Induction of matrix metalloproteinase-9 and -2 activity in mouse blastocyst by fibronectin-integrin interaction

Fibronectin, a major extracellular matrix, plays an important role in embryo implantation by mediating embryo adhesion and outgrowth. In this work, mouse blastocysts produced pro-matrix metalloproteinase-9, pro-matrix metalloproteinase-2 and 64 ku matrix metalloproteinase-2 when they were co-cultured with fibronectin. In contrast, mouse blastocysts did not produce these proteinases without fibronectin. Focal adhesion kinase is a fundamental molecule of integrin signaling pathway and its antisense oligodeoxynucleiotide inhibited blastocyst matrix metalloproteinases expression induced by fibronectin. The results indicated that fibronectin triggered matrix metalloproteinase-9 and -2 expression in mouse blastocyst through its integrin receptors and subsequent signaling pathway, which enhanced the synchronization of blastocyst invasiveness and uterine receptivity and ensured the accuracy of events relative to implantation in timing and spatiality.     

Effect of matrix metallo-proteinase-26 (MMP-26) during embryo implantation in the mouse

Matrix metalloproteinase-26 (MMP-26, endometase and matrilysin-2), a novel member of the MMPs family, is detected not only in the placenta and uterus, but is widely expressed in malignant tumors from different sources as well as in diverse tumor cell lines. However, the function of MMP-26 in the reproductive system has never been reported. Expression of MMP-26 in mouse embryos and the function of the MMP-26 antibody during mouse embryo implantation was examined for the first time by injecting the uterine horn, immunohistochemistry,in situ hybridization, co-culture of mouse blastocysts and uterine monolayer epithelial cells, Western blot, RT-PCR, Northern blot and zymography. Our results show that there is strong expression of MMP-26 mRNA and protein in the mouse embryo. Furthermore, the MMP-26 antibody dramatically inhibited mouse embryo implantation and significantly inhibited adhesion and outgrowth of mouse blastocysts onin vitro uterine monolayer epithelial cells. At the same time, the MMP-26 antibody inhibited the expression of integrin αV mRNA and protein in a dose-dependent manner. These data suggest that MMP-26 may play a role in some of the tissue-remodeling events associated with the invasion of the endometrium by trophoblast cells and facilitate successfully embryo implantation.     

非妊娠小鼠子宫内膜白血病抑制因子基因表达研究

白血病抑制因子（LIF）能抑制胚胎干细胞的分化和保持其增殖能力,并与胚泡着床及胚胎早期发育有着密切的关系。经实验证实,LIF基因在子宫内膜中的表达具有高度的时间限制性。采用反转录聚合酶链式反应技术（RT－RCR）对小鼠子宫内膜LIF基因表达进行了研究,结果显示：妊娠第4天小鼠的子宫内膜组织的样本在电泳图的538bp处出现清晰的电泳带,表明该组织有LIF基因的强表达;而在所检测的13只非妊娠小鼠子宫内膜的组织样本中均未发现LIF基因表达讨论了小鼠动情周期中各阶段的孕激素水平对子宫内膜LIF基因表达产生的影响,提出了一定的孕激素水平是启动LIF基因表达的必要条件     

差异展示法鉴定GA3诱导的水稻差异表达的mRNA

赤霉素是植物生长发育过程中一类重要的调节激素，在水稻不育系上喷施合适浓度的ＧＡ３（赤霉素的一种）可以克服其包颈现象。运用反转录和聚合酶链式反应建立了一套旨在分离差异表达ｃＤＮＡ的差异展示方法。     

Regulation of mouse blastocyst adhesion,outgrowth and matrix metalloproteinase—2 by focal adhesion kinase

