BaP通过Wnt5a信号通路干扰孕鼠围着床期子宫内膜细胞凋亡

发布时间：2018-03-14 09:39

本文选题：苯并芘　切入点：Wnt信号通路　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:苯并芘(BaP)是一种广泛存在于环境中的雌激素干扰物。课题组前期的研究发现,苯并芘暴露会导致小鼠的妊娠率下降,胚胎着床点明显减少,雌孕激素及其受体发生异常改变,证实苯并芘对胚胎着床产生影响,但其的机制还需要进一步探究。胚胎的成功着床取决于胚泡的侵入能力和子宫的功能状态,子宫内膜细胞凋亡的平衡对围着床期子宫功能的建立至关重要,而这个过程涉及多条通路,其中Wnt信号通路是比较受到关注的。本研究旨在探讨苯并芘对围着床期小鼠子宫内膜Wnt信号通路及细胞凋亡的影响,为进一步研究苯并芘的生殖毒性提供实验依据。方法:实验采用前期已经证实的BaP有效剂量0.2mg/kg/d,以灌胃的方式,建立妊娠第1天(D1)至第6天(D6)的染毒孕鼠模型,分别于孕4,5,6天收取子宫内膜材料,进行以下实验:(1)Western blot,免疫组化的方法检测子宫内膜容受性的分子HoxA10的蛋白表达。(2)Real-time PCR,Western blot和IHC检测Wnt5a,β-catenin和GSK-3β的表达情况。(3)TUNEL法检测子宫内膜组织细胞的凋亡,Western blot检测BAX,BCL2,Caspase3表达。体外培养人子宫内膜基质细胞,建立BaP染毒细胞模型,进行以下实验:(1)Western blot检测Wnt信号通路相关基因表达情况,免疫荧光检测β-catenin表达。(2)流式细胞术检测子宫内膜基质细胞凋亡情况及凋亡相关蛋白表达。(3)在BaP染毒同时给予Wnt5a重组蛋白或Wnt5a抑制剂处理,Western blot检测Wnt信号通路相关基因及凋亡相关基因的表达情况结果:(1)苯并芘暴露的小鼠,子宫内膜组织容受性相关基因HoxA10表达下降;Wnt5a和β-catenin蛋白表达上升;抗凋亡基因BCL2表达上升,促凋亡基因BAX表达下降,TUNEL检测子宫内膜细胞的凋亡下降。(2)体外培养人子宫内膜基质细胞经BaP处理后,Wnt信号通路的改变与体内实验基本一致,流式细胞术检测结果显示细胞凋亡率下降。(3)人子宫内膜基质细胞给予Wnt5a重组蛋白处理后,Wnt5a蛋白表达升高,凋亡相关基因表达下降,与BaP处理结果相似;在Wnt5a和BaP处理组分别给予Wnt5a抑制剂处理,可部分恢复苯并芘诱导的上述改变。结论:由上述的实验结果得出结论:苯并芘通过干扰Wnt信号通路中Wnt5a和β-catenin的表达,打破了Wnt信号通路对子宫内膜细胞凋亡平衡的调控。
[Abstract]:Objective: benzopyrene (Benzo pyrene) is a kind of estrogenic interference widely found in the environment. Our previous study found that benzopyrene exposure led to a decrease in pregnancy rate and a significant decrease in embryo implantation sites in mice. The abnormal changes of estrogen and progesterone receptor confirm that benzopyrene has an effect on embryo implantation, but its mechanism needs to be further explored. The successful implantation of embryo depends on the invading ability of blastocyst and the functional state of uterus. The balance of endometrial cell apoptosis is essential to the establishment of uterine function in the peri-bed phase, a process that involves multiple pathways. The effect of benzopyrene on Wnt signaling pathway and apoptosis in endometrium of surrounding bed mice was studied. Methods: in order to further study the reproductive toxicity of benzopyrene, the model of pregnant mice was established by intragastric administration of 0.2mg / kg 路kg / d of BaP, which had been confirmed in the previous period, from the first day of gestation to the sixth day of gestation. Endometrial materials were collected on the 4th and 6th day of pregnancy. The protein expression of endometrial receptive molecule HoxA10 was detected by immunohistochemistry. Western blot and IHC were used to detect the expression of Wnt5a, 尾 -catenin and GSK-3 尾. Western blot was used to detect the apoptosis of endometrial tissue cells. Western blot was used to detect the expression of BAXBCL2Caspase3. Culture of human endometrial stromal cells in vitro, The cell model of BaP was established. The following experiments were carried out to detect the expression of genes related to the Wnt signaling pathway by Western blot. Detection of apoptosis of endometrial stromal cells and expression of apoptosis-related protein by flow cytometry) Detection of Wnt signaling pathway by Wnt5a recombinant protein or Wnt5a inhibitor at the same time of BaP exposure. Expression of genes and apoptosis-related genes in mice exposed to benzo pyrene, The expression of receptivity related genes (HoxA10) and 尾 -catenin (尾 -catenin) in endometrial tissue decreased, and the expression of anti-apoptotic gene (BCL2) increased. The expression of pro-apoptotic gene BAX was decreased and Tunel was used to detect the apoptosis of endometrial cells. (2) the changes of Wnt signaling pathway in human endometrial stromal cells treated with BaP in vitro were basically consistent with those in vivo. The results of flow cytometry showed that the apoptosis rate of human endometrial stromal cells was decreased. (3) the expression of Wnt5a protein increased and the expression of apoptosis-related genes decreased after treated with Wnt5a recombinant protein, which was similar to that of BaP treatment. In Wnt5a and BaP groups, the above changes induced by benzopyrene could be partially recovered by Wnt5a inhibitor treatment. Conclusion: benzopyrene may interfere with the expression of Wnt5a and 尾 -catenin in Wnt signaling pathway by interfering with the above results. It breaks down the regulation of Wnt signaling pathway on the balance of endometrial cell apoptosis.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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