微丝相关的微囊藻毒素毒性机理研究
发布时间：2018-09-19 18:05
【摘要】：目的：水体富营养化导致蓝藻水华频繁发生。蓝藻水华不仅严重降低水体利用价值，而且很多蓝藻（主要是铜绿微囊藻）还能产生毒素——微囊藻毒素（Microcystin, MC），对人类健康构成威胁，其中存在较多、毒性较大的是微囊藻毒素LR（Microcystin-LR, MC-LR）和RR。同位素示踪发现MC进入动物体内后主要分布在肝脏，提示肝脏可能是它的主要靶器官，可引起肝细胞损伤、原发性肝癌、肝细胞肿胀和坏死。MC肝脏毒性的分子机制到目前为止已有了比较广泛的研究，也取得了一定的研究成果，但其具体机制仍存在很多疑问，尤其是细胞骨架在MC毒性效应中的作用。因此，我们选择正常人来源的肝细胞系HL7702作为研究对象来研究细胞骨架中微丝参与MC-LR的具体毒性机理。 方法：1、用MTT法检测MC-LR对HL7702细胞增殖的影响，不同浓度的MC-LR（0-10μM）染毒处理HL7702细胞。然后，，根据MTT结果确定后续实验的染毒浓度。 2、用微丝特异染色剂荧光标记的鬼笔环肽对细胞进行染色，在激光共聚焦显微镜下观察其形态的变化。 3、再用荧光定量PCR和蛋白免疫印迹法分析微丝相关基因转录、翻译和磷酸化水平的变化，以及MAPK信号通路在此过程中的作用。 结果：1、不同浓度MC-LR（0、0.001、0.01、0.1、1、10μM）暴露24h后，HL7702细胞的数量随着MC-LR染毒浓度的升高而逐渐减少，在染毒浓度达到1μM时开始有统计学意义。 2、MC-LR处理后，HL7702细胞的大小及形态仍保持完整，但微丝的正常纤维状结构逐渐消失，可见明显的致密束形成，微丝结构破坏、重组。 3、MC-LR处理后，微丝相关基因的转录和翻译水平没有发生明显改变，也不具有统计学意义。 4、MC-LR处理细胞后，微丝相关蛋白的磷酸化水平发生明显改变，p-VASP（Ser239）、p-VASP（Ser157）和p-Ezrin（Thr567）在染毒浓度达到10μM时显著升高并具有统计学意义，p-Cofilin（Ser3）无明显改变。 5、MC-LR处理后，MAPK信号通路中P38、ERK1/2和JNK的蛋白表达没有明显改变，但其磷酸化水平与MC-LR有剂量依赖关系，在高浓度时显著升高并具有统计学意义。 6、P38和ERK1/2抑制剂组的蛋白磷酸化水平向对照组恢复，与不加抑制剂的MC-LR处理组相比显著降低并具有统计学意义，说明P38和ERK1/2抑制剂能显著抑制MC-LR诱导的微丝相关蛋白过磷酸化，而JNK抑制剂无明显效果。 结论：1、MC-LR能抑制HL7702细胞增殖，并引起微丝结构的紊乱和重组。 2、MC-LR可引起微丝相关蛋白VASP和Ezrin过磷酸化。 3、MC-LR可激活MAPK信号通路，且该信号通路中的P38和ERK1/2参与了MC-LR诱导的微丝相关蛋白过磷酸化。 4、MC-LR的肝细胞毒性可能是通过PP2A抑制、MAPK信号通路（尤其是P38和ERK1/2）的激活，引起微丝相关蛋白的过磷酸化，最终导致微丝结构的破坏和重组、肝细胞损伤。
[Abstract]:Objective: water eutrophication leads to frequent occurrence of cyanobacteria Shui Hua. The cyanobacteria Shui Hua not only seriously reduces the water use value, but also many cyanobacteria (mainly microcystis aeruginosa) can produce the toxin, microcystins (Microcystin, MC), which is a threat to human health. Microcystins LR (Microcystin-LR, MC-LR) and RR. are more toxic. Isotopic tracer showed that MC was mainly distributed in the liver after entering the animal body, suggesting that the liver might be its main target organ, which could cause hepatocyte damage and primary liver cancer. The molecular mechanism of hepatocyte swelling and necrosis. The molecular mechanism of hepatotoxicity has been extensively studied, and some research results have been made, but there are still many questions about its specific mechanism. Especially the role of cytoskeleton in MC toxicity. Therefore, we selected the normal human liver cell line HL7702 as the research object to study the specific mechanism of the involvement of microfilaments in the cytoskeleton of MC-LR. Methods MTT assay was used to detect the effect of MC-LR on the proliferation of HL7702 cells. HL7702 cells were treated with different concentrations of MC-LR (0-10 渭 M). Then, according to the results of MTT, the concentration of the following experiment was determined. 2. The cells were stained with the fluorescence labeled phalanophorin, a microfilament-specific staining agent. The morphologic changes of microfilaments were observed under confocal laser microscope. 3. The changes of transcription, translation and phosphorylation of microfilament-related genes were analyzed by fluorescence quantitative PCR and Western blotting. And the role of MAPK signaling pathway in this process. Results the number of HL7702 cells gradually decreased with the increase of MC-LR concentration after 24 hours of exposure to different concentrations of MC-LR (0. 