【作者】 李舟；

【导师】 朱桂金；

【作者基本信息】 华中科技大学 ， 妇产科学， 2006， 博士

【摘要】 第一部分:JNKs蛋白在各阶段植入前小鼠胚胎中的表达目的:了解JNKs蛋白在各阶段小鼠着床前胚胎中的表达规律:方法:实验动物为昆明种小鼠,经过药物促排卵后,分别获取妊娠0.5天(合子),1.5天(二细胞期),2.5天(融合期桑椹胚),3.5天(早期囊胚)的胚胎;使用JNK多克隆抗体,通过免疫荧光法结合激光共聚焦技术,在蛋白水平上定性且半定量检测个阶段鼠胚内JNKs蛋白的表达,并且初步找出其中规律结果与结论:1.在各阶段的小鼠胚胎细胞的胞浆内均有JNKs蛋白的表达;2.随着胚胎的发育,JNKs蛋白表达呈上升趋势,其中在妊娠1.5天(2细胞阶段)到妊娠2.5天(融合期桑椹胚阶段)上升最为明显(荧光强度3176.9±446vs 1297.2±392.3,P<0.05):第二部分外界培养环境与小鼠早期胚胎细胞内JNKs分子的激活目的:探讨体外培养环境及其内各种刺激因素对小鼠胚胎细胞内JNKs分子激活的影响方法:1.分别获取体内及体外的妊娠第3.5天的小鼠胚胎(早期囊胚阶段),以免疫荧光和Western-Blotting的检测方法,比较两者细胞内JNKs分子的激活(磷酸化)水平;2.以免疫荧光法比较不同温度下(35摄氏度,37摄氏度,39摄氏度)培养9小时后小鼠着床前胚胎(早期囊胚期)细胞内JNKs分子的激活水平。3.以免疫荧光法比较在3种不同培养液(CZB,G-1,G-2)中培养48小时后的小鼠胚胎细胞内JNKs分子的激活水平;4.以免疫荧光法比较在不同培养密度下(<3个/25ul;15-25个/25ul与>50个/ul)培养48小时后小鼠胚胎细胞内JNKs分子的激活水平。结果:1.体外培养的小鼠胚胎细胞内JNKs分子的激活水平显著高于体内发育者。(免疫荧光法与Western-Bloting法比较均有统计学意义)2.而在39摄氏度培养下9小时后的鼠胚细胞内JNKs分子的激活水平明显高于35与37摄氏度组(P<0.05);3.而使用CZB培养液培养的胚胎细胞内激活的JNKs分子的荧光密度显著高于G-2培养液组(P<0.05)4.而在25微升的液滴中,当培养密度〉50个胚胎/液滴时,细胞内激活的JNKs分子的含量显著增加,较另外两组差异有统计学意义(P<0.05)结论:1.体外培养的小鼠胚胎细胞内JNKs分子的激活水平高于体内发育者;2.培养温度,培养液的成分以及培养密度的变化局可以影响小鼠胚胎细胞内JNKs分子的激活。第三部分:JNKs分子的激活与小鼠早期胚胎的发育与凋亡目的:探讨JNKs分子的激活与小鼠胚胎细胞凋亡之间的关系:方法:1.比较在培养液内加入不同浓度(10uM,20uM与50uM)或在不同时间内(妊娠1.5天到2.5天与妊娠2.5天到妊娠3.5天)加入JNKs激活的特异性抑制剂SP600125对小鼠胚胎囊胚形成率的影响;2.取妊娠3.5天的小鼠早期囊胚,以TUNEL标记法比较培养液加入或不加入SP600125的情况下,热处理(41摄氏度)6小时后小鼠囊胚细胞的凋亡比例;3.取妊娠1.5天的小鼠胚胎,以线粒体膜电位荧光探针JC-1染色法,比较加入或不加入SP600125的情况下,热处理(41摄氏度)1小时后胚胎细胞内线粒体膜电位的改变;4.同上法,比较加入或不加入SP600125的情况下,热处理3小时后小鼠胚胎细胞内Caspase-3蛋白表达水平的差异;结果:1.培养液中添加不同浓度SP600125后,囊胚形成率有所上升,其中当SP600125浓度为20uM是囊胚形成率最高,达72.3%,培养液中在妊娠1.5-2.5天时(卵裂球融合前)添加SP600125比妊娠2.5-3.5天时(卵裂球融合后)添加SP600125的囊胚形成率更高,但以上差异均未见统计学意义;2.在培养液中加入20uM浓度的SP600125时,可显著降低热处理后小鼠囊胚中调亡的细胞比例(39.27±7.15 Vs 64.97±8.23,P<0.05):3.在培养液中加入20uM浓度的SP600125时,热处理1小时后胚胎膜电位丧失的线粒体比例显著下降(P<0.05)4.在培养液中加入20uM浓度的SP600125时,热处理后3小时的胚胎细胞内Caspase蛋白表达的水平明显下降(P<0.05)结论:1.JNKs分子的激活可能影响小鼠胚胎进一步的发育潜力;2.外界刺激引起的JNKs分子的激活可以促进小鼠胚胎细胞的凋亡过程;3.JNKs促进胚胎细胞凋亡的机制可能与线粒体依赖的凋亡途径有关。更多还原

