TIMP-4 mRNA在小鼠子宫中的表达和激素调节

为研究TIMP-4 mRNA在子宫中的变化规律,采用半定量RT-PCR方法,观察了TIMP-4 mRNA在出生后小鼠子宫不同发育期、动情周期、妊娠早期以及假孕早期子宫中的表达和激素调节。结果发现,TIMP-4 mRNA在出生后不同发育期的小鼠子宫中、动情周期的任何时期、妊娠第1天以及假孕第1天的子宫中都不表达,但在妊娠和假孕第2天、第3天、第4天的子宫中都表达。以上结果提示,TIMP-4 mRNA在妊娠小鼠子宫中的表达可能不依赖存活胚胎的激活,而可能受母体妊娠分泌物的上调。     

Expression of vascular endothelial growth factor in rat uterus during peri-implantation

The first distinct mark of rodent implantation is the increased vascular permeability and significant angiogenesis at the sites of blastocyst implantation, but its mechanism is not clearly defined. Vascular endothelial growth factor (VEGF) is the key mediator for angiogenesis during embryogenesis and adult span and also serves as a vascular permeability factor. The aim of this study is to explore VEGF regulation mechanism and the possible role that VEGF plays in implantation by studying the VEGF expression and angiogenesis in the rat uterus during estrous cycle, ovarioectomized and peri-implantation stages usingin situ message RNA hybridization and confocal laser scanning techniques. The results indicated that VEGF was regulated by ovarian steroid hormones. VEGF expression before implantation was localized at luminal epithelium, shifted to stroma as implantation initiated and extensively located at the decidualizing stroma region after implantation. Bandeiraea simplicifolia-1 (BS-1) agglutinin and antibody against von Willebrand factor (vWF) were used to mark the endothelial cells and blood vessels. The results showed that the active angiogenesis occurred during the implantation process and this effect was probably mediated by VEGF. The results suggest that under the regulation of ovarian steroid hormones, VEGF plays an essential role in angiogenesis and increasing vascular permeability in endometrium, which are necessary for successful implantation.     

BMP2，BMP4及BMP信号通路相关成员在发情周期小鼠子宫中的表达

研究目的：研究骨形态发生蛋白（BMP）2,4及BMP信号通路相关成员在发情周期小鼠子宫中的表达模式。创新要点：运用实时荧光定量聚合酶链式反应（PCR）系统地研究了Bmp2和Bmp4及其BMP信号通路相关成员在小鼠子宫中mRNA水平表达模式,同时运用免疫组化方法研究了BMP2蛋白在小鼠子宫中的定位模式。研究方法：重要结论：收集发情周期各个时期小鼠子宫,一侧子宫角贮存于液氮或-80℃冰箱用于实时荧光定量PCR,另一侧子宫角用40mg／ml多聚甲醛固定用于BMP2蛋白免疫组化定位。实时荧光定量PCR结果表明,Bmp2的表达水平在发情前期显著高于发情期和发情后期（P〈0．05）,Bmp4的表达水平呈现显著波动,但Bmp2与Bmp4差异不显著（P〉0．05）。Bmprla和Bmpr2的表达水平在整个发情周期无显著变化（P〉0．05）。然而,Bmprlb的mRNA水平在发情期显著下降（P〈0．05）,在发情后期上升。此外,Bmprlb的mRNA水平显著低于相应时期Bmprla和Bmpr2的mRNA水平（P〈0．05）。三种R—Smads差异地表达于小鼠子宫,并且Smad1和Smad5的表达水平显著高于Smad8（P〈0．05）。此外,Smad4的表达水平在整个发情周期无显著变化。免疫组化结果显示,BMP2蛋白在整个发情周期差异表达,并主要定位于子宫腔上皮细胞和腺上皮细胞。我们的结果提供了BMP2和BMP4及其BMP信号通路相关成员mRNA水平表达变化信息,这些数据为论证BMP在子宫内膜中的作用如子宫内膜的退化与重塑提供量化和有用的信息。     

LPA3-mediated lysophosphatidic acid signalling in embryo implantation and spacing

