本文選題：六價鉻 + 人支氣管上皮細胞�。� 參考：《江蘇大學(xué)》2017年碩士論文

【摘要】：目的:六價鉻是與肺癌等肺部疾病相關(guān)的重要人類致癌物質(zhì)。暴露于六價鉻可誘導(dǎo)人肺上皮細胞的DNA損傷以及惡性轉(zhuǎn)化。盡管人們對六價鉻的致癌機理進行了廣泛的研究,其分子機制尚未完全明確,而且對于六價鉻誘導(dǎo)的細胞轉(zhuǎn)化是否可能伴隨侵入特性以促進轉(zhuǎn)移還不清楚。因此,本研究旨在通過構(gòu)建六價鉻體外誘導(dǎo)人支氣管上皮細胞Beas-2B惡性轉(zhuǎn)化細胞模型,探討:1.六價鉻對Beas-2B增殖活性的影響;2.六價鉻短期誘導(dǎo)對人支氣管細胞Beas-2B遷移、侵襲以及相關(guān)蛋白的影響;3.六價鉻長期誘導(dǎo)對人支氣管上皮細胞Beas-2B遷移、侵襲以及相關(guān)蛋白的影響;4.六價鉻長期誘導(dǎo)人支氣管上皮細胞Beas-2B是否發(fā)生惡性轉(zhuǎn)化;5.抑癌基因LKB1在六價鉻誘導(dǎo)的穩(wěn)定轉(zhuǎn)化細胞(Beas-2B-Cr)遷移、侵襲過程中的作用及機制。從而進一步明確六價鉻在細胞水平和分子水平的可能的致癌機制,揭示六價鉻誘導(dǎo)惡性轉(zhuǎn)化過程中細胞遷移、侵襲的機制,闡明LKB1在細胞惡性轉(zhuǎn)化研究中的作用,從而為重金屬致癌分子機制的研究提供良好的模型,也為化學(xué)物質(zhì)的致癌性檢測以及職業(yè)性肺癌的治療和預(yù)防提供了理論支持和科學(xué)依據(jù)。方法:1.以不同濃度的重鉻酸鉀(K_2Cr_2O_7)溶液(0-20μM;0-5μM;0-1μM)處理人支氣管上皮細胞不同時間(6、12、24、48、72h),MTT法和平板克隆實驗檢測六價鉻對細胞增殖活性的影響,篩選六價鉻長期誘導(dǎo)的濃度;2.低濃度重鉻酸鉀(K_2Cr_2O_7)溶液(0.125μM、0.25μM)短期誘導(dǎo)細胞(24h、48h)后,Transwell實驗檢測六價鉻誘導(dǎo)對Beas-2B細胞遷移、侵襲能力的影響,蛋白免疫印跡法檢測肺癌抑癌基因LKB1以及遷移、侵襲相關(guān)蛋白FAK、Src、MMP-2、GSK3β、β-catenin、HEF1等表達情況;3.軟瓊脂克隆實驗檢測六價鉻(0.125μM、0.25μM)長期誘導(dǎo)(20、40和60代)人支氣管上皮細胞Beas-2B的惡性轉(zhuǎn)化情況,Transwell實驗和蛋白免疫印跡法分別檢測惡性轉(zhuǎn)化過程中六價鉻對細胞遷移、侵襲能力以及相關(guān)蛋白FAK、Src、MMP-2、GSK3β、β-catenin、HEF1等表達的影響;4.軟瓊脂克隆形成實驗和裸鼠成瘤實驗檢測六價鉻轉(zhuǎn)化細胞Beas-2B-Cr的惡性轉(zhuǎn)化情況,蛋白質(zhì)組學(xué)相關(guān)實驗檢測六價鉻轉(zhuǎn)化細胞Beas-2B-Cr差異蛋白的表達;5.Transwell和蛋白免疫印跡法檢測六價鉻轉(zhuǎn)化細胞Beas-2B-Cr的遷移、侵襲能力以及相關(guān)蛋白表達情況;6.分別利用過表達質(zhì)粒STK11誘導(dǎo)LKB1上調(diào)和si RNA基因干擾方法下調(diào)LKB1表達的情況下,考察LKB1對細胞遷移、侵襲能力的影響以及LKB1與FAK、Src、MMP-2、GSK3β、β-catenin、HEF1等蛋白之間的關(guān)系。結(jié)果:1.高濃度六價鉻化合物(5-20μM)對人支氣管上皮細胞的增殖活性影響較大,且成時間濃度依賴性(P0.05),為了消除由于增殖抑制對實驗的干擾,選用兩個低濃度六價鉻0.125μM和0.25μM作為長期誘導(dǎo)以及遷移、侵襲實驗的作用濃度;2.Transwell實驗結(jié)果顯示六價鉻化合物短期作用24h和48h能夠誘導(dǎo)細胞的遷移(P0.01),并且隨著作用濃度的升高,細胞遷移的能力也隨之增強;蛋白免疫印跡實驗結(jié)果表明LKB1蛋白水平隨著六價鉻濃度的升高而下降,而遷移、侵襲相關(guān)蛋白FAK、Src、MMP-2、GSK3β、β-catenin、HEF1等蛋白水平升高;3.細胞在經(jīng)過六價鉻誘導(dǎo)連續(xù)傳代20代后可在半固體瓊脂上形成克隆,獲得錨著獨立性生長能力,且具有濃度依賴關(guān)系(P0.05),克隆的數(shù)量和大小隨誘導(dǎo)代數(shù)的增加而升高。在長期誘導(dǎo)過程中,六價鉻始終誘導(dǎo)Beas-2B細胞的遷移、侵襲能力,且成濃度依賴性。LKB1蛋白表達在細胞轉(zhuǎn)化過程中始終受到抑制,而遷移、侵襲相關(guān)蛋白表達升高;4.從六價鉻誘導(dǎo)60代細胞在軟瓊脂上形成的克隆,擴大培養(yǎng)獲得的細胞株Beas-2B-Cr可以在軟瓊脂中形成克隆,能夠在裸鼠體內(nèi)成瘤。該細胞株遷移、侵襲能力與對照組相比明顯增強(P0.001),細胞中LKB1蛋白表達與對照組相比明顯下降,但是與遷移、侵襲相關(guān)的蛋白表達高于對照組;5.雙向熒光差異凝膠電泳的結(jié)果表明六價鉻轉(zhuǎn)化細胞Beas-2B-Cr與對照組細胞相比有159個差異表達的蛋白點,質(zhì)譜結(jié)果顯示35個差異表達顯著的蛋白中包括下調(diào)的抑癌基因LKB1;6.在LKB1表達低的細胞Beas-2B-Cr中,質(zhì)粒轉(zhuǎn)染過表達LKB1,可以抑制細胞遷移、侵襲能力,誘導(dǎo)FAK、Src、MMP-2、GSK3β、β-catenin、HEF1等蛋白表達下降;基因干擾LKB1下調(diào)蛋白表達,可以促進細胞遷移、侵襲能力的增強,抑制FAK、Src、MMP-2、GSK3β、β-catenin、HEF1等蛋白表達;結(jié)論:六價鉻對人支氣管上皮細胞Beas-2B的增殖活性抑制具有時間濃度依賴關(guān)系;六價鉻轉(zhuǎn)化細胞Beas-2B-Cr可以在軟瓊脂中形成克隆,細胞具有腫瘤細胞特征;六價鉻轉(zhuǎn)化細胞Beas-2B-Cr在異體移植的小鼠體內(nèi)誘導(dǎo)腫瘤的發(fā)生,細胞具有成瘤性;低濃度六價鉻能夠誘導(dǎo)人支氣管上皮細胞Beas-2B的惡性轉(zhuǎn)化;六價鉻長期暴露可以誘導(dǎo)Beas-2B細胞遷移和侵襲能力,并且能夠抑制細胞中LKB1以及激活遷移侵襲相關(guān)蛋白的表達;遷移、侵襲相關(guān)蛋白FAK、Src、MMP-2、GSK3β、β-catenin、HEF1等都是LKB1的下游蛋白并受其調(diào)控,從而對Beas-2B-Cr細胞的遷移、侵襲產(chǎn)生影響。
[Abstract]:Objective: Six valent chromium is an important human carcinogen associated with lung diseases such as lung cancer. Exposure to six valence chromium can induce DNA damage and malignant transformation of human lung epithelial cells. Although the carcinogenic mechanism of six valent chromium is widely studied, its molecular mechanism is not completely clear, and the transformation of six valent chromium induced cell transformation is not completely clear. It is not clear that invasive properties may be associated with the promotion of metastasis. Therefore, the purpose of this study is to explore the effect of six valent chromium on the Beas-2B malignant transformation cell