14-3-3蛋白在小鼠受精卵及2-细胞期胚胎中的表达与定位

目的:研究小鼠受精卵及2-细胞期胚胎中14-3-3蛋白的表达及亚细胞定位。方法:采用Western blot方法鉴定小鼠受精卵和2-细胞期胚胎14-3-3蛋白的表达,利用间接免疫荧光技术观察其在细胞中的定位。结果:在小鼠受精卵和2-细胞期胚胎中,全部表达14-3-3蛋白,并且为同一亚型;14-3-3蛋白主要定位于受精卵的细胞质及2-细胞期胚胎中的细胞质和细胞核。结论:14-3-3蛋白正常表达于小鼠受精卵及2-细胞期胚胎中,其定位可能影响胚胎的早期发育。     

14-3-3蛋白在小鼠受精卯及2-细胞期胚胎中的表达与定位

目的：研究小鼠受精卵及2-细胞期胚胎中14-3-3蛋白的表达及亚细胞定位。方法：采用Western blot方法鉴定小鼠受精卵和2-细胞期胚胎14-3-3蛋白的表达，利用间接免疫荧光技术观察其在细胞中的定位。结果：在小鼠受精卵和2-细胞期胚胎中，全部表达14-3-3蛋白，并且为同一亚型：14-3-3蛋白主要定位于受精卵的细胞质及2-细胞期胚胎中的细胞质和细胞核。结论：14-3-3蛋白正常表达于小鼠受精卵及2-细胞期胚胎中，其定位可能影响胚胎的早期发育。     

PKC、PKB通过p21~(CIP1/WAF1)蛋白对小鼠受精卵G2期向M期转换的影响

目的:探讨在小鼠受精卵中,PKC及PKB是如何通过p21CIP1/WAF1蛋白影响小鼠受精卵G2/M的转换。方法:以小鼠受精卵为材料,通过Westernblot及免疫荧光的方法,检测经PKC及PKB抑制剂处理后的p21蛋白表达及定位。结果:经PKC抑制剂(PMA)处理后的小鼠G2期受精卵中p21蛋白表达增加,而经PKB抑制剂(LY294002)处理后的p21蛋白表达没有变化,但是其在亚细胞的定位发生了改变。结论:PKC通过改变p21蛋白表达,影响小鼠受精卵G2/M转换。而PKB通过改变p21蛋白在小鼠受精卵中的亚细胞定位,影响受精卵G2/M转换。     

p70S6K在小鼠受精卵第一次有丝分裂周期中的表达

目的:研究p70S6K在小鼠受精卵第一次有丝分裂周期中的表达。方法:采用Westernblot和RT-PCR方法,检测在小鼠受精卵第一次有丝分裂的G1、S、G2以及M期中p70S6K在mRNA水平以及蛋白水平的表达。结果:在mRNA水平,p70S6K的表达在各期无明显变化,在蛋白水平,磷酸化状态的p70S6K在S期升高,表明从S期p70S6K的活性升高。结论:p70S6K在小鼠卵子受精后磷酸化程度增加,因而有可能参与了小鼠受精卵的早期发育。     

蛋白激酶B(AKT)抑制剂对小鼠1-细胞期胚胎分裂的影响

目的:研究蛋白激酶B(AKT)在小鼠1-细胞期受精卵中的作用。方法:激酶活性分析检测2种AKT的抑制剂在小鼠1-细胞期胚胎中对MPF活性的调节;G2/M期的细胞计数检测AKT抑制剂对小鼠1-细胞期胚胎细胞分裂的影响;Western blotting检测AKT抑制剂对小鼠1-细胞期胚胎中pCDC2Tyr15的去磷酸化状态的改变。结果:AKT抑制剂抑制了MPF活性、G2/M期的转换,pCDC2Tyr15的去磷酸化状态。结论:AKT存在于小鼠1-细胞期胚胎中并通过调节MPF活性和pCDC2Tyr15的去磷酸化状态调节细胞分裂的进程。     