Every successful pregnancy requires proper embryo implantation. Low implantation rate is a major problem during infertility treatments using assisted reproductive technologies. Here we report a newly discovered molecular influence on implantation through the lysophosphatidic acid (LPA) receptor LPA3 (refs 2-4). Targeted deletion of LPA3 in mice resulted in significantly reduced litter size, which could be attributed to delayed implantation and altered embryo spacing. These two events led to delayed embryonic development, hypertrophic placentas shared by multiple embryos and embryonic death. An enzyme demonstrated to influence implantation, cyclooxygenase 2 (COX2) (ref. 5), was downregulated in LPA3-deficient uteri during pre-implantation. Downregulation of COX2 led to reduced levels of prostaglandins E2 and I2 (PGE2 and PGI2), which are critical for implantation. Exogenous administration of PGE2 or carbaprostacyclin (a stable analogue of PGI2) into LPA3-deficient female mice rescued delayed implantation but did not rescue defects in embryo spacing. These data identify LPA3 receptor-mediated signalling as having an influence on implantation, and further indicate linkage between LPA signalling and prostaglandin biosynthesis.     

Secretion and regulatory mechanism of leptin during pregnancy in the golden hamster （Mesocricetus auratus）

Leptin, a 16 kDa polypeptide mainly secreted by white adipose tissue, has important function on the regulation of food intake and energy expenditure[1]. High concentration of leptin in the circulation suppresses food- intake, increases energy expenditure and therefore decreases the body weight; Accordingly, low concentration of leptin in the circulationstimulates food intake, decreases energy expenditure andincreases the body weight[1]. Normally, leptin level in thecirculation correlates with …     

Expression of Smad2 and Smad4 in rhesus monkey endometrium during the menstrual cycle and early pregnancy

Expression of Smad2 and Smad4 mRNAs in the endometrium of rhesus monkey on Days 8, 20 and 28 of the normal menstrual cycle and on Days 12, 18 and 26 of early pregnancy was detected using in situ hybridization. The results showed that Smad2 and Smad4 mRNAs were mainly localized in luminal epithelium and glandular epithelium. The expression of Smad2 mRNA in glandular epithelium was sustained at moderate level on Days 8, 20 and 28 of the menstrual cycle, while the expression of Smad4 gradually increased with the menstrual cycle. Both Smad2 and Smad4 mRNAs in functionalis glandular epithelium were expressed at the highest levels on Day 12 of early pregnancy, while in basalis glandular epithelium the most abundant expression of both Smads occurred on Days 12 and 18 of pregnancy. On Day 26, both Smads mRNAs were expressed at the lowest levels either in functionalis or in basalis. The data suggest that the epithelium is the major compartment where TGF-βs/activins exert their biological effects via Smads, and that Smad4 may play a role in the maintenance of endometrial gland function during secreting period of the menstrual cycle. During lacunar stage of early pregnancy, Smad2 and Smad4 are implicated in the tissue remodeling of endometrial functionalis and basalis, and during early villous stage both Smads are functional primarily in basalis.     

Expression of LIF,VEGF, CD57 and CD68 after the transfer of rat embryos to mouse uteri

The high failure rate of interspecific preganacy is a major obstacle to the successful interspectific cloning of mammals,To in vestigate the reasons for the failure of inter-specfic pregnancy between rats and mice,we transferred rat blastocysts into mouse uteri on the third day of pseudopreg -nancy (D3),oure previous study showed that intact rat embryos could still be observed in mouse uteri on D9.In the present study ,we found that expression of CD57 and CD68 increased significantly at the maternal -fetal interface fol-lowing the transfer of rat embryos,Similarly ,Leukaemia inhibitory factor(LIF) expression increased ,but vascular endothelial growth factor(VEGF) expession decreased,In a co-culture system ,the percentage of rat ectoplacental cones (EPCs) with adhesion and outgrowth and outgrowth area on mouse uterine decidual cells were less than that of mouse EPCs,These results indicate that an increase in the immunological rejection response and a decrease in the in vasiveness of rat embryos may be important reasons for the failure of interspecific pregnancy between rat and mouse.     

