趋势,到妊娠第5天达到最高,后渐降.免疫组织化学分析显示p16INK4a蛋白在子宫内膜的表达规律与mRNA结果一致.子宫角注射p16INK4a抗体后胚泡着床数明显减少.以上结果提示,P161INK4a在妊娠早期子宫内膜持续表达,可能参与胚泡着床.     

半夏蛋白的抗兔胚泡着床作用

半夏蛋白有很强的抗兔胚泡着床作用,子宫内注射500μg,抗着床率达100%。经半夏蛋白作用后的子宫内膜能使被移植的正常胚泡不着床。在子宫内经半夏蛋白孵育的胚泡移植到同步的假孕子宫,着床率随孵育时间延长而降低。用辣根过氧化物酶标记半夏蛋白的定位实验表明该蛋白结合在子宫内膜腺管的上皮细胞膜上。已知半夏蛋白有类似凝集素的活性,能与甘露聚糖结合,它的抗着床作用可能是由于该蛋白结合了母体和(或)子体细胞膜上的某些糖结构,改变了细胞膜的生物学行为所致。     

PTEN在早孕小鼠子宫内膜的表达及其对胚泡着床的影响

本研究旨存检测肿瘤抑制基因PTEN(phosphatase andtensinhomologdeletedonchromosometen)在早孕小鼠子宫内膜中的表达规律,探讨PTEN在小鼠胚胎着床过程中的作用.采用实时荧光定量聚合酶联反应(real.time fluorescent quantitative PCR.FQ.PCR)和免疫组织化学方法分别检测未孕及孕1、3、4、5、7 d小鼠子宫内膜PTEN mRNA和蛋白的表达;子宫角注射PTEN反义寡核苷酸观察胚泡着床数.FQ-PCR结果显示,妊娠小鼠子宫内膜组织PTENmRNA的表达高于未妊娠小鼠,且随着妊娠天数的增加表达逐渐增强,到妊娠第5天达最高.免疫组织化学分析显示,PTEN蛋白在子宫内膜的表达规律与mRNA结果一致.子宫角注射PTEN反义寡核苷酸后胚泡着床数明显减少.结果提示,PTEN在妊娠早期子宫内膜持续表达,可能参与了胚泡着床.     

着床期小鼠子宫内膜中EGF及其受体转录和表达状况的研究

为探讨表皮生长因子（epidermal growth factor,EGF)在胚泡着床过程中的作用。本文应用原位杂交和免疫组织化学方法,检测了EGE及其受体在胚泡着床前后小鼠子宫内膜中的转录和表达。结果显示：未孕和受精后第4-5天,子宫内膜表面上皮和腺上皮细胞仍呈EGF,EGFR原位杂交和免疫组化阴性着色,受精后第4-5天子宫内膜基质细胞EGF及其受体转录和表达较未孕期增强,受精后第6天,EGF及其受体免疫组化和原位杂交阳性着色主要分布于初级蜕膜带（primary decidual zone,PDZ);随着胚泡植入的进行,PDZ区蜕膜细胞EGF及其受体的转录和表达明显减少,而PDZ周围蜕膜细胞EGF及其受体的转录和表达增强,结果提示,EGF是小鼠胚泡着床过程中的一个重要调节因子。     

Molecular cloning and functional expression of the second mouse nm23/NDP kinase gene, nm23-M2.

A new murine cDNA of nm23/NDP kinase was isolated. A RT-PCR product was obtained from the normal mouse liver mRNA with primers designed for the human nm23-H2 gene. The product was used as a probe to screen a cDNA library from the murine melanoma cell line, B16, and two clones containing the entire open reading frame were obtained. It was predicted that the DNA sequence encoded 152 amino acids which was 98% identical to the nm23-H2 protein. The entire nm23-M1 and -M2 gene-coding regions were translated as fusion proteins with a glutathione S-transferase. These fusion proteins displayed NDP kinase activities.     

PRUNE and NM23-M1 expression in embryonic and adult mouse brain

A genetic interaction between PRUNE and NM23/NDPK has been postulated in Drosophila melanogaster. Many have focused on Drosophila for the genetic combination between PRUNE “knock down” and AWD/NM23 fly mutants bearing the P97S mutation (K-pn, Killer of PRUNE mutation). We postulated a role for PRUNE-NM23 interactions in vertebrate development, demonstrating a physical interaction between the human PRUNE and NM23-H1 proteins, and partially characterizing their functional significance in cancer progression. Here, we present an initial analysis towards the functional characterization of the PRUNE-NM23 interaction during mammalian embryogenesis. Our working hypothesis is that PRUNE, NM23-H1 and their protein-protein interaction partners have important roles in mammalian brain development and adult brain function. Detailed expression analyses from early mouse brain development to adulthood show significant co-expression of these two genes during embryonic stages of brain development, especially focusing on the cortex, hippocampus, midbrain and cerebellum. We hypothesize that their abnormal expression results in an altered pathway of activation, influencing protein complex formation and its protein partner interactions in early embryogenesis. In the adult brain, their function appears concentrated towards their enzyme activities, wherein biochemical variations can result in brain dysfunction.     

Knockout mice as model systems for studying nm23/NDP kinase gene functions. Application to the nm23-M1 gene

Mice carrying a homozygous germ-line mutation in the nm23-M1 gene that eliminates its protein expression and drives expression of -galactosidase by nm23-M1 promoter have been generated. nm23-M1 gene inactivation is not teratogenic and the pups can grow to adult age without apparent health problems. However, they undergo a growth retardation and knocked out females cannot feed their pups. Both effects are background dependent. -galactosidase mapping of nm23-M1 promoter activation during embryogenesis shows that the nm23-M1 gene is principally expressed in epithelial layer of tissues which require inductive epithelial–mesenchymal interactions for their formation. In conclusion, invalidated mice could be interesting models to analyze the role of nm23-M1 on signal transduction pathway regulation, or cancer induction and proliferation.