【作者】 徐辉；

【导师】 刘起展； 李忠； 王守林； 周建伟；

【作者基本信息】 南京医科大学 ， 劳动卫生与环境卫生学， 2009， 硕士

【摘要】 砷是自然界分布极广的类金属元素,亚砷酸钠(NaAsO2)是砷元素在环境中主要的存在形式之一。流行病学资料表明,长期摄入砷化物可导致多种癌症发病率升高,如皮肤癌、肺癌、膀胱癌和肝癌等。肺组织是砷对人体产生毒作用的重要靶器官之一,特别是有色金属采矿冶炼工人和含砷农药制造工人长期吸入低浓度无机砷而导致肺癌发病率升高是一种严重危害工人健康的职业病。砷化物虽然是人类确定致癌物,但其致癌的分子机制尚未完全清楚。研究认为,正常细胞恶性转化的机理之一是致癌因素作用于正常细胞后,引起原癌基因表达过强或/和抗癌基因表达过低、细胞信号转导异常、细胞周期紊乱,从而引起细胞不可控制地过度生长,导致细胞恶性转化,最终产生癌症。其中,正常细胞周期紊乱及其所致的细胞增殖异常是癌症发生的基础。国内外已有研究报道不同浓度NaAsO2可引起细胞增殖与凋亡的双相效应,即低浓度引起细胞增殖升高而凋亡降低,而高浓度则引起细胞增殖降低而凋亡升高;并发现活性氧(ROS)和丝裂素活化蛋白激酶(MAPKs)信号通路在不同浓度NaAsO2所致细胞双相效应中发挥重要作用,但其具体的分子机制尚不清楚。本研究拟探讨不同浓度NaAsO2对人胚肺成纤维细胞(HELF)细胞增殖和细胞周期的变化,以及对JNK激活、c-Jun激活和相关周期蛋白cyclin D1表达的影响及其相互关系;并应用JNK特异性抑制SP600125预处理、RNAi技术下调JNK和c-Jun蛋白而阻滞JNK/c-Jun信号通路后再观察低浓度NaAsO2对HELF细胞增殖和细胞周期的影响。从而探讨JNK/c-Jun信号通路在低浓度NaAsO2所致HELF细胞增殖异常和细胞周期紊乱中的作用。方法一、不同浓度NaAsO2对HELF细胞增殖和细胞周期的影响为了解NaAsO2对HELF细胞增殖的影响,分别用0.0、0.1、0.5和5.0μM不同浓度NaAsO2处理HELF细胞12、24和48 h后,CCK-8法检测HELF细胞增殖率;为了解NaAsO2对HELF细胞周期的影响,分别用0.0、0.1、0.5和5.0μM不同浓度NaAsO2处理HELF细胞24 h后,溴化丙啶(PI)染色所收集细胞,流式细胞仪检测细胞周期。二、不同浓度NaAsO2对cylclin D1蛋白表达水平的影响为了解NaAsO2对HELF细胞周期素cyclin D1蛋白表达的影响,分别用0.0、0.1、0.5和5.0μM不同浓度NaAsO2处理HELF细胞6、12和24 h后,提取HELF细胞总蛋白,用western blot方法检测cyclin D1蛋白表达水平。三、不同浓度NaAsO2对JNK/c-Jun信号通路的影响为检测NaAsO2对HELF细胞JNK/c-Jun信号通路的影响,分别用0.0、0.1、0.5和5.0μM不同浓度NaAsO2处理HELF细胞6、12和24 h后,提取HELF细胞总蛋白,用western blot方法分别检测JNK、磷酸化JNK、c-Jun和磷酸化c-Jun的表达水平。四、JNK/c-Jun信号通路在低浓度NaAsO2所致HELF细胞增殖异常和细胞周期紊乱中的作用为探讨JNK/c-Jun信号通路是否参与了低浓度NaAsO2诱导HELF细胞增殖异常和细胞周期紊乱的过程,用20μM JNK抑制剂SP600125预处理HELF细胞30 min或20 nM JNK siRNA和c-Jun siRNA分别转染HELF细胞24 h而阻滞JNK/c-Jun信号通路后,再分别用0.0、0.1、0.5和5.0μM NaAsO2处理HELF细胞24 h,分别用CCK-8法检测细胞增殖率,流式细胞仪检测细胞周期。