The interaction of extracellular matrix-integrin markedly influences the adhesion,outgrowth,differentiation and expression of serine proteinases by the blastocyst,so it is regarded as a vital factor in blastocyst implantation.Although the mechanism of extracellular interactions between extracellular matrix and integrins has been well elucidated,the roles of the signaling molecules in the extracellular matrix-integrin signal transduction pathway in blastocyst implantation are unknown.This limits the understanding of blastocyst implantation and ECM-integrin signal transduction pathway.In the present study,in vitro blastocyst culture and indirect immunocytochemistry,matrix metalloproteinases(MMPs) zymography and antisense oligodeoxynucleotide(ODN) were used to investigate the expression of a fundamental molecule of integrin-dependent signal transduction pathways,focal adhesion kinase(FAK),in mouse blastocysts and its influence on mouse blastocyst adhesion,outgrowth and MMP-2.The results showed that mouse blastocysts expressed FAK.FAK protein was clustered in the peripheral migrating trophoblast cells and dispersed in the central area of blastocyst outgrowth.Fibronectin triggered pro-MMP-2 and 64kD MMP-2 activities.The antisense ODN to FAK attnuated pro-MMP-2 and 64kD MMP-2 activites which decreased abruptly and tended to disappear with increasting concentrations of the antisense ODN.Both mouse blastocyst adhesion and outgrowth on fibronectin were also influenced by the antisense ODN.Up to 20μg/mL of the antisense ODN concentration,the adhesion and out-growth rates were decreased in a dose-dependent manner.The results indicated that FAK influenced mouse blastocyst adhesion,outgrowth and MMP-2 activity by intracellular signal transduction.In other words,FAK regulates mouse implantation in terms of blastocyst adhesive and invasive abilities.     

维生素D受体蛋白及其mRNA在人输卵管黏膜上皮的表达

目的:探讨人输卵管黏膜上皮维生素D受体(VDR)蛋白及其mRNA的表达.方法:30例输卵管标本取自具有正常妊娠生育史,因子宫肌瘤、子宫腺肌病等行腹式子宫切除术,一并切除输卵管的妇女.输卵管取材包括峡部、壶腹部和伞部,按子宫内膜组织学分期分为增生早期组6例、增生中晚期组9例、分泌早期组7例、分泌中晚期组8例.应用免疫组织化学法和逆转录聚合酶链反应法检测人输卵管黏膜上皮VDR的表达.结果:人输卵管黏膜上皮中检测到VDR蛋白及其mRNA表达.VDR蛋白主要表达于输卵管上皮细胞的细胞核中,部分间质平滑肌细胞核内亦有表达.相同时期各不同部位输卵管上皮细胞VDR蛋白表达无明显差别(P＞0.05),随月经周期变化,各不同部位输卵管上皮细胞VDB蛋白表达发生改变,在增生早期和分泌中晚期较低,与之比较,在增生中晚期和分泌早期其表达明显增高(P＜0.01),至分泌早期达最高值;壶腹部输卵管黏膜上皮组织VDR mRNA表达在增生早期和分泌中晚期较低,在增生中晚期和分泌早期明显增高(P＜0.01).结论:人输卵管黏膜上皮组织存在VDR蛋白及其mRNA表达,在增生中晚期和分泌早期其表达明显增高,且具有周期性变化.     

巨噬细胞移动抑制因子在宫颈癌组织中的表达及临床意义

目的探讨巨噬细胞移动抑制因子（MIF）在宫颈癌组织中的表达,评价其在宫颈癌发生、发展中的作用。方法采用免疫组化SP法检测42例宫颈癌组织及20例正常宫颈组织中MIF的表达。结果MIF在宫颈癌中的阳性表达率明显高于正常宫颈组织（P〈0．05）;MIF表达与宫颈癌的分化程度、临床分期及淋巴结转移密切相关（P〈0．05）,与病理类型及肿瘤直径无关联性（P〉0．05）。结论MIF可能在宫颈癌的发生及进展中起着重要的作用。     