001 / 0. 01) and 10 渭 M (0. 001 / 0. 01) and began to have statistical significance when the concentration reached 1 渭 M. the size and morphology of HL-7702 cells remained intact after treatment with MC-LR. However, the normal fibrous structure of the microfilaments gradually disappeared, and obvious dense bundles were formed, the microfilament structure was destroyed, and the transcriptional and translation levels of the microfilament-related genes were not significantly changed after the treatment of MC-LR. The cells were treated with MC-LR. The phosphorylation level of microfilament-associated proteins was significantly changed when the concentration of p-VASP (Ser239) p-VASP (Ser157) and p-Ezrin (Thr567) reached 10 渭 M. there was no significant change in the phosphorylation of p-Cofilin (Ser3). 5The protein surface of P38ERK1 / 2 and JNK in the signal pathway of P38ERK1 / 2 and JNK was treated with MC-LR. Da has not changed significantly. However, the phosphorylation level of P38 and ERK1/2 inhibitor group was significantly increased in a dose-dependent manner, and was significantly increased at high concentration. 6 the protein phosphorylation level of P38 and ERK1/2 inhibitor groups recovered to the control group. P38 and ERK1/2 inhibitors could significantly inhibit MC-LR induced microfilament-associated protein hyperphosphorylation, but JNK inhibitors had no significant effect. Conclusion MC-LR can inhibit the proliferation of HL7702 cells and cause the disruption and recombination of microfilament structure. 2MC-LR can induce the hyperphosphorylation of VASP and Ezrin, and MC-LR can activate the MAPK signaling pathway. The P38 and ERK1/2 involved in the MC-LR induced hyperphosphorylation of microfilament-associated protein. 4 the hepatocytotoxicity of MC-LR may be due to the inhibition of the activation of MAPK signaling pathway (especially P38 and ERK1/2) by PP2A. It leads to the hyperphosphorylation of microfilament-associated proteins, resulting in the destruction and recombination of microfilament structures and liver cell damage.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R96
本文编号：2250916
[Abstract]:Objective: water eutrophication leads to frequent occurrence of cyanobacteria Shui Hua. The cyanobacteria Shui Hua not only seriously reduces the water use value, but also many cyanobacteria (mainly microcystis aeruginosa) can produce the toxin, microcystins (Microcystin, MC), which is a threat to human health. Microcystins LR (Microcystin-LR, MC-LR) and RR. are more toxic. Isotopic tracer showed that MC was mainly distributed in the liver after entering the animal body, suggesting that the liver might be its main target organ, which could cause hepatocyte damage and primary liver cancer. The molecular mechanism of hepatocyte swelling and necrosis. The molecular mechanism of hepatotoxicity has been extensively studied, and some research results have been made, but there are still many questions about its specific mechanism. Especially the role of cytoskeleton in MC toxicity. Therefore, we selected the normal human liver cell line HL7702 as the research object to study the specific mechanism of the involvement of microfilaments in the cytoskeleton of MC-LR. Methods MTT assay was used to detect the effect of MC-LR on the proliferation of HL7702 cells. HL7702 cells were treated with different concentrations of MC-LR (0-10 渭 M). Then, according to the results of MTT, the concentration of the following experiment was determined. 