微絲相關(guān)的微囊藻毒素毒性機(jī)理研究
發(fā)布時(shí)間：2018-09-19 18:05
【摘要】：目的：水體富營(yíng)養(yǎng)化導(dǎo)致藍(lán)藻水華頻繁發(fā)生。藍(lán)藻水華不僅嚴(yán)重降低水體利用價(jià)值，而且很多藍(lán)藻（主要是銅綠微囊藻）還能產(chǎn)生毒素——微囊藻毒素（Microcystin, MC），對(duì)人類健康構(gòu)成威脅，其中存在較多、毒性較大的是微囊藻毒素LR（Microcystin-LR, MC-LR）和RR。同位素示蹤發(fā)現(xiàn)MC進(jìn)入動(dòng)物體內(nèi)后主要分布在肝臟，提示肝臟可能是它的主要靶器官，可引起肝細(xì)胞損傷、原發(fā)性肝癌、肝細(xì)胞腫脹和壞死。MC肝臟毒性的分子機(jī)制到目前為止已有了比較廣泛的研究，也取得了一定的研究成果，但其具體機(jī)制仍存在很多疑問(wèn)，尤其是細(xì)胞骨架在MC毒性效應(yīng)中的作用。因此，我們選擇正常人來(lái)源的肝細(xì)胞系HL7702作為研究對(duì)象來(lái)研究細(xì)胞骨架中微絲參與MC-LR的具體毒性機(jī)理。 方法：1、用MTT法檢測(cè)MC-LR對(duì)HL7702細(xì)胞增殖的影響，不同濃度的MC-LR（0-10μM）染毒處理HL7702細(xì)胞。然后，，根據(jù)MTT結(jié)果確定后續(xù)實(shí)驗(yàn)的染毒濃度。 2、用微絲特異染色劑熒光標(biāo)記的鬼筆環(huán)肽對(duì)細(xì)胞進(jìn)行染色，在激光共聚焦顯微鏡下觀察其形態(tài)的變化。 3、再用熒光定量PCR和蛋白免疫印跡法分析微絲相關(guān)基因轉(zhuǎn)錄、翻譯和磷酸化水平的變化，以及MAPK信號(hào)通路在此過(guò)程中的作用。 結(jié)果：1、不同濃度MC-LR（0、0.001、0.01、0.1、1、10μM）暴露24h后，HL7702細(xì)胞的數(shù)量隨著MC-LR染毒濃度的升高而逐漸減少，在染毒濃度達(dá)到1μM時(shí)開(kāi)始有統(tǒng)計(jì)學(xué)意義。 2、MC-LR處理后，HL7702細(xì)胞的大小及形態(tài)仍保持完整，但微絲的正常纖維狀結(jié)構(gòu)逐漸消失，可見(jiàn)明顯的致密束形成，微絲結(jié)構(gòu)破壞、重組。 3、MC-LR處理后，微絲相關(guān)基因的轉(zhuǎn)錄和翻譯水平?jīng)]有發(fā)生明顯改變，也不具有統(tǒng)計(jì)學(xué)意義。 4、MC-LR處理細(xì)胞后，微絲相關(guān)蛋白的磷酸化水平發(fā)生明顯改變，p-VASP（Ser239）、p-VASP（Ser157）和p-Ezrin（Thr567）在染毒濃度達(dá)到10μM時(shí)顯著升高并具有統(tǒng)計(jì)學(xué)意義，p-Cofilin（Ser3）無(wú)明顯改變。 5、MC-LR處理后，MAPK信號(hào)通路中P38、ERK1/2和JNK的蛋白表達(dá)沒(méi)有明顯改變，但其磷酸化水平與MC-LR有劑量依賴關(guān)系，在高濃度時(shí)顯著升高并具有統(tǒng)計(jì)學(xué)意義。 6、P38和ERK1/2抑制劑組的蛋白磷酸化水平向?qū)φ战M恢復(fù)，與不加抑制劑的MC-LR處理組相比顯著降低并具有統(tǒng)計(jì)學(xué)意義，說(shuō)明P38和ERK1/2抑制劑能顯著抑制MC-LR誘導(dǎo)的微絲相關(guān)蛋白過(guò)磷酸化，而JNK抑制劑無(wú)明顯效果。 結(jié)論：1、MC-LR能抑制HL7702細(xì)胞增殖，并引起微絲結(jié)構(gòu)的紊亂和重組。 2、MC-LR可引起微絲相關(guān)蛋白VASP和Ezrin過(guò)磷酸化。 3、MC-LR可激活MAPK信號(hào)通路，且該信號(hào)通路中的P38和ERK1/2參與了MC-LR誘導(dǎo)的微絲相關(guān)蛋白過(guò)磷酸化。 4、MC-LR的肝細(xì)胞毒性可能是通過(guò)PP2A抑制、MAPK信號(hào)通路（尤其是P38和ERK1/2）的激活，引起微絲相關(guān)蛋白的過(guò)磷酸化，最終導(dǎo)致微絲結(jié)構(gòu)的破壞和重組、肝細(xì)胞損傷。
[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.
【學(xué)位授予單位】：寧波大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R96
本文編號(hào)：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.
【學(xué)位授予單位】：寧波大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R96
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