model of human bronchial epithelial cells in vitro, and to explore the effect of 1. six valence chromium on the proliferation of Beas-2B, and the short-term induction of Beas-2B migration, invasion and related proteins in human bronchiolar cells by six valent chromium. Influence; 3. six valence chromium long induced the effect of Beas-2B migration, invasion and related proteins on human bronchial epithelial cells; 4. six valence chromium long induced the malignant transformation of human bronchial epithelial cells, and the role and mechanism of 5. tumor suppressor gene LKB1 in the migration of stable transforming cells (Beas-2B-Cr) induced by six valence chromium, the role and mechanism of the invasion process. To further clarify the possible carcinogenic mechanism of six valence chromium at the cell level and molecular level, reveal the mechanism of cell migration and invasion during the malignant transformation of six valence chromium, and clarify the role of LKB1 in the study of cell malignant transformation, thus providing a good model for the research of the molecular mechanism of heavy metal carcinogenesis and the carcinogenesis of chemical substances. The theoretical support and scientific basis for the treatment and prevention of occupational lung cancer were provided. Methods: 1. the effects of six valent chromium on cell proliferation activity were detected by different concentrations of potassium dichromate (K_2Cr_2O_7) solution (0-20 M; 0-5 u M; 0-1 M) at different time (6,12,24,48,72h), MTT and flat clones. The long-term induced concentration of six valent chromium was screened; 2. low concentration potassium dichromate (K_2Cr_2O_7) solution (0.125, M, 0.25 M) was used for short-term induction of cell (24h, 48h), and Transwell test was used to detect the effect of six valent chromium on the migration of Beas-2B cells and the invasion ability. Protein immunoblotting was used to detect the lung cancer suppressor gene LKB1 and migration, and the invasion related proteins FAK, Src, MMP. The expression of -2, GSK3 beta, beta -catenin, HEF1, and 3. soft agar cloning test was used to detect the malignant transformation of Beas-2B in human bronchial epithelial cells (20,40 and 60 generations) by six valent chromium (0.125 mu M, 0.25 M). Transwell test and protein immunoblotting were used to detect the migration, invasiveness and correlation of six valent chromium in malignant transformation, respectively. The effects of protein FAK, Src, MMP-2, GSK3 beta, beta -catenin, HEF1, etc., 4. soft agar cloning and tumorigenesis test for the detection of the malignant transformation of Beas-2B-Cr in the six valence chromium transformed cells, the expression of Beas-2B-Cr differential protein in the six valent chromium transformed cells by proteomic related experiments; and the detection of 5.Transwell and protein immunoblotting The migration, invasiveness and related protein expression of six valent chromium transformed cells Beas-2B-Cr; 6. the effects of LKB1 on cell migration, invasion ability and LKB1 and FAK, Src, MMP-2, GSK3 beta, beta, beta, and other proteins were investigated by using overexpression plasmid STK11 to induce up regulation of LKB1 and the expression of Si RNA gene interference, respectively. Results: 1. high concentration of six valence chromium compounds (5-20 mu M) had great influence on the proliferation activity of human bronchial epithelial cells, and became time dependent (P0.05). In order to eliminate the interference caused by proliferation inhibition, two low concentrations six valence chromium 0.125 M and 