JNKs分子在小鼠着床前胚胎中表达、激活及其与细胞凋亡的关系

发布时间：2018-05-20 14:00

本文选题：JNKs + 植入前胚胎　；参考：《华中科技大学》2006年博士论文

【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±446 vs 1297.2±392.3,P0.05): 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(P0.05); 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(P0.05) 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(P0.05) 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,P0.05): 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(P0.05) 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(P0.05) 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。
[Abstract]:Part I: the expression of JNKs protein in pre implantation mouse embryos at different stages.
Objective: to understand the expression pattern of JNKs protein in mouse preimplantation embryos at different stages.
Methods: the experimental animals were Kunming mice. After the ovulation induction, the 0.5 days of pregnancy (zygote), 1.5 days (two cell stage), 2.5 days (fusion stage morula) and 3.5 days (early blastocyst) were obtained respectively. Using JNK polyclonal antibody, the laser confocal technique was combined with immunofluorescence, and the protein level was qualitatively and semi quantified at the level of protein. The expression of JNKs protein in mouse embryos was preliminarily identified.
Results and conclusions:
1. JNKs protein was expressed in the cytoplasm of mouse embryonic cells at all stages.
2. with the development of embryo, the expression of JNKs protein showed an upward trend, in which the 1.5 day of pregnancy (2 cell stage) to 2.5 days of pregnancy (fusion stage morula stage) was the most obvious (fluorescence intensity 3176.9 + 446).
Vs 1297.2 + 392.3, P0.05):
The second part is the activation of JNKs molecules in the environment and the early embryonic cells of mice.
Objective: To investigate the effects of various stimulating factors on the activation of JNKs in mouse embryonic cells.
Method:
1. the mouse embryos (early blastocyst stage) on day 3.5 of the body and in vitro were obtained respectively, and the activation (phosphorylation) level of JNKs molecules in the two cells was compared by immunofluorescence and Western-Blotting detection.
2. the activation level of JNKs in the mouse preimplantation embryos (early blastocyst stage) was compared by immunofluorescence at different temperatures (35 degrees Celsius, 37 degrees centigrade, 39 degrees centigrade) for 9 hours.
3. immunofluorescence method was used to compare the activation level of JNKs molecules in mouse embryo cells cultured in 3 different culture media (CZB, G-1, G-2) for 48 hours.
4. immunofluorescence method was used to compare 3 /25ul, 15-25 /25ul and 50 /ul under different culture densities.
The activation level of JNKs in mouse embryonic cells was cultured after 48 hours.
Result:
1. the activation level of JNKs in the mouse embryo cells in vitro was significantly higher than that in the body. (both the immunofluorescence method and the Western-Bloting method were statistically significant).
2., the activation level of JNKs in mouse embryo cells after 9 hours of culture at 39 degrees Celsius was significantly higher than that in 35 and 37 degrees Celsius (P0.05).
3., the fluorescence density of JNKs molecules activated by CZB medium was significantly higher than that of G-2 medium (P0.05).
4., the content of JNKs molecules activated in the cells increased significantly when the culture density was 50 embryos / droplets in the 25 micro - lift droplets, and the difference was statistically significant compared with the other two groups (P0.05).
Conclusion:
1. in vitro, the activation level of JNKs in mouse embryo cells was higher than that in vivo.
2. the changes in the culture temperature, the composition of the culture medium and the density of culture can affect the activation of JNKs molecules in mouse embryonic cells.
The third part: the activation of JNKs and the development and apoptosis of early mouse embryos.
Objective: To investigate the relationship between activation of JNKs and apoptosis of mouse embryonic cells.
Method:
1. the effects of adding different concentrations (10uM, 20uM and 50uM) in the culture medium or at different times (1.5 to 2.5 days of pregnancy and 2.5 days of pregnancy to 3.5 days of pregnancy) were compared with the specific inhibitor SP600125, which was activated by JNKs, on the rate of blastocyst formation in mouse embryos.
2. the early blastocysts of mice were taken for 3.5 days of pregnancy, and the apoptosis ratio of blastocyst cells in mice after heat treatment (41 degrees centigrade) 6 hours was compared with the TUNEL labeling method.
3. the mouse embryos of 1.5 days of pregnancy were stained with mitochondrial membrane potential fluorescence probe JC-1, and the changes of mitochondrial membrane potential in the embryonic cells after 1 hours of heat treatment (41 degrees centigrade) were compared with or without SP600125.
4. in the same way, the expression level of Caspase-3 protein in mouse embryonic cells after 3 hours of heat treatment was compared with that of SP600125.
Result:
1. after adding different concentrations of SP600125 in the culture medium, the rate of blastocyst formation was increased. When the concentration of SP600125 was 20uM, the rate of blastocyst formation was the highest, up to 72.3%. The formation rate of the blastocyst with SP600125 added to the SP600125 was higher, but the above difference was higher in the culture medium at 1.5-2.5 days of pregnancy (before blastomere fusion) (before blastomere fusion) (after blastomere fusion). The difference was not statistically significant.
2. when adding 20uM concentration of SP600125 in culture medium, the percentage of cells in the blastocyst of heat treated mice decreased significantly (39.27 + 7.15 Vs 64.97 + 8.23, P0.05).
3. when the 20uM concentration of SP600125 was added to the culture medium, after 1 hours of heat treatment, the mitochondrial proportion of the membrane potential loss decreased significantly (P0.05).
4. when the 20uM concentration of SP600125 was added to the culture medium, the expression level of Caspase protein in the embryonic cells decreased significantly after heat treatment for 3 hours (P0.05).
Conclusion:
The activation of 1.JNKs molecules may affect the further developmental potential of mouse embryos.
2. activation of JNKs molecules induced by external stimulation can promote the apoptosis process of mouse embryonic cells.
The mechanism of 3.JNKs promoting embryonic apoptosis may be related to mitochondria dependent apoptotic pathway.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R321-33

【共引文献】