六价铬体外诱导人支气管上皮细胞Beas-2B恶性转化相关机理初步研究

发布时间：2018-05-20 23:38

本文选题：六价铬 + 人支气管上皮细胞　；参考：《江苏大学》2017年硕士论文

【摘要】：目的:六价铬是与肺癌等肺部疾病相关的重要人类致癌物质。暴露于六价铬可诱导人肺上皮细胞的DNA损伤以及恶性转化。尽管人们对六价铬的致癌机理进行了广泛的研究,其分子机制尚未完全明确,而且对于六价铬诱导的细胞转化是否可能伴随侵入特性以促进转移还不清楚。因此,本研究旨在通过构建六价铬体外诱导人支气管上皮细胞Beas-2B恶性转化细胞模型,探讨:1.六价铬对Beas-2B增殖活性的影响;2.六价铬短期诱导对人支气管细胞Beas-2B迁移、侵袭以及相关蛋白的影响;3.六价铬长期诱导对人支气管上皮细胞Beas-2B迁移、侵袭以及相关蛋白的影响;4.六价铬长期诱导人支气管上皮细胞Beas-2B是否发生恶性转化;5.抑癌基因LKB1在六价铬诱导的稳定转化细胞(Beas-2B-Cr)迁移、侵袭过程中的作用及机制。从而进一步明确六价铬在细胞水平和分子水平的可能的致癌机制,揭示六价铬诱导恶性转化过程中细胞迁移、侵袭的机制,阐明LKB1在细胞恶性转化研究中的作用,从而为重金属致癌分子机制的研究提供良好的模型,也为化学物质的致癌性检测以及职业性肺癌的治疗和预防提供了理论支持和科学依据。方法:1.以不同浓度的重铬酸钾(K_2Cr_2O_7)溶液(0-20μM;0-5μM;0-1μM)处理人支气管上皮细胞不同时间(6、12、24、48、72h),MTT法和平板克隆实验检测六价铬对细胞增殖活性的影响,筛选六价铬长期诱导的浓度;2.低浓度重铬酸钾(K_2Cr_2O_7)溶液(0.125μM、0.25μM)短期诱导细胞(24h、48h)后,Transwell实验检测六价铬诱导对Beas-2B细胞迁移、侵袭能力的影响,蛋白免疫印迹法检测肺癌抑癌基因LKB1以及迁移、侵袭相关蛋白FAK、Src、MMP-2、GSK3β、β-catenin、HEF1等表达情况;3.软琼脂克隆实验检测六价铬(0.125μM、0.25μM)长期诱导(20、40和60代)人支气管上皮细胞Beas-2B的恶性转化情况,Transwell实验和蛋白免疫印迹法分别检测恶性转化过程中六价铬对细胞迁移、侵袭能力以及相关蛋白FAK、Src、MMP-2、GSK3β、β-catenin、HEF1等表达的影响;4.软琼脂克隆形成实验和裸鼠成瘤实验检测六价铬转化细胞Beas-2B-Cr的恶性转化情况,蛋白质组学相关实验检测六价铬转化细胞Beas-2B-Cr差异蛋白的表达;5.Transwell和蛋白免疫印迹法检测六价铬转化细胞Beas-2B-Cr的迁移、侵袭能力以及相关蛋白表达情况;6.分别利用过表达质粒STK11诱导LKB1上调和si RNA基因干扰方法下调LKB1表达的情况下,考察LKB1对细胞迁移、侵袭能力的影响以及LKB1与FAK、Src、MMP-2、GSK3β、β-catenin、HEF1等蛋白之间的关系。结果:1.高浓度六价铬化合物(5-20μM)对人支气管上皮细胞的增殖活性影响较大,且成时间浓度依赖性(P0.05),为了消除由于增殖抑制对实验的干扰,选用两个低浓度六价铬0.125μM和0.25μM作为长期诱导以及迁移、侵袭实验的作用浓度;2.Transwell实验结果显示六价铬化合物短期作用24h和48h能够诱导细胞的迁移(P0.01),并且随着作用浓度的升高,细胞迁移的能力也随之增强;蛋白免疫印迹实验结果表明LKB1蛋白水平随着六价铬浓度的升高而下降,而迁移、侵袭相关蛋白FAK、Src、MMP-2、GSK3β、β-catenin、HEF1等蛋白水平升高;3.细胞在经过六价铬诱导连续传代20代后可在半固体琼脂上形成克隆,获得锚着独立性生长能力,且具有浓度依赖关系(P0.05),克隆的数量和大小随诱导代数的增加而升高。在长期诱导过程中,六价铬始终诱导Beas-2B细胞的迁移、侵袭能力,且成浓度依赖性。LKB1蛋白表达在细胞转化过程中始终受到抑制,而迁移、侵袭相关蛋白表达升高;4.从六价铬诱导60代细胞在软琼脂上形成的克隆,扩大培养获得的细胞株Beas-2B-Cr可以在软琼脂中形成克隆,能够在裸鼠体内成瘤。该细胞株迁移、侵袭能力与对照组相比明显增强(P0.001),细胞中LKB1蛋白表达与对照组相比明显下降,但是与迁移、侵袭相关的蛋白表达高于对照组;5.双向荧光差异凝胶电泳的结果表明六价铬转化细胞Beas-2B-Cr与对照组细胞相比有159个差异表达的蛋白点,质谱结果显示35个差异表达显著的蛋白中包括下调的抑癌基因LKB1;6.在LKB1表达低的细胞Beas-2B-Cr中,质粒转染过表达LKB1,可以抑制细胞迁移、侵袭能力,诱导FAK、Src、MMP-2、GSK3β、β-catenin、HEF1等蛋白表达下降;基因干扰LKB1下调蛋白表达,可以促进细胞迁移、侵袭能力的增强,抑制FAK、Src、MMP-2、GSK3β、β-catenin、HEF1等蛋白表达;结论:六价铬对人支气管上皮细胞Beas-2B的增殖活性抑制具有时间浓度依赖关系;六价铬转化细胞Beas-2B-Cr可以在软琼脂中形成克隆,细胞具有肿瘤细胞特征;六价铬转化细胞Beas-2B-Cr在异体移植的小鼠体内诱导肿瘤的发生,细胞具有成瘤性;低浓度六价铬能够诱导人支气管上皮细胞Beas-2B的恶性转化;六价铬长期暴露可以诱导Beas-2B细胞迁移和侵袭能力,并且能够抑制细胞中LKB1以及激活迁移侵袭相关蛋白的表达;迁移、侵袭相关蛋白FAK、Src、MMP-2、GSK3β、β-catenin、HEF1等都是LKB1的下游蛋白并受其调控,从而对Beas-2B-Cr细胞的迁移、侵袭产生影响。
[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.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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