0.25 M were selected as long-term induction, migration and invasion experiments. The results of 2.Transwell test showed that the short-term effect of six valence chromium compounds on 24h and 48h could induce cell migration (P0.01), and the ability of cell migration increased with the increase of action concentration. The results of protein immunoblot test showed that the level of LKB1 protein decreased with the increase of the concentration of six valence chromium, and the migration, invasion of the related eggs. White FAK, Src, MMP-2, GSK3 beta, beta -catenin, HEF1, and other protein levels rise; 3. cells can be cloned on semi solid agar after six valent chromium induction after 20 successive generations of chromium, which can anchor independent growth ability and have concentration dependence (P0.05). The number and size of clones increase with the increase of induced algebra. In the long term induction process, the number and size of the clones are increased. Six valence chromium always induces migration and invasiveness of Beas-2B cells, and the expression of concentration dependent.LKB1 protein is always inhibited during cell transformation, while migration, invasion related protein expression rises; 4. from six valent chromium, induced by 60 generation of cells on soft agar, and the expanded cell line Beas-2B-Cr can be soft. The formation of clones in agar was formed in nude mice. The cell migration and invasion ability were significantly enhanced with the control group (P0.001). The expression of LKB1 protein in the cells decreased significantly compared with the control group, but the protein expression related to migration and invasion was higher than that of the control group; the results of 5. bi-directional fluorescence differential gel electrophoresis showed that the conversion of six valence chromium was found. There were 159 differentially expressed protein points in cell Beas-2B-Cr compared with the control group. Mass spectrometry results showed that 35 differentially expressed proteins included down regulated tumor suppressor gene LKB1; 6. in Beas-2B-Cr with low LKB1 expression, plasmid transfected with LKB1 could inhibit cell migration, invasion ability, induced FAK, Src, MMP-2, GSK3 beta, and beta -caten. The expression of in, HEF1 and other proteins decreased, and the gene interference of LKB1 down regulated protein expression, which could promote cell migration, enhance the invasion ability, inhibit the expression of FAK, Src, MMP-2, GSK3 beta, beta -catenin, HEF1 and other proteins. Conclusion: Six valence chromium has time dependence on the proliferation inhibition of Beas-2B in human bronchial epithelial cells; six valence chromium conversion cells Beas-2B -Cr can form clones in soft agar, and the cells have tumor cell characteristics. Six chromium transformed cells Beas-2B-Cr induce tumor occurrence in allograft mice, and the cells have tumorigenicity. Low concentration of six valence chromium can induce malignant transformation of Beas-2B in human bronchial epithelial cells, and six valence chromium long-term exposure can induce Beas-2B cell migration. Migration and invasion ability can inhibit the expression of LKB1 in cells and activation of migration and invasion related proteins; migration, invasion of related protein FAK, Src, MMP-2, GSK3 beta, beta -catenin, HEF1 are all downstream proteins of LKB1 and are regulated by them, thus affecting the migration and invasion of Beas-2B-Cr cells.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R734.2

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