本文選題：微囊藻毒素 + 細(xì)胞凋亡��； 參考：《復(fù)旦大學(xué)》2011年碩士論文

【摘要】：近年來,水華現(xiàn)象在世界各地頻繁發(fā)生,給人類健康帶來嚴(yán)重的威脅。微囊藻毒素(Microcystin, MC)是一類藍(lán)藻水華產(chǎn)生的具有生物毒性的單環(huán)七肽化合物,至今己發(fā)現(xiàn)80余種異構(gòu)體。MC性質(zhì)穩(wěn)定,難以降解,現(xiàn)行自來水處理工藝不能將其有效去除。此外,MC還具有生物富集作用,可通過食物鏈進(jìn)入人體。MC通過飲水或飲食對(duì)人體健康構(gòu)成的危害已經(jīng)引起世界各國的廣泛關(guān)注。MC生物毒性作用的靶器官主要為肝臟。有研究表明,MC的毒性效應(yīng)表現(xiàn)為劑量依賴性的雙向毒性,即高劑量的MC能引起細(xì)胞程序性死亡或凋亡,而低劑量的MC則促進(jìn)細(xì)胞增殖以及腫瘤的形成。由于MC對(duì)人群的危害更多地集中于通過飲水或飲食長期低劑量的暴露,且越來越多的流行病學(xué)資料顯示其與腫瘤發(fā)生的相關(guān)性,因此,MC的慢性毒性尤其是潛在的致癌性成為研究的熱點(diǎn)之一。目前有關(guān)MC的毒性機(jī)制研究大多集中于其誘導(dǎo)細(xì)胞凋亡的分子機(jī)制,而其促癌機(jī)制的研究國內(nèi)外并不多見。動(dòng)物實(shí)驗(yàn)研究結(jié)果顯示,MC是腫瘤發(fā)生的促進(jìn)劑而非引發(fā)劑,而失控的細(xì)胞增殖是在腫瘤形成過程中至關(guān)重要,因此,研究MC的促細(xì)胞增殖效應(yīng)對(duì)于闡明其促癌機(jī)制具有重要意義。 本課題基于目前MC的研究現(xiàn)狀以及存在的問題,選取毒性最強(qiáng)、產(chǎn)生量最大和危害最嚴(yán)重的微囊藻毒素MC-LR,染毒原代大鼠肝臟細(xì)胞,從細(xì)胞生物學(xué)與分子生物學(xué)的角度研究MC-LR對(duì)原代大鼠肝臟細(xì)胞的毒性效應(yīng)。系統(tǒng)性探討了MC-LR特異性肝臟毒性的作用特征,在明確MC-LR具有誘導(dǎo)細(xì)胞凋亡和促進(jìn)細(xì)胞增殖的雙向毒性及其劑量范圍的基礎(chǔ)上,進(jìn)一步分析MC-LR促細(xì)胞增殖效應(yīng)的可能機(jī)制,為闡明MC與肝癌高發(fā)之間的關(guān)系提供實(shí)驗(yàn)基礎(chǔ)和理論依據(jù)；同時(shí),也為進(jìn)一步深入探討MC的促癌分子機(jī)制提供研究線索。 研究采用膠原酶灌流法建立了原代大鼠肝臟細(xì)胞培養(yǎng)模型,通過監(jiān)測(cè)培養(yǎng)液中肝細(xì)胞受損特征性酶譜一谷丙轉(zhuǎn)氨酶(ALT)和谷草轉(zhuǎn)氨酶(AST)的水平,以及對(duì)肝細(xì)胞表面特異性標(biāo)志物的分析,證實(shí)分離得到的肝細(xì)胞完整性良好且純度達(dá)到近100%。從而建立了適用于課題研究的實(shí)驗(yàn)?zāi)Ｐ?為后續(xù)實(shí)驗(yàn)現(xiàn)象的觀察和指標(biāo)的測(cè)定奠定了基礎(chǔ)。 通過染毒10-5～10-12 mol/L梯度濃度的MC-LR,觀察其在較長染毒時(shí)間范圍內(nèi)(24 h-72 h)的毒性作用特征,發(fā)現(xiàn)MC-LR對(duì)原代大鼠肝臟細(xì)胞存在劑量依賴性的雙向毒性作用,即μM級(jí)別的MC-LR對(duì)原代大鼠肝臟細(xì)胞具有細(xì)胞毒性作用,表現(xiàn)為細(xì)胞狀態(tài)惡化、細(xì)胞活率顯著下降、細(xì)胞凋亡率明顯上升；而pM～nM級(jí)別的MC-LR具有促進(jìn)細(xì)胞增殖效應(yīng),表現(xiàn)為細(xì)胞活率增加、細(xì)胞周期中S期細(xì)胞比例增加,DNA合成速率增加。 進(jìn)一步對(duì)MC-LR導(dǎo)致的原代大鼠肝臟細(xì)胞增殖效應(yīng)進(jìn)行研究,發(fā)現(xiàn)MAPK家族成員磷酸化狀態(tài)、細(xì)胞內(nèi)ROS含量以及抗氧化系統(tǒng)均與這一效應(yīng)有關(guān)。具體表現(xiàn)為：MAPK家族的磷酸化狀態(tài)與低劑量(10-9～10-12 mol/L) MC-LR的促細(xì)胞增殖效應(yīng)密切相關(guān),在細(xì)胞增殖活躍的時(shí)問(48 h以內(nèi)),ERK1/2的磷酸化活性顯著增加,而JNK1/2和p38的磷酸化活性受到了明顯的抑制；10-9～10-12mol/L濃度的MC-LR染毒后,細(xì)胞內(nèi)ROS水平出現(xiàn)了溫和的變化,低水平ROS對(duì)大鼠肝臟細(xì)胞反復(fù)而溫和的刺激與MC-LR促增殖效應(yīng)的時(shí)間區(qū)間(48 h以內(nèi))存在很大的重疊性,且與細(xì)胞周期、MAPK結(jié)果相一致。
[Abstract]:In recent years, the phenomenon of water bloom occurs frequently in all parts of the world and poses a serious threat to human health. Microcystin (MC) is a kind of biologically toxic mono ring seven peptide produced by cyanobacteria bloom. Up to now, more than 80 isomers have been found to be stable and difficult to degrade. The current tap water treatment process can not be effective. In addition, MC also has bioaccumulation, and can enter the human body's health by drinking water or diet through the food chain through the food chain. The target organs of the world's widespread concern about the biological toxicity of.MC are mainly liver. Studies have shown that the toxic effects of MC are dose dependent bi-directional toxicity, that is, high dose of MC. The amount of MC can cause programmed cell death or apoptosis, while low doses of MC promote cell proliferation and tumor formation. Because the harm of MC to the population is more concentrated on long-term low dose exposure through drinking water or diet, and more and more epidemiological data show its correlation with the occurrence of swollen tumors, so the chronic toxicity of MC is especially significant. The potential carcinogenicity has become one of the hotspots of research. At present, the research on the toxicity mechanism of MC is mostly focused on the molecular mechanism of inducing cell apoptosis, and the research on the mechanism of cancer promoting is not common at home and abroad. The results of animal experiments show that MC is a promoter rather than a initiator, and the proliferation of out of control cells is in the swelling. Therefore, studying the effect of MC on cell proliferation is of great significance in elucidating its mechanism of promoting cancer.