钙离子敏感受体在大鼠子宫中的表达

本实验为了探讨钙离子敏感受体(Calcium-sensing receptor,CaR)与胚胎着床及蜕膜化的关系,建立了大鼠早期妊娠、假孕、延迟着床和人工诱导蜕膜化模型,收集各种模型的子宫材料,利用免疫组化方法检测了CaR在大鼠子宫中的表达。结果表明,CaR在妊娠第6~9天的子宫蜕膜上强表达;假孕第6~9天和延迟着床的大鼠子宫腔上皮下基质中无明显的免疫信号,而延迟着床激活胚胎周围的腔上皮下基质中有强的免疫信号。蜕膜化子宫中整个蜕膜区呈强的免疫染色信号。从而推断,CaR蛋白的表达与胚胎着床及蜕膜化过程密切相关。     

Frizzed家族基因mRNA在小鼠动情周期卵巢组织中的表达规律

运用Real-time Q-PCR方法分析了Frizzed1-10mRNA在小鼠动情周期卵巢中的相对表达水平.结果发现10个Frizzed家族基因中,除Frizzed8外,其他均有不同水平的表达.其中Frizzed1和2在动情前期表达水平显著高于其他3个时期水平,约是间情期水平的4倍(P<0.05);动情前期和间情期的Frizzed3表达水平约是另2个时期的1.5倍(P<0.05);Frizzed4在动情期表达水平显著高于其他3个时期,约是间情期的3倍(P<0.05);间情期Frizzed5表达水平显著高于动情前期和动情期,约是动情期的5倍(P<0.05);Frizzed7表达类似于Frizzed5,间情期表达水平约是动情前期的4倍(P<0.05);Frizzed10的表达在动情前期表达水平约是动情期的3倍(P<0.05);Frizzed6和9在4个时期的表达无显著差别(P<0.05).以上结果表明:Frizzeds在4个动情时期卵巢中广泛而差异表达,并推测Frizzed1—4可能在动情周期卵泡发育过程中具有重要作用.     

Hormonal regulation of K+-channel messenger RNA in rat myometrium during oestrus cycle and in pregnancy

The dramatic enhancement of smooth-muscle excitability in the uterus which occurs at oestrus and at term in pregnant rats is closely related to increased blood oestrogen concentrations. How oestrogen alters the electrical properties of myometrial cells is unclear, although electrical coupling between cells has been shown to increase. Many examples are known of changes in cellular excitability involving modification of existing ion channels by second-messenger pathways. Steroid hormones, in contrast, are generally thought to influence cellular processes mainly through effects on gene expression, inducing the synthesis of new proteins. Previous work, using an oocyte translation system, has shown that a very slowly activating, voltage-dependent K+ current can be expressed from the poly(A)+ RNA of oestrogen-treated rat uteri. We report here that the messenger RNA species producing this channel is rapidly and reversibly induced in the presence of oestrogen, as shown by the appearance and disappearance of this mRNA during the oestrous cycle, its emergence at the end of pregnancy, and its presence or absence following hormonal treatments. These results suggest that oestrogen controls the expression of a voltage-dependent ion channel in uterine smooth muscle cells.     

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。     

小白鼠动情周期及妊娠早期（1—6天）子宫雌激素受体含量的变化

本文实验初步证明,妊娠D_1天的小鼠子宫ER。含量稍高(142.4±42.1 fmol mg蛋白)。D、降至117.7±45.4fmol/mg蛋白。D_3和D_4分别上升到158.9±40.3fmol/mg蛋白及162.1±34.8fmol/mg蛋白.D_5降为137.7±52.7fmol/mg蛋白(P<0.01)。D_6急增至182.4±44.9fmol/mg蛋白(P<0.01),达到妊娠D_1～D_6的峰值,在动情周期中,前期ER(?)含量为188.6±49.2fmol/mg蛋白,动情期则降至109.7±47.1fmol/mg蛋白(P<0.01)。后期较低(106.6±28.4fmol/mg蛋白),间期又回升到163.5±41.2fmol/mg蛋白,反映了ER_c的规律性变化是受内源性雌激素周期性地调控的,妊娠D_1的小鼠子宫湿重较高(126.7±56.8mg).此后D_2～D_4呈下降趋势。D_5极显著地上升至138.0±29.4mg。D_6又急下降(P<0.01)。在动情周期中,小鼠子宫湿重从前期的67.6±36.5mg上升至动情期的124.2±72.4mg。后期降为74.1±57.3mg。间期则降至最低水平。     