2. The cells were stained with the fluorescence labeled phalanophorin, a microfilament-specific staining agent. The morphologic changes of microfilaments were observed under confocal laser microscope. 3. The changes of transcription, translation and phosphorylation of microfilament-related genes were analyzed by fluorescence quantitative PCR and Western blotting. And the role of MAPK signaling pathway in this process. Results the number of HL7702 cells gradually decreased with the increase of MC-LR concentration after 24 hours of exposure to different concentrations of MC-LR (0. 001 / 0. 01) and 10 渭 M (0. 001 / 0. 01) and began to have statistical significance when the concentration reached 1 渭 M. the size and morphology of HL-7702 cells remained intact after treatment with MC-LR. However, the normal fibrous structure of the microfilaments gradually disappeared, and obvious dense bundles were formed, the microfilament structure was destroyed, and the transcriptional and translation levels of the microfilament-related genes were not significantly changed after the treatment of MC-LR. The cells were treated with MC-LR. The phosphorylation level of microfilament-associated proteins was significantly changed when the concentration of p-VASP (Ser239) p-VASP (Ser157) and p-Ezrin (Thr567) reached 10 渭 M. there was no significant change in the phosphorylation of p-Cofilin (Ser3). 5The protein surface of P38ERK1 / 2 and JNK in the signal pathway of P38ERK1 / 2 and JNK was treated with MC-LR. Da has not changed significantly. However, the phosphorylation level of P38 and ERK1/2 inhibitor group was significantly increased in a dose-dependent manner, and was significantly increased at high concentration. 6 the protein phosphorylation level of P38 and ERK1/2 inhibitor groups recovered to the control group. P38 and ERK1/2 inhibitors could significantly inhibit MC-LR induced microfilament-associated protein hyperphosphorylation, but JNK inhibitors had no significant effect. Conclusion MC-LR can inhibit the proliferation of HL7702 cells and cause the disruption and recombination of microfilament structure. 2MC-LR can induce the hyperphosphorylation of VASP and Ezrin, and MC-LR can activate the MAPK signaling pathway. The P38 and ERK1/2 involved in the MC-LR induced hyperphosphorylation of microfilament-associated protein. 4 the hepatocytotoxicity of MC-LR may be due to the inhibition of the activation of MAPK signaling pathway (especially P38 and ERK1/2) by PP2A. It leads to the hyperphosphorylation of microfilament-associated proteins, resulting in the destruction and recombination of microfilament structures and liver cell damage.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R96
【参考文献】
相关期刊论文 前4条
1 徐浩翔;闫言;;波形蛋白表达和功能的研究进展[J];北京大学学报(医学版);2009年05期
2 潘燕;韩婧;张晔;李学军;;Vimentin在肿瘤转移中的作用及药物研究进展[J];生理科学进展;2010年06期
3 傅文宇,王咪咪,徐立红;微囊藻毒素LR对大鼠肝细胞Caspase-3酶活性的影响[J];水生生物学报;2004年04期
4 傅文宇;徐立红;;微囊藻毒素致毒机理的研究进展[J];浙江大学学报(医学版);2006年03期
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