Girdin蛋白在小鼠受精卵微丝聚集调控作用的初步研究

目的:探讨Girdin蛋白在调控小鼠受精卵早期发育微丝聚集中的作用。方法:采用免疫荧光染色观察Girdin蛋白和F-actin在小鼠受精卵中的共定位情况。激光共聚焦显微镜观察Girdin表达敲低后小鼠受精卵分裂形态及分裂率。结果:在小鼠受精卵中存在Girdin蛋白并且Girdin蛋白与F-actin存在共定位。敲低Girdin蛋白的表达,小鼠受精卵出现不规则分裂,10μmol/L Girdin si RNA处理组小鼠受精卵有28.93%到达2-细胞期,而注射20μmol/L Girdin si RNA组小鼠受精卵只有15.1%到达2-细胞期。并且Girdin表达敲低的受精卵微丝不能正确聚集。结论:Girdin蛋白在调控小鼠受精卵早期分裂微丝聚集中发挥作用。     

CDC25B蛋白S149/S321位点突变对小鼠1-细胞期受精卵发育的影响

目的:研究小鼠CDC25B蛋白S149位点对小鼠1-细胞期受精卵发育的影响及S149位点与S321位点的关系。方法:构建pBSK-CDC25B-S149A和pBSK-CDC25B-S149A/S321A突变体,体外转录成mRNA;采用小鼠超排卵技术取G1期受精卵,显微注射CDC25B-WT-mRNA和突变CDC25B-S149A-mRNA、CDC25B-S149A/S321A-mRNA,观察其对受精卵发育、M期促进因子(MPF)活性及CDC2-pTyr15磷酸化状态的影响。结果:CDC25B-S149A-mRNA注射组受精卵卵裂率明显高于CDC25B-WT-mRNA注射组,MPF活性提前1 h达到高峰;而联合突变体CDC25B-S149A/S321A-mRNA注射组卵裂率又显著高于CDC25B-S149A-mRNA注射组。结论:在小鼠1-细胞期受精卵有丝分裂过程中,蛋白激酶A(PKA)对小鼠CDC25B蛋白S149位点的磷酸化修饰是控制受精卵G2/M转换的重要方式,并且S149位点与S321位点可能具有协同作用。     

PI3K催化亚基p110α小干扰RNA对小鼠受精卵发育的影响

目的:利用特异性小发夹RNA(shRNA)表达质粒,研究IA型磷脂酰肌醇3-激酶(PI3Ks)催化亚基p110α对小鼠受精卵发育的影响。方法:把p110αshRNA表达质粒显微注射到受精卵中。确定能干扰p110α表达的shRNA的最适浓度。通过检测RNA干扰后的p110αmRNA水平和蛋白表达情况,证明干扰是否成功。在确定成功干扰p110α后,观察受精卵的形态变化、分裂率以及对MPF活性的影响。结果:p110α表达受到干扰后,MPF的活性明显降低,受精卵第一次有丝分裂受到干扰,卵裂率明显降低。结论:p110α可能通过影响MPF的活性,调控小鼠受精卵的早期发育,为进一步探讨PI3K信号转导通路在小鼠胚胎中的作用奠定了基础。     

卵裂期胚胎可溶性人类白细胞抗原G的表达及其与胚胎发育的关系

目的观察卵裂期胚胎可溶性人类白细胞抗原G (soluble human leukocyte antigen G, sHLA-G)的表达,并探讨sHLA-G的表达与胚胎发育的关系.方法采用免疫组化标记法,检测177个卵裂期胚胎sHLA-G的表达情况.结果 177个卵裂期胚胎中,101个卵裂期胚胎有sHLA-G蛋白表达,sHLA-G的总表达率为57.1%(101/177).其中双原核受精卵发育形成的卵裂期胚胎sHLA-G的表达率为66.2 %(90/136),三原核受精卵发育形成的卵裂期胚胎sHLA-G的表达率为26.8 %(11/41),两者比较,差异有统计学意义(P<0.01).双原核受精卵发育形成的1级卵裂期胚胎sHLA-G 的表达阳性率为64.3%(18/28),2级卵裂期胚胎为91.7%(66/72),3级卵裂期胚胎为16.7%(6/36).3者比较,差异有统计学意义(P<0.01).三原核受精卵发育形成的Ⅰ级卵裂期胚胎sHLA-G的阳性率为88.9%(32/36),与双原核受精卵发育形成的Ⅰ级卵裂期胚胎比较,差异有统计学意义(P<0.01).双原核受精卵发育形成的≤4个细胞的胚胎sHLA-G的表达率为56.7%(34/60), 5个细胞及6个细胞的胚胎为67.9%(36/53),7个细胞及8个细胞的胚胎为87.0%(20/23),3者比较,差异无统计学意义(P>0.05).结论卵裂期胚胎有sHLA-G蛋白表达;且sHLA-G表达与胚胎发育有关.     