Fas在云芝糖肽诱导HL-60细胞凋亡中的作用

目的：探讨云芝糖肽（PSP）诱导HL-60细胞凋亡的Fas死亡受体信号转导途径．方法：观察PSP处理后HL-60细胞的形态变化，并采用流式细胞仪及免疫印迹检测细胞Fas，FADD蛋白的表达及Caspase-8凋亡酶的激活．结果：100，400g／mLPSP处理HL-60细胞48h后，细胞出现明显凋亡特征，同时伴随Caspase-8酶原被激活和Fas抗原的表达量增加，表现为剂量依赖关系．400g／mLPSP处理36h时Caspase-8酶原开始被剪切激活，48h时激活更为显著．各实验组FADD的表达量基本没有变化．结论：Fas死亡受体信号可能参与了PSP诱导HL-60细胞凋亡．     

伤寒沙门菌激活TRAIL信号通路促进巨噬细胞凋亡

为探究伤寒沙门菌对巨噬细胞凋亡及肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-related apoptosis inducing ligand,TRAIL)信号通路的影响,对伤寒沙门菌感染THP-1细胞早期和后期提取细胞RNA进行转录组测序(RNA-seq).通过定量PCR(qPCR)技术检测凋亡相关基因的转录水平,对RNA-seq结果进行验证,流式细胞术和蛋白质免疫印迹法检测THP-1细胞的凋亡和凋亡相关蛋白;加入TRAIL抗体结合TRAIL蛋白后,流式细胞术和蛋白质免疫印迹法检测感染早期THP-1细胞凋亡的变化.结果显示,伤寒沙门菌感染THP-1细胞后,凋亡相关基因caspase-3、caspase-8、caspase-9在感染后期转录水平显著增加,Tnfsf10、Tnfrsf10b、Tnf、Fas在感染早期和后期转录均增加,细胞凋亡率明显增加,caspase-3、caspase-8、caspase-9蛋白活化增加,TRAIL、DR5蛋白表达增加;与PBS对照组相比,加入TRAIL抗体后caspase-3蛋白活化明显减弱,细胞凋亡率明显下降.综上,伤寒沙门菌通过部分激活TRAIL信号通路来诱导巨噬细胞凋亡.     

As4S4诱导宫颈癌Hela细胞凋亡及相关分子机制研究

研究AS4S4对宫颈癌Hela细胞生长抑制厦诱导凋亡作用，并探讨可能的分子机制。以不同浓度的AS4S4分不同时间段处理Hela细胞，用四甲基偶氮唑蓝（MTT）法测定细胞增殖抑制率；采用流式细胞术测定细胞凋亡；Western blotting测凋亡相关蛋白Bcl-2，Caspase3表达的变化。结果表明，经AS4S4处理的Hela细胞，增殖受到抑制，作用呈明显的时效和量效关系，细胞凋亡率明显增高并呈浓度依赖性；Western blotting示Bcl-2表达下降，Caspase3表达增加。因此，AS4S4对Hela细胞具有增殖抑制和诱导凋亡作用，其机制与下调Bcl-2蛋白、上调Caspase3蛋白表达有关。     

青龙衣中胡桃醌对A-549细胞周期及凋亡影响

探讨青龙衣中胡桃醌（Juglone）对体外培养的人肺癌细胞A-549生长抑制作用及对细胞凋亡和周期的影响.采用MTT法检测青龙衣中胡桃醌对A-549细胞的生长抑制作用,采用激光共聚焦扫描显微镜观察凋亡形态,流式细胞仪分析胡桃醌对A-549细胞周期的影响.MTT法试验结果显示,胡桃醌对体外培养的人肺癌细胞A-549具有明显的生长抑制作用,处理24、48、72 h的IC50值为分别为3.4×10^-5、1.8×10^-5、2.6×10^-6mol/L.凋亡形态观察结果显示,胡桃醌能诱导A-549细胞的凋亡,同时胡桃醌能剂量依赖性的降低A-549细胞的G1期细胞的比例,升高G2期和S期细胞比例,呈G2期和S期阻滞作用.胡桃醌对A-549细胞具有明显的生长抑制作用,阻止周期进程,诱导A-549细胞凋亡.     