逆转录聚合酶链式反应快速检测水稻条叶枯病毒

逆转录聚合酶链式反应（Ｒｅｖｅｒｓｅ Ｔｒａｓｃｒｉｐｔｉｏｎ ａｎｄ Ｐｏｌｙｍｅｒａｓｅ Ｃｈａｉｎ Ｒｅａｃｔｉｏｎ,ＲＴ－ＰＣＲ）具有灵敏度高,准确性好的特点,因而被广泛应用于植物病毒检测。在ＲＴ反应中,ＲＮＡ样品的快速制备尤为重要用异丙醇二步沉淀法获得ＲＮＡ,快速检测水地片中ＲＳｔＶ伯ＲＴ－ＰＣＲ方法,可使测定时间缩短至６ｈ,并可从０．０７８ｍｇ感病叶片中检测出ＲＳｔＶ。     

RT—PCR检测草鱼呼肠孤病毒的方法研究

草鱼呼肠孤病毒（Grass carp reovirus,GCRV）为草鱼出血病的病原．本实验根据GenBank中GCRV和其他水生呼肠孤病毒毒株的第六基因片段,在其保守区设计了一对GCRV特异性引物,建立了快速检测GCRV的逆转录聚合酶链式反应（RT—PCR）方法．该PCR体系中,上下游引物的最适终浓度为120nmol／L,最适退火温度为52℃．PCR特异性试验表明：所设计的引物只能扩增GCRV的核酸,而不能扩增嗜水气单胞菌BSK-10、WSSV以及正常CIK细胞的DNA或RNA．敏感性试验表明,当GCRV的反转录模板稀释至5-7时,PCR结果还为阳性．用所建立的RT—PCR方法对5份样品进行检测,结果表明本研究建立的RT—PCR检测方法可靠且可行．     

Effect of Steel factor and leukaemia inhibitory factor on murine primordial germ cells in culture.

Despite the importance of germ cells to the survival of species, surprisingly little is known about their embryological origin, proliferation, migration and entry into mitotic arrest or meiosis. Mutations in the murine Dominant White Spotting (W) and Steel genes, which respectively encode the c-kit tyrosine kinase receptor and the c-kit ligand (or Steel factor), impair the development of primordial germ cells (PGCs) in vivo, as well as haematopoietic stem cells and neural crest-derived melanoblasts. Here we use a monoclonal antibody against c-kit tyrosine kinase receptor and recombinant Steel factor to study the c-kit receptor-ligand system in cultured PGCs. In addition, we show that leukaemia inhibitory factor (also known as differentiation inhibitory activity), a factor secreted by STO fibroblasts, can stimulate proliferation of primordial germ cells in vitro.     

人白血病抑制因子真核表达载体的构建及在哺乳动物细胞中的表达

应用RT-PCR方法从人子宫内膜组织总RNA扩增出hLIF的全长基因,然后将其克隆至pcDNA3上,成功构建了重组真核表达载体pcDNA3/hLIF.利用脂质体介导将这一表达载体导入COS-7细胞和CHO-K1细胞,分别获得了hLIF的瞬时表达和稳定表达,为进一步进行hLIF生物学功能研究和hLIF在哺乳动物细胞中的高表达研究奠定了基础.     

Regulation of mouse blastocyst adhesion,outgrowth and secretion of matrix metalloproteinase-2 by cGMP and nitric oxide <Emphasis Type="Italic">in vitro</Emphasis>

Nitric oxide (NO) is a multifunctional messenger molecule produced through oxidation of L-arginine to L-citrulline by enzyme NO synthase (NOS). In the current study, mouse blastocysts were cultured in the different media, and the implantation capacity of blastocyst was evaluated by evaluating the percentage of embryos adhesion and outgrowth after culture for 12, 24 or 48 h. Matrix metalloproteinase-2 (MMP-2) mRNA was detected by RT-PCR, and MMP-2 protein was detected by gelatin zymography. Inhibition of blastocyst adhesion and outgrowth was observed in embryo cultured with 500 μmol/L NOS inhibitor N^G-mono-methyI-L-arginine (L-NMMA) alone; however, 100 μmol/L S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, and 20μmol/L cGMP analogue, 8-Br-cGMP could block this inhibition. The expression and production of MMP-2 in the blastocysts were suppressed by L-NMMA, and SNAP or 8-br-cGMP could reverse this suppression. These results suggest that NO induces embryo implantation by cGMP signaling pathway.     

