本文選題：三丁基錫 + 凋亡��； 參考：《浙江大學(xué)》2012年博士論文

【摘要】：三丁基錫(Tributyltin, T B T)是具有極強毒性的環(huán)境污染物,已經(jīng)有大量報道顯示TBT能引起哺乳類動物免疫、神經(jīng)、生殖以及肝毒性,而凋亡的誘導(dǎo)一直被認為是TBT產(chǎn)生毒性最主要的機制。以往大多數(shù)研究都將TBT誘導(dǎo)凋亡的機制集中在Bax和Bcl-2調(diào)控的線粒體凋亡途徑。然而,凋亡的調(diào)控是一個錯綜復(fù)雜的網(wǎng)絡(luò),越來越多研究指出在這條通路的上游以及轉(zhuǎn)導(dǎo)的過程還受到其它信息分子的調(diào)控,尚待更深入探究。此外,以往對TBT誘導(dǎo)凋亡機制的探討多采用體外培養(yǎng)的細胞模型,很少有研究將體外研究結(jié)果延伸到動物活體實驗作進一步證實。 本研究首先以人羊膜FL細胞為靶細胞,在TBT誘導(dǎo)凋亡中,圍繞蛋白磷酸酶2A(otein phosphatase2A, PP2A)檢測與其在凋亡調(diào)控中密切相關(guān)的因素如微管骨架和MAPKs家族的改變、探討PP2A對微管骨架和MAPKs調(diào)控作用,并進一步確定參與TBT誘導(dǎo)凋亡的MAPKs家族成員及其作用的靶分子。在此基礎(chǔ)上,以小鼠肝臟為靶器官,將體外實驗的結(jié)果在體內(nèi)進行更進一步證實,通過體內(nèi)外實驗結(jié)果的比較,確定TBT細胞毒性作用的靶分子,為TBT致毒機理闡述提供有力的理論依據(jù)。 主要結(jié)果： 1.TBT在誘導(dǎo)FL細胞凋亡中,明顯抑制PP2A酶活性,并下調(diào)其調(diào)節(jié)亞基B55α表達和催化亞基C Leu309位點甲基化修飾水平。 2.TBT促使PP2A從微管解離,并導(dǎo)致FL細胞微管骨架解聚。 3.TBT活化FL細胞JNK和p38及其下游轉(zhuǎn)錄因子c-Jun和ATF-2。 4.TBT通過下調(diào)FL細胞Raf表達,使其下游激酶MEK1/2和ERK1/2以及轉(zhuǎn)錄因子c-Myc脫磷酸,導(dǎo)致ERK通路被抑制。 5.抑制JNK的活化能夠明顯抑制TBT對FL細胞凋亡的誘導(dǎo),但p38被抑制對此過程沒有影響。 6.抑制JNK的活化明顯抑制TBT對caspase-3的激活,但對Bcl-2的表達和磷酸化水平,以及Bax表達和定位沒有影響。 7.經(jīng)口攝入TBT抑制小鼠肝細胞PP2A酶活性,并使小鼠肝細胞ROS水平升高。 8.經(jīng)口攝入TBT激活小鼠肝細胞ERK、JNK和p38MAPKs。 9.經(jīng)口攝入TBT誘導(dǎo)小鼠肝細胞凋亡主要體現(xiàn)在DNA鏈的斷裂(TUNEL法)、染色質(zhì)濃縮并發(fā)生邊聚化、以及凋亡關(guān)鍵調(diào)控蛋白Bax/Bcl-2比值上調(diào)和執(zhí)行酶caspase-3激活。 主要結(jié)論： 1在體內(nèi)和體外研究中,TBT誘導(dǎo)凋亡的方式不同。體外實驗中TBT誘導(dǎo)FL細胞可能至少部分以線粒體非依賴途徑發(fā)生凋亡,而在體內(nèi)實驗TBT誘導(dǎo)小鼠肝細胞發(fā)生線粒體依賴途徑凋亡。 2TBT在體內(nèi)和體外研究中誘導(dǎo)凋亡方式的不同必然與體內(nèi)外凋亡的調(diào)控機制相關(guān)。體外實驗TBT誘導(dǎo)FL細胞凋亡主要通過PP2A的抑制和隨之引起微管解聚以及JNK/c-Jun的活化,活化的JNK/c-Jun直接激活caspase-3誘導(dǎo)凋亡；而體內(nèi)實驗TBT誘導(dǎo)肝細胞凋亡的可能機制是PP2A的抑制和ROS的上調(diào)共同激活MAPKs家族,尤其是JNK,最終通過Bax/Bcl-2介導(dǎo)的線粒體途徑啟動凋亡。 3比較體內(nèi)外研究結(jié)果,發(fā)現(xiàn)PP2A的抑制和JNK的活化在TBT誘導(dǎo)的體內(nèi)外凋亡中均起重要作用,而且可能處于凋亡途徑的上游,是TBT細胞毒性作用的關(guān)鍵靶分子。
[Abstract]:Three Tributyltin (T B T) is a highly toxic environmental pollutant. There have been a large number of reports that TBT can cause immunity, nerve, reproduction and liver toxicity of mammalian animals. The induction of apoptosis has been considered as the most important mechanism for the toxicity of TBT. Most of the previous studies have focused on the mechanism of TBT induced apoptosis in Bax and B. Cl-2 regulates the apoptosis pathway of mitochondria. However, the regulation of apoptosis is an intricate network. More and more studies have pointed out that the upstream and transduction processes of this pathway are also regulated by other information molecules. In addition, in the past, the mechanism of apoptosis induced by TBT is mostly used in vitro cultured cell models. Few studies have extended the in vitro study to animal living experiments for further confirmation.
In this study, human amniotic FL cells are used as the target cells. In the TBT induced apoptosis, the factors that are closely related to the protein phosphatase 2A (otein phosphatase2A, PP2A), such as the microtubule skeleton and the MAPKs family, are closely related to the apoptosis regulation, and the regulation of PP2A to microtubule skeleton and MAPKs is discussed, and the M on TBT to induce apoptosis is further determined. APKs family members and their target molecules. On this basis, the mouse liver as the target organ, the results of the experiment in vitro are further confirmed in the body. Through the comparison of the experimental results in vitro and in vivo, the target molecules of the toxicity of TBT cells are determined, which provides a powerful theoretical basis for the toxic mechanism of TBT.