Aurora kinase B,epigenetic state of centromeric heterochromatin and chiasma resolution in oocytes

Aurora kinases comprise a family of phosphoproteins performing multiple functions in mitosis and meiosis. Because Aurora kinase B (AURKB) expression is altered in aged oocytes and there is only limited information on its function in meiosis, it was decided to study the spatial distribution and co-localization of AURKB with other regulatory proteins at centromeres during mouse oocyte maturation. AURKB associates with chromosomes after germinal vesicle breakdown, is enriched at centromeres from prometaphase I and transits to the spindle midzone at late anaphase I. Preferential inhibition of AURKB by low concentrations of ZM 447439 inhibitor prevents polar body formation and affects spindle formation and chromosome congression at meiosis I, associated with expression of BubR1 checkpoint protein at kinetochores. Release of cohesion between sister chromatids appears inhibited resulting in failure of chiasma resolution in oocytes progressing to anaphase I. Concomitantly, the inhibitor reduces histone H3 lysine 9 trimethylation at centromeric heterochromatin and affects chromosome condensation. The cytokinesis arrest protects young, healthy oocytes from errors in chromosome segregation although increasing polyploidy. This study shows that changes in activity of AURKB may increase risks for chromosome non-disjunction and aneuploidy in mammalian oocytes, irrespective of age.     

整合素β1与FN黏附对卵巢癌A2780细胞增殖及凋亡的影响

目的:观察整合素β1介导的细胞与纤维粘连蛋白(FN)黏附对卵巢癌A2780细胞增殖及凋亡的影响,初步探索整合素β1的信号传导机制。方法:体外培养卵巢癌A2780细胞。实验分组:对照组(多聚赖氨酸铺板)、实验A组(整合素β1的配体FN铺板)、实验B组(FN铺板+整合素β1单克隆抗体)。CCK-8检测3组细胞的增殖率;PI单染技术检测细胞周期;Anexin-V/PI双染检测顺铂诱导的卵巢癌细胞凋亡率;实时定量RT-PCR检测Aurora B及Survivin基因mRNA水平的变化。结果:实验A组与对照组、实验B组比较,细胞增殖率升高,凋亡率下降,S+G2/M期细胞比例增加,Aurora B及Sur-vivin mRNA表达水平升高,均有显著差异(P<0.05)。结论:整合素β1介导的A2780细胞与FN黏附可促进细胞增殖、抑制细胞凋亡,其作用机制可能与诱导Aurora B及Sur-vivin的表达有关。     

Aurora kinase inhibitor AZD1152 has an additional effect of platinum on a sequential application at the human ovarian cancer cell line SKOV3

Purpose

The treatment of ovarian tumors is carried out with platinum medicine which can lead to incompatibilities or resistances. Thus, it is of great interest to check new medicine suitability for its application. AZD1152 is an Aurora kinase inhibitor predominantly works against Aurora kinase B involved in the chromosome segregation. Cells become polyploidy and reduce the proliferation by this impairment. To investigate whether AZD1152, may play a role in the treatment of ovarian carcinoma we serving it to the cisplatinum-resistant cell line SKOV3 alone and in combination with platinum.

Methods

We look at the proliferation, the ploidy, the phases of cell cycle and the apoptosis activity of the cells.

Results and conclusion

We could show that the combination of both medicines in the preclinical experiment produces a working advantage.     

The impact of vitrification on murine germinal vesicle oocyte In vitro maturation and aurora kinase A protein expression

Purpose

Investigate the effect of vitrification on in vitro maturation (IVM) and expression of Aurora kinases A, B, and C in germinal vesicle (GV)-stage oocytes.

Methods

GV-stage oocytes from B6D2F1 female mice 7–11 weeks of age were vitrified after collection, thawed, and matured in vitro for 0, 4, 8, and 12 h (hrs). The rate of germinal vesicle breakdown (GVBD), spindle apparatus assembly, and Aurora kinase mRNA and protein expression during IVM was measured.

