| Abstract: | Glyphosate 中文名為嘉磷賽，在世界各地被廣泛使用的有機磷除草劑，尤其是針對基改作物，有更好的效果。於2015年3月，世界衛生組織 World Health Organization (WHO) 的國際癌症研究機構 International Agency for Research on Cancer (IARC) 根據流行病學、動物及體外研究判定為可能性人類致癌物2A 級。一些研究表明，攝入大量 glyphosate 除草劑的患者有心律失常，呼吸衰竭，精神惡化和發展低血壓的風險。為了探討 glyphosate 針對免疫系統的急性毒性損傷，本研究中使用小鼠巨噬細胞 RAW 264.7 cells 觀察細胞凋亡及細胞自噬。過去的研究已通過 MTT 測定細胞存活率，初步了解 glyphosate 所誘發的細胞毒性。經流式細胞儀分析，發現 glyphosate 能引發基因毒性、活性氧傷害、粒線體膜電位損傷；本論文首先經流式細胞儀分析，發現 glyphosate 能引發細胞凋亡及壞死、自噬溶酶體表現、表面抗原的活化；再來經由 caspase 活性測定，發現了內外源凋亡路徑的活化，最後通過西方墨點法探討潛在機制，發現glyphosate 能活化Bcl-2 family、p-p53、PARP1、p-JNK、Beclin 1、LC3 I/II、p62。綜合上述顯示 glyphosate 可能通過 ROS 對粒線體造成損傷，粒線體損傷的進一步降低抗凋亡蛋白：Bcl-2 的表現，同時活化了促凋亡蛋白Bax、Bad、Bid，接著通過內源性凋亡路徑活化 caspase -9, -3；另一方面，通過活化死亡家族受體：Fas (CD95) 啟動了 caspase-8,-3外源性凋亡路徑，最後通過凋亡蛋白 p53 及 DNA 修復酶蛋白 PARP 1 的表現量推論 glyphosate 引發基因毒性及細胞凋亡。除了細胞凋亡，本研究於更短時間的傷害內，觀察到了細胞自噬。經預處理 SP600125 (JNK 抑制劑) 後，發現了glyphosate 能通過磷酸化 JNK 促使 Bcl-2 與 Beclin 1 複合體產生解離，活化 Beclin 1 促使 LC3 I 脂化成 LC3 II 及 p62 的啟動，形成自噬囊泡體，再來與溶酶體結合成自噬溶酶體，完成細胞自噬反應。
Glyhosate-containing herbicides are the most frequently used agrochemicals in the world, particularly on genetically modified plants. In march 2015 the World Health Organization''s International Agency for Research on Cancer classified Glyphosate as " probably carcinogenic to humans (Group 2A) " based on epidemiological studies, animal studies, and in vitro studies. Some studies showed that patients who ingested large number of glyhosate herbicides were in danger of arrhythmia, respiratory failure, mental deterioration, and developing hypotension. In order to find out whether glyphosate could induce acute toxicity to the immune system. In this research, apoptosis and autophagy were found in RAW264.7 cells. In my previous study, the cellular viability, intracellular reactive oxygen species, mitochondrial membrane potential, genotoxicity were assessed by MTT assay, DCFH-DA assay, and JC-1 assay, MN assay. In this study, apoptosis, caspase family and death receptor activity were assessed by Annexin-V & Propidium iodide assay, Caspase assay, CD marker (Fas) assay. Respectively, activation of autophagosome and autolysosome , antigen presentation that about autolysosome were assessed by Cyto-ID assay, Acridine orange assay, Lyso-green assay,MHC II assay. The potential mechanism was detected by western blot. We found that glyphosate could activite Bcl-2 family, p-p53, PARP1, p-JNK, Beclin 1, LC3I/II, p62. These results demonstrate that glyhosate probable induce mitochondrial membrane potential destruction by reactive oxygen species. At the same time, we found that the anti-apoptosis protein Bcl-2 production degradation; pro-apoptosis protein : Bax、Bad、Bid activation were upgraded. Next, we found that glyhosate could induce apoptosis intrinsic pathway by activating caspase-9,-3. On the other hand, the death receptor : Fas (CD95) was activated to promote apoptosis extrinsic pathway by activating caspase -8,-3. Finally, we could find p53 phosphorylation and PARP 1 degradation that induced genotoxicity and apoptosis. Glyhosate also induced autophagy in RAW 264.7 cells. In the shorter time, pretreated with SP600125 ,we insured that glyhosate could break the link between Bcl-2 and Beclin 1 by phosphorylation of JNK. Promoting autophagosome relative protein LC3 I/II and P62 activation. Next, autophagosome was fusion with lysosome to become autolysosome. |