Based on the current research status of MC and the existing problems, we select the most toxic, the largest and most serious microcystin MC-LR, dye the primary rat liver cells, and study the toxic effect of MC-LR on the primary rat liver cells from the angle of cell biology and molecular biology. The MC-LR specificity is systematically discussed. On the basis of identifying the bidirectional toxicity and dose range of MC-LR inducing cell apoptosis and promoting cell proliferation, the possible mechanism of MC-LR promoting cell proliferation effect is further analyzed to provide experimental basis and theoretical basis for clarifying the relationship between MC and high incidence of liver cancer. MC provides a clue to the molecular mechanism of cancer promoting.
The primary rat liver cell culture model was established by collagenase perfusion method. By monitoring the level of ALT and AST, and the analysis of the specific markers on the surface of the liver cells, the integrity and purity of the liver cells were obtained. Near 100%., an experimental model suitable for research was established, which laid a foundation for subsequent observation and index determination.
The toxicity of 10-5 to 10-12 mol/L gradient concentration was observed. The toxic effects of MC-LR on the primary rat liver cells were observed in a long period of time (24 h-72 h), and it was found that MC-LR had a dose-dependent two-way toxic effect on the primary rat liver cells. That is, the MC-LR of the M grade MC-LR has cytotoxic effect on the primary rat liver cells, which shows the cell status. The rate of cell viability decreased significantly and the rate of cell apoptosis increased obviously, while the MC-LR of pM ~ nM level had the effect of promoting cell proliferation, the increase of cell viability, the increase in the proportion of S cells in the cell cycle and the increase of DNA synthesis rate.
Further studies on the proliferation of primary rat liver cells induced by MC-LR show that the state of phosphorylation of MAPK family members, the content of ROS in the cells and the antioxidant system are all related to this effect. The specific expression is that the phosphorylation status of the MAPK family is closely related to the proliferation effect of low dose (10-9 to 10-12 mol/L) MC-LR. When the cell proliferation was active (within 48 h), the phosphorylation activity of ERK1/2 increased significantly, while the phosphorylation activity of JNK1/2 and p38 was significantly inhibited, and the intracellular ROS level appeared mild changes after 10-9 ~ 10-12mol/L concentration of MC-LR, and low level ROS had repeated and mild stimulation with MC-LR to promote proliferation of rat liver cells. The time interval of the effect (within 48 h) is very overlapped, and is consistent with the cell cycle and MAPK results.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363

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本文編號(hào)：1890912