灯盏乙素对乳腺癌细胞中lncRNAs表达的影响

检测灯盏乙素对乳腺癌MDA-MB-231细胞中的长链非编码RNA(LncRNAs)的相对表达量的影响,进一步探索灯盏乙素对肿瘤的作用机制.采用实时荧光定量PCR的方法,检测对照组与不同剂量组的灯盏乙素处理后的乳腺肿瘤细胞中lncRNAs MALAT1、NEAT1和p53基因mRNA的相对表达量,并观察细胞存活率.结果发现,灯盏乙素在低剂量(1、10、25μmol/L)时促进细胞增殖,而在高剂量50μmol/L以上时抑制细胞增殖.灯盏乙素在低剂量时使得乳腺癌MDA-MB-231细胞中MALAT1,NEAT1和p53基因的表达水平下降,而在100μmol/L的高剂量时MALAT1,NEAT1和p53基因的表达水平上升.灯盏乙素影响乳腺癌MDA-MB-231细胞中LncRNAs MALAT1和NEAT1基因以及p53基因的表达,并且存在剂量反应关系,低剂量与高剂量呈现出相反的表达量变化.     

皮蛋模拟胃肠道消化物对HepG2细胞的影响

皮蛋是中国传统食品,其水解物具有抗氧化、抗炎和抗癌等功效,具有良好的医学前景,但目前皮蛋发挥抗癌功效的途径及作用机制尚不清楚。探究了皮蛋模拟胃肠道消化物(preserved eggs simulated gastrointestinal digests,PESD)对人肝癌HepG2细胞增殖和细胞周期的影响。将皮蛋经体外模拟胃肠道消化处理后,进行细胞实验,用CCK-8法进行细胞增殖分析,研究PESD对HepG2细胞存活率的影响,采用碘化丙啶(propidium iodide, PI)染色标记的方法,通过流式细胞术测定PESD对HepG2细胞周期的影响,用western blot法测定细胞周期相关调控蛋白的表达。研究发现:PESD以剂量依赖性方式对HepG2细胞增殖进行抑制,IC₅₀为4.17mg/mL;PESD处理显著增加了S期细胞的占比(P<0.05),使HepG2细胞阻滞于S期;4mg/mL的PESD处理HepG2细胞24h后,细胞中cyclin A、cyclin E、ATM、chk2和CDC25A的蛋白表达水平上升(P<0.05),而CDK2蛋白表达水平下降(P<0.05)。研究认为,皮蛋模拟胃肠道消化物是以剂量依赖性的方式来抑制人肝癌HepG2细胞的增殖,主要是通过ATM-chk2-CDC25A信号通路,上调cyclin A和cyclin E蛋白的表达,下调CDK2蛋白的表达来实现人肝癌HepG2细胞阻滞于S期,影响人肝癌HepG2细胞周期进程,抑制癌细胞的增殖。研究结果旨在为皮蛋抗癌作用机制的阐明提供一定的理论基础。     

CPUY013对胰腺癌Capan2的生长抑制与诱导凋亡作用

探讨CPUY013对体外培养人胰腺癌细胞Capan2生长抑制及诱导凋亡作用.采用MTT法、克隆原形成法检测CPUY013对Capan2细胞生长的抑制作用,采用流式细胞仪分析CPUY013对细胞周期的影响,Western Blot法检测TopoⅠ、野生型p53、caspase-3、bcl-2和bax蛋白表达的变化.MTT法、集落形成试验结果显示,CPUY013对体外培养的人胰腺癌细胞Capan2有明显的生长抑制作用,处理72 h的IC50值为7.3×10^-7mol/L（MTT法）,CPUY013在8.0×10^-8mol/L浓度可以明显抑制Ca-pan2细胞的集落形成,抑制率为69.6%.同时,CPUY013可剂量依赖地降低Capan2细胞G1期细胞的比例,升高S期细胞的比例,呈现明显的S期阻滞.CPUY013可下调TopoⅠ蛋白的表达,上调细胞中p53、caspase-3、bax蛋白表达,下调bcl-2蛋白表达,并呈剂量依赖性.CPUY013对Capan2细胞具有明显的生长抑制作用,阻滞细胞周期进程,诱导Capan2细胞凋亡,其可能与降低细胞内TopoⅠ蛋白表达,增加p53、caspase-3、bax蛋白表达,降低bcl-2蛋白表达有关.