PP2A和JNK在TBT诱导的体内外细胞凋亡中的重要作用

发布时间：2018-05-26 02:03

本文选题：三丁基锡 + 凋亡　；参考：《浙江大学》2012年博士论文

【摘要】：三丁基锡(Tributyltin, T B T)是具有极强毒性的环境污染物,已经有大量报道显示TBT能引起哺乳类动物免疫、神经、生殖以及肝毒性,而凋亡的诱导一直被认为是TBT产生毒性最主要的机制。以往大多数研究都将TBT诱导凋亡的机制集中在Bax和Bcl-2调控的线粒体凋亡途径。然而,凋亡的调控是一个错综复杂的网络,越来越多研究指出在这条通路的上游以及转导的过程还受到其它信息分子的调控,尚待更深入探究。此外,以往对TBT诱导凋亡机制的探讨多采用体外培养的细胞模型,很少有研究将体外研究结果延伸到动物活体实验作进一步证实。本研究首先以人羊膜FL细胞为靶细胞,在TBT诱导凋亡中,围绕蛋白磷酸酶2A(otein phosphatase2A, PP2A)检测与其在凋亡调控中密切相关的因素如微管骨架和MAPKs家族的改变、探讨PP2A对微管骨架和MAPKs调控作用,并进一步确定参与TBT诱导凋亡的MAPKs家族成员及其作用的靶分子。在此基础上,以小鼠肝脏为靶器官,将体外实验的结果在体内进行更进一步证实,通过体内外实验结果的比较,确定TBT细胞毒性作用的靶分子,为TBT致毒机理阐述提供有力的理论依据。主要结果： 1.TBT在诱导FL细胞凋亡中,明显抑制PP2A酶活性,并下调其调节亚基B55α表达和催化亚基C Leu309位点甲基化修饰水平。 2.TBT促使PP2A从微管解离,并导致FL细胞微管骨架解聚。 3.TBT活化FL细胞JNK和p38及其下游转录因子c-Jun和ATF-2。 4.TBT通过下调FL细胞Raf表达,使其下游激酶MEK1/2和ERK1/2以及转录因子c-Myc脱磷酸,导致ERK通路被抑制。 5.抑制JNK的活化能够明显抑制TBT对FL细胞凋亡的诱导,但p38被抑制对此过程没有影响。 6.抑制JNK的活化明显抑制TBT对caspase-3的激活,但对Bcl-2的表达和磷酸化水平,以及Bax表达和定位没有影响。 7.经口摄入TBT抑制小鼠肝细胞PP2A酶活性,并使小鼠肝细胞ROS水平升高。 8.经口摄入TBT激活小鼠肝细胞ERK、JNK和p38MAPKs。 9.经口摄入TBT诱导小鼠肝细胞凋亡主要体现在DNA链的断裂(TUNEL法)、染色质浓缩并发生边聚化、以及凋亡关键调控蛋白Bax/Bcl-2比值上调和执行酶caspase-3激活。主要结论： 1在体内和体外研究中,TBT诱导凋亡的方式不同。体外实验中TBT诱导FL细胞可能至少部分以线粒体非依赖途径发生凋亡,而在体内实验TBT诱导小鼠肝细胞发生线粒体依赖途径凋亡。 2TBT在体内和体外研究中诱导凋亡方式的不同必然与体内外凋亡的调控机制相关。体外实验TBT诱导FL细胞凋亡主要通过PP2A的抑制和随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诱导凋亡；而体内实验TBT诱导肝细胞凋亡的可能机制是PP2A的抑制和ROS的上调共同激活MAPKs家族,尤其是JNK,最终通过Bax/Bcl-2介导的线粒体途径启动凋亡。 3比较体内外研究结果,发现PP2A的抑制和JNK的活化在TBT诱导的体内外凋亡中均起重要作用,而且可能处于凋亡途径的上游,是TBT细胞毒性作用的关键靶分子。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R114

【参考文献】