本文選題：苯并芘　切入點(diǎn)：Wnt信號通路　出處：《重慶醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:苯并芘(BaP)是一種廣泛存在于環(huán)境中的雌激素干擾物。課題組前期的研究發(fā)現(xiàn),苯并芘暴露會導(dǎo)致小鼠的妊娠率下降,胚胎著床點(diǎn)明顯減少,雌孕激素及其受體發(fā)生異常改變,證實(shí)苯并芘對胚胎著床產(chǎn)生影響,但其的機(jī)制還需要進(jìn)一步探究。胚胎的成功著床取決于胚泡的侵入能力和子宮的功能狀態(tài),子宮內(nèi)膜細(xì)胞凋亡的平衡對圍著床期子宮功能的建立至關(guān)重要,而這個過程涉及多條通路,其中Wnt信號通路是比較受到關(guān)注的。本研究旨在探討苯并芘對圍著床期小鼠子宮內(nèi)膜Wnt信號通路及細(xì)胞凋亡的影響,為進(jìn)一步研究苯并芘的生殖毒性提供實(shí)驗(yàn)依據(jù)。方法:實(shí)驗(yàn)采用前期已經(jīng)證實(shí)的BaP有效劑量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,分別于孕4,5,6天收取子宮內(nèi)膜材料,進(jìn)行以下實(shí)驗(yàn):(1)Western blot,免疫組化的方法檢測子宮內(nèi)膜容受性的分子HoxA10的蛋白表達(dá)。(2)Real-time PCR,Western blot和IHC檢測Wnt5a,β-catenin和GSK-3β的表達(dá)情況。(3)TUNEL法檢測子宮內(nèi)膜組織細(xì)胞的凋亡,Western blot檢測BAX,BCL2,Caspase3表達(dá)。體外培養(yǎng)人子宮內(nèi)膜基質(zhì)細(xì)胞,建立BaP染毒細(xì)胞模型,進(jìn)行以下實(shí)驗(yàn):(1)Western blot檢測Wnt信號通路相關(guān)基因表達(dá)情況,免疫熒光檢測β-catenin表達(dá)。(2)流式細(xì)胞術(shù)檢測子宮內(nèi)膜基質(zhì)細(xì)胞凋亡情況及凋亡相關(guān)蛋白表達(dá)。(3)在BaP染毒同時給予Wnt5a重組蛋白或Wnt5a抑制劑處理,Western blot檢測Wnt信號通路相關(guān)基因及凋亡相關(guān)基因的表達(dá)情況結(jié)果:(1)苯并芘暴露的小鼠,子宮內(nèi)膜組織容受性相關(guān)基因HoxA10表達(dá)下降;Wnt5a和β-catenin蛋白表達(dá)上升;抗凋亡基因BCL2表達(dá)上升,促凋亡基因BAX表達(dá)下降,TUNEL檢測子宮內(nèi)膜細(xì)胞的凋亡下降。(2)體外培養(yǎng)人子宮內(nèi)膜基質(zhì)細(xì)胞經(jīng)BaP處理后,Wnt信號通路的改變與體內(nèi)實(shí)驗(yàn)基本一致,流式細(xì)胞術(shù)檢測結(jié)果顯示細(xì)胞凋亡率下降。(3)人子宮內(nèi)膜基質(zhì)細(xì)胞給予Wnt5a重組蛋白處理后,Wnt5a蛋白表達(dá)升高,凋亡相關(guān)基因表達(dá)下降,與BaP處理結(jié)果相似;在Wnt5a和BaP處理組分別給予Wnt5a抑制劑處理,可部分恢復(fù)苯并芘誘導(dǎo)的上述改變。結(jié)論:由上述的實(shí)驗(yàn)結(jié)果得出結(jié)論:苯并芘通過干擾Wnt信號通路中Wnt5a和β-catenin的表達(dá),打破了Wnt信號通路對子宮內(nèi)膜細(xì)胞凋亡平衡的調(diào)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R114

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Molsidomine and N-omega-nitro-L-arginine methyl ester inhibit implantation and apoptosis in mouse endometrium[J];Acta Pharmacologica Sinica;2003年12期

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本文編號：1610644