Main results:
1.TBT significantly inhibited the activity of PP2A enzyme in the induction of FL cell apoptosis, and down regulated the expression of regulatory subunit B55 alpha and the methylation level of the catalytic subunit C Leu309 site.
2.TBT causes PP2A to dissociate from microtubules and leads to depolymerization of FL cell microtubules.
3.TBT activated FL cells JNK and p38 and their downstream transcription factors c-Jun and ATF-2.
4.TBT depresses phosphorylation of downstream kinase MEK1/2 and ERK1/2 and transcription factor c-Myc by downregulating the expression of Raf in FL cells, resulting in inhibition of ERK pathway.
5. inhibition of JNK activation significantly inhibited TBT induced apoptosis in FL cells, but p38 inhibited the process.
6. inhibition of JNK activation significantly inhibited the activation of Caspase-3 by TBT, but had no effect on Bcl-2 expression and phosphorylation level, as well as Bax expression and localization.
7. oral intake of TBT inhibited the activity of PP2A enzyme and increased the level of ROS in mouse hepatocytes.
8. oral intake of TBT activates mouse hepatocytes ERK, JNK and p38MAPKs..
9. the hepatocyte apoptosis induced by oral intake of TBT was mainly manifested in the DNA strand breaks (TUNEL), chromatin concentration and polycondensation, and the up regulation of the key regulatory protein Bax/Bcl-2 ratio and the activation of the enzyme caspase-3.
The main conclusions are as follows:
1 in the study in vivo and in vitro, TBT induced apoptosis in different ways. In vitro, TBT induced FL cells to apoptosis at least partly by mitochondrial non dependent pathway, while TBT induced mitochondria dependent apoptosis in mouse hepatocytes in vivo.
The different ways of inducing apoptosis in 2TBT in vivo and in vitro are bound to be related to the regulation mechanism of apoptosis in vitro and in vitro. In vitro, TBT induced apoptosis of FL cells mainly through the inhibition of PP2A and the consequent depolymerization of microtubules and the activation of JNK/c-Jun. Activated JNK/c-Jun directly activates caspase-3 to induce apoptosis; in vivo experiment TBT induces the liver. The possible mechanism of apoptosis is the inhibition of PP2A and the up-regulation of ROS to co activate the MAPKs family, especially JNK, and eventually initiate apoptosis through the mitochondrial pathway mediated by Bax/Bcl-2.
3 comparing the results in vivo and in vivo, it is found that the inhibition of PP2A and the activation of JNK play an important role in TBT induced apoptosis in vitro and in vivo, and may be in the upstream of the apoptotic pathway, which is the key target for the toxicity of TBT cells.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R114

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相關(guān)期刊論文 前1條

1 汪保安;李明;母義明;呂朝暉;李江源;;環(huán)境污染物三丁基氯化錫和氯化三苯錫對大鼠睪丸Leydig細胞的影響[J];中華男科學(xué)雜志;2006年06期

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本文編號：1935519