【Abstract】 PART I. Expression of JNKs Proteins in Different Stages of Preimplantation Mouse EmbryosObjective: To investigate the expression of JNKs proteins in different stages of preimplantation mouse embryos.Materials and Methods:E0.5,1.5,2.5and 3.5 mouse embryos were gathered after PMSG and HCG administration. Immunofluorescence and laser confocal microcopy technique were applied to detect the expression of JNKs protein in different stage embryos.Result and Conclusion:1. JNKs protein were expressed in all stages of preimplantation mouse embryos;2. Expression of the protein were increased with the embryos stage progressed., and this increase was significant in E1.5-E2.5(P<0.05);PART II. The Influence of In-Vitro Environment and Stress from the Culture System to the Activation of JNKs ProteinObjective: To investigate the influence of In-Vitro environment and stress from the culture system to the activation of JNKs protein;Materials and Methods: Immunofluorescence and Wester-Blotting technique were used to compared the activation (phosphorylation) of JNKs protein in mouse embryos grow in vitro or in vivo; Immunofluorescencewas used to compared the activated level of JNKs protein in different culture contion as following: (1)Culture in different temperature (35°c,37 °c and 39 °c);(2)Culture in different media(CZB,G-l and G-2);(3)Cultured I different density(<3 embryos/25ul, 15-25 embryos/25ul and >50 embryo/25ul)Result: (1)Embryo grew in vitro seemed in a higher level of JNKs activation than the ones grew in vivo;(2) Embryo cultured in 39c seemed in a higher level of JNKs activation than the ones grew in other temperatures;(3) Embryo cultured in CZB seemed in a higher level of JNKs activation than the ones grew in other media; (4) Embryo cultured in a density of >50/ul showed a higher level of JNKs activation than the ones cultured in other density.Conclusion: (1)JNKs protein were more easily activated in embryos grew in vitro than in vivo;(2)Stress from culture system had a influence in activation of JNKs proteins. PART III. Relationship of JNKs Activation and Apoptosis of Mouse Embryo cellObjective: To investigate weather and how JNKs activation contributed to thedevelopment and apoptosis of mouse embryo cell.Materials and Methods: (1)Compare the addition of SP600125,a specific inhibitor of JNKs activation, in different concentration or different time to the development of mouse embryo development; (2)Detect the influence of Sp600125 to the apoptosis rate of blastocyst cells suffered in a heat shock of 41c ,6h.;(3)Detect the influence of SP600125 to the mitochondrial membrane potential of the E1.5 mouse embryos suffer a heat shock of 41c ,lh;(4)Detect the influence of SP600125 to Caspase-3 level in E1.5 mouse embryos suffer a heat shock of 41c ,3h;Result: (1) Embryos treated bySP600125 in a concentration of 20uM and in E1.5-2.5 show a better blastcyst formation rate than others; (2)Blastcyst treated by SP600125 showed a significant lower apoptosis rate than positive control after a heat shock of 6h; (3) E1.5 mouse embryos treated by SP600125 have more high membrane potential mitochondrial than positive control after heat shock of 1h; (4) E1.5 mouse embryos treated by SP600125 showed a higher expressed level of Caspase-3 than positive control after heat shock of 3h;Conclusion: Activation of JNKs protein perhaps negatively influence the development potential of preimplantation mouse embryos and can promote the apoptosis program , which had a relationship with the mitochondrial-dependent parthway.更多还原