Results

Oocyte vitrification was associated with significant delays in both GVBD and normal spindle apparatus assembly at 4 and 8 h of IVM (p < 0.05). There was no difference in mRNA levels between control and vitrified oocytes for any of the Aurora kinases. Aurora A protein levels were reduced in vitrified compared to control oocytes at 0 h (p = 0.008), and there was no difference at 4 and 8 h (p = 0.08 and 0.69, respectively) of IVM.

Conclusions

Vitrified oocytes have delayed GVBD and normal spindle assembly during in vitro maturation. Reduced levels of Aurora A protein immediately post-thaw may be associated with the impaired oocyte maturation manifested by the delayed progression through meiosis I and II, and the atypical timing of the formation of meiotic spindles in vitrified GV-stage oocytes.     

磷脂酰肌醇3激酶在卵巢上皮性癌组织中的表达及其抑制剂对卵巢上皮性癌细胞生长抑制作用的研究

目的 探讨细胞信号转导信使磷脂酰肌醇3激酶(PI3K)在卵巢上皮性癌(卵巢癌)组织中的表达及其意义;并探讨PI3K抑制剂LY294002联合顺铂对卵巢癌细胞株的生长抑制作用.方法 应用蛋白印迹法和RT-PCR技术检测正常卵巢组织(正常组,20份)、卵巢良性上皮性肿瘤组织(良性组,6份)、卵巢交界性上皮性肿瘤组织(交界性组,6份)和卵巢癌组织(卵巢癌组,39份)中PI3K p85亚单位蛋白和mRNA的表达.将SKOV3细胞分为对照组(只加培养液)、LY294002组(加1、10、30、50、100 μmol/L LY294002)、顺铂组(加0.33、1.25、2.5、5、10 μmol/L顺铂)和联合组(加50 μmol/L LY294002+10 μmol/L顺铂),四甲基偶氮唑蓝还原法测定不同浓度的LY294002及顺铂对SKOV3细胞的生长抑制作用.结果 (1)PI3K p85亚单位蛋白的表达阳性率:正常组和良性组均为0,交界性组为2/6,卵巢癌组为85%(33/39),正常组、良性组、交界性组分别与卵巢癌组比较,差异均有统计学意义(P＜0.01).(2)PI3K p85亚单位mRNA的表达水平:正常组为0.178±0.102,良性组为0.643±0.112,交界性组为0.847±0.058,卵巢癌组为1.689±0.423,正常组、良性组、交界性组分别与卵巢癌组比较,差异均有统计学意义(P＜0.01).卵巢癌组织中,PI3K p85亚单位蛋白表达阳性率及mRNA的表达水平,不同年龄、病理类型间比较,差异均无统计学意义(P＞0.05);而不同病理分化程度、手术病理分期间比较,差异均有统计学意义(P＜0.05).(3)LY294002及顺铂呈剂量依赖性地抑制SKOV3细胞的生长.LY294002组(50 μmol/L)、顺铂组(10 μmol/L)、联合组SKOV3细胞的抑制率分别为(46.0±2.0)%、(44.4±3.2)%、(57.1±4.1)%,联合组SKOV3细胞的抑制率高于LY294002组及顺铂组(P＜0.01).结论 PI3K p85亚单位在卵巢癌组织中呈高表达,与病理分化程度、手术病理分期有关.LY294002与顺铂联合用药对卵巢癌细胞生长的抑制具有协同效应.     

Altered endothelin receptor binding in response to nitric oxide synthase inhibition in the pregnant rat

The authors evaluate the expression of endothelin-1 (ET-1) and its receptors in the uterus and placenta during maternal nitric oxide synthase (NOS) inhibition. Timed-pregnant rats received L-NAME (2.5 mg/kg/h) or saline from day 14 to 21 of gestation. Uterine and placental tissues collected on day 21 were assayed for preproET-1, ET( A), and ET(B) mRNA expression; localization and expression of ET-1 and receptor proteins; and receptor activity. NOS inhibition did not affect preproET-1 mRNA expression in the placenta or uterus. ET(A) expression decreased in the uterine free wall, but no other changes in receptor mRNA expression were observed in the uterus or placenta. ET-1 and receptor proteins were unchanged. Placental ET(A) and ET(B) receptor binding decreased. Uterine ET(A) receptor binding decreased in the placental bed. ET-1, a prominent mediator during NOS inhibition, is not of uterine or placental origin. Reduced receptor binding activity is the primary means by which these tissues regulate their response to ET-1 in the setting of NOS inhibition.     