Comparative study on the effect of integrin αvβ3 on secretion of matrix metalloproteinases in human normal and carcinoma trophoblasts

The invasion of cytotrophoblast cells into the maternal endometrium during embryonic implantation is very similar to the metastasis of carcinoma cells. However, the significant difference is that the former is a highly controlled process. In this report, the effects of integrin αvβ3 on the secretion of matrix metalloproteinase (MMP) were compared between normal cytotrophoblast cells and choriocarcinoma cells by RT-PCR, gelatin zymography and immunocytochemistry. The results reveal that both normal cytotrophoblast cells and choriocarcinoma cells can express integrin αvβ3. The secretion of MMPs in normal cytotrophoblast ceils is up-regulated by anti-αvβ3 antibody, whereas, decreased in choriocarcinoma cells. It was suggested that αvβ3 can modulate the expression of MMPs in trophoblasts, and this action is carried out through distinct mechanisms in normal and carcinoma cytotrophoblast cells.     

Effect of UPP on the expression of VEGF and its receptors in mouse uterus during peri-implantation

On day 3 of gestation ,one uterine horn of female pregnant mouse was injected intraluminally with 5 μL 0.1μg/mL lactacystin,a specific inhibitor of ubiquitin-pro-teasome pathway (UPP),while the contralateral horn served as control ,Animals were sacrificed by cervical dislocation on day 5,6,7 of gestation ,respectively,Then the number of implanted embryos in each uterine horn was calcuated,and the expression of VEGF and its receptors was examined,The data showed that the number of implanted embryos was decreased significantly after treatment with lactacystin ,The results of RT-PCR and Western blot indicated that expression of VEGF and its receptors at mRNA and protein levels was significantly decreased in the treated uterus,menawhile,the expression of HIF-1α(the α subunit of HIF ,a transcrip-tional factor of VEGF) was reduced at both mRNA and protein levels,These data suggested that the effect of UPP on VEGF expression was realized through regulating HIF-1α expression .In addition ,UPP is likely to take part in the modulation of VEGF receptors expression ,These changes may be one of the reasons for the reduction of implanted embryos.     

超声波对小鼠活动行为的影响及其驱鼠机制研究

为了探究超声波对小鼠行为的影响以及超声波驱鼠器的作用机制,建立评价超声波驱鼠器效果的简便方法,使用市售超声波驱鼠器进行旷场驱离小鼠实验,通过自发活动、高架十字实验和蔗糖水偏好实验统计了小鼠在超声波发生器与超声波驱鼠器胁迫下的行为学变化.结果显示,超声波驱鼠器连续10 d处理,具有驱离小鼠效果.超声波发生器和超声波驱鼠器连续10 d处理都会引起小鼠运动和探索能力下降,诱发其焦虑和抑郁情绪,并且超声波发生器处理引发的上述变化更为显著.本研究可为超声波驱鼠机制提供数据支持,也可为相关驱鼠器的效果评价与研发提供技术参考.     

不同基质对人工湿地脱氮效果和硝化及反硝化细菌分布的影响

对组合基质和煤渣基质潜流式人工湿地的脱氮效果进行了对比研究,结果表明:组合基质人工湿地的脱氮效果较好,氨氮和总氮的平均去除率达到81.9%和75.7%,而煤渣基质人工湿地的去除率相对较低,氨氮和总氮的平均去除率仅为59.6%和51.8%.为进一步探讨2组人工湿地脱氮效果差异显著的原因,研究了不同基质潜流式人工湿地系统内...     

利用16S-23S rDNA间隔区快速鉴定猪链球菌和马链球菌兽疫亚种

利用16S-23S rDNA间隔区(ISR)长度和序列的多态性,结合16S rDNA和23S rDNA的保守性,分别在16S rDNA末端和23S rDNA前端保守区设计上下游引物,进行PCR扩增.结果为21株猪链球菌均扩增出长度约为1200bp的片段,8株马链球菌兽疫亚种均扩出约1300 bp的片段,其他属菌株和阴性...