卵巢上皮性癌组织中转移抑制基因ME491/CD63与整合素α5蛋白和mRNA的表达及其意义

目的探讨转移抑制基因ME491/CD63、整合素(integrin)α5蛋白和mRNA在卵巢上皮性癌(卵巢癌)组织中的表达及其意义.方法应用RT-PCR技术和原位杂交方法检测正常卵巢组织(Ⅰ组,30份)、卵巢良性上皮性肿瘤组织(Ⅱ组,10份)、卵巢交界性上皮性肿瘤组织(Ⅲ组,6份)和卵巢癌组织(Ⅳ组,48份)中ME491/CD63及integrin α5蛋白和mRNA的表达,并分析其与患者各临床病理指标的关系.结果 (1)ME491/CD63与integrin α5 mRNA表达水平Ⅰ组均为1.7±0.3,Ⅱ组均为1.5±0.3,Ⅲ组均为1.1±0.5,Ⅳ组均为0.6±0.4.各组间分别比较,差异均有统计学意义(P<0.01).卵巢癌组织中,ME491/CD63和integrin α5 mRNA表达水平,不同年龄、病理类型间比较,差异均无统计学意义(P>0.05);而不同病理分化程度、手术病理分期及有无淋巴结转移间比较,差异均有统计学意义(P<0.01).(2)ME491/CD63和integrin α5蛋白表达阳性率Ⅰ组和Ⅱ组均为100%,Ⅲ组均为50%;Ⅳ组均为31%,其中手术病理分期为Ⅰ期者均为7/7,Ⅱ期均为6/9,Ⅲ～Ⅳ期均为6%.Ⅰ、Ⅱ组分别与Ⅲ、Ⅳ组比较,差异均有统计学意义(P<0.05,P<0.01).卵巢癌组织中,ME491/CD63和integrin α5蛋白表达阳性率,不同年龄、病理类型间比较,差异均无统计学意义(P>0.05);而不同病理分化程度、手术病理分期及有无淋巴结转移间比较,差异均有统计学意义(P<0.05).结论ME491/CD63和integrin α5蛋白和mRNA在卵巢癌组织中呈低表达,其表达缺失与病理分化程度、手术病理分期及淋巴结转移有关.     

整合素连接激酶反义寡核苷酸对卵巢上皮性癌细胞生长的抑制作用

目的 探讨整合素连接激酶(ILK)反义寡核苷酸(ASODN)对卵巢上皮性癌(卵巢癌)细胞生长的抑制作用.方法 将ILK-ASODN转染入卵巢癌细胞株H08910细胞,阻断其表达ILK.实验分为6组,分别为空白对照组(A组)、脂质体对照组(B组)、ILK-正义寡核苷酸(SODN)100 nmol/L组(C组)和ILK-ASODN 60 nmol/L组(D组)、ILK-ASODN 80 nmol/L组(E组)、ILK-ASODN 100 nmol/L组(F组).采用RT-PCR技术和蛋白印迹法检测各组H08910细胞ILK mRNA和蛋白表达量的变化;水溶性四氮唑1(WST-1)法及流式细胞术评价H08910细胞转染ILK-ASODN后对该细胞体外增殖的抑制作用及对该细胞周期和凋亡的影响.结果 ILK-ASODN转染后H08910细胞ILK mRNA表达量明显下降,D、E、F 3个实验组分别为0.307±0.011、0.198±0.008、0,与A、B两对照组(分别为0.343±0.006、0.342±0.009)比较,差异均有统计学意义(P＜0.05).ILK-ASODN转染后H08910细胞ILK蛋白表达量也明显下降,D、E、F 3个实验组分别为26.3±0.8、20.6±0.4、0;细胞生长受到明显抑制,细胞凋亡率明显增高,3个实验组分别为7.31%、8.84%、11.27%;G0/G1期细胞增多,3个实验组分别为49.25%、56.28%、67.61%.以上指标分别与A、B组比较,差异均有统计学意义(P＜0.01).结论 ILK-ASODN转染卵巢癌H08910细胞后可以明显抑制其生长.