五、JNK/c-Jun信号通路在低浓度NaAsO2诱导cyclin D1蛋白表达升高中的作用为探讨低浓度NaAsO2是否通过激活JNK/c-Jun信号通路而介导cyclin D1蛋白表达水平升高,用20μM JNK抑制剂SP600125预处理HELF细胞30 min或20 nM JNK siRNA和c-Jun siRNA分别转染HELF细胞24 h而阻滞JNK/c-Jun信号通路后,再分别用0.1和0.5μM NaAsO2处理HELF细胞12 h,收集HELF细胞总蛋白,用western blot方法检测JNK、磷酸化JNK、c-Jun、磷酸化c-Jun和cyclin D1蛋白表达水平。结果一、不同浓度NaAsO2对HELF细胞增殖和细胞周期的影响(一)不同浓度NaAsO2对HELF细胞增殖的影响HELF细胞相对增殖率在低浓度0.1和0.5μM NaAsO2组处理12、24和48 h时均显著高于对照组(P<0.05或<0.01),其增殖高峰表现在0.1μM NaAsO2组和24 h时间点。而高浓度5.0μM NaAsO2组HELF细胞相对增殖率在不同时间均显著低于对照组(P<0.05)。结果说明低浓度NaAsO2可引起HELF细胞增殖升高,而高浓度NaAsO2则引起HELF细胞增殖降低。(二)不同浓度NaAsO2对HELF细胞周期的影响HELF细胞暴露于低浓度0.1和0.5μM NaAsO2组24 h后,G1期细胞比例显著低于对照组(P<0.01),S期细胞比例则显著高于对照组(P<0.01);而高浓度5.0μM NaAsO2组G2/M期细胞比例显著高于对照组(P<0.01)。结果说明低浓度NaAsO2可诱导HELF细胞由G1期向S期跃迁,促进细胞增殖;而高浓度NaAsO2则可引起HELF细胞G2/M期阻滞,抑制细胞增殖。二、不同浓度NaAsO2对cylclin D1蛋白表达水平的影响HELF细胞暴露于低浓度0.1和0.5μM NaAsO2组6、12和24 h后,均不同程度地引起cyclin D1蛋白表达水平明显升高;而5μM NaAsO2未引起cyclin D1蛋白表达水平明显升高。结果说明低浓度NaAsO2可引起cyclin D1蛋白表达水平明显升高。三、不同浓度NaAsO2对JNK/c-Jun信号通路的影响(一)不同浓度NaAsO2对磷酸化JNK表达水平的影响HELF细胞暴露于低浓度0.1和0.5μM NaAsO2组6、12和24 h后,磷酸化JNK表达水平明显高于对照组;而高浓度5.0μM NaAsO2未引起JNK磷酸化水平明显升高。结果说明低浓度NaAsO2可不同程度诱导JNK信号通路激活,且变化规律与cyclin D1蛋白表达升高变化基本一致。(二)不同浓度NaAsO2对磷酸化c-Jun表达水平的影响HELF细胞暴露于低浓度0.1和0.5μM NaAsO2组6、12和24 h后,均引起c-Jun磷酸化水平明显升高;而高浓度5.0μM NaAsO2未引起c-Jun磷酸化水平明显升高。结果说明低浓度NaAsO2可引起c-Jun激活,其变化规律与JNK信号通路激活和cyclin D1蛋白表达升高基本一致。四、JNK/c-Jun信号通路在低浓度NaAsO2所致HELF细胞增殖异常和细胞周期紊乱中的作用(一)JNK/c-Jun信号通路阻滞对低浓度NaAsO2所致HELF细胞增殖异常的影响HELF细胞相对增殖率在SP600125预处理时低浓度0.1和0.5μM NaAsO2组均显著低于同一浓度无抑制剂处理组(P<0.01),但与自身空白对照组无显著性差别(P>0.05);HELF细胞相对增殖率在转染JNK siRNA和c-Jun siRNA时低浓度0.1和0.5μM NaAsO2组细胞相对增殖率均显著低于同一浓度无siRNA转染处理组(P<0.01),而与空白对照组无显著性差别(P>0.05)。结果说明JNK/c-Jun信号通路在低浓度NaAsO2所致HELF细胞增殖异常过程中发挥重要作用。(二) JNK/c-Jun蛋白下调对低浓度NaAsO2所致HELF细胞周期紊乱的影响HELF细胞S期细胞百分比在用20 nM JNK siRNA转染时0.1和0.5μM NaAsO2组明显低于同一浓度未用siRNA转染组(P<0.05),而与其自身对照组无显著性差别(P>0.05);类似地,HELF细胞S期细胞百分比在用20 nM c-Jun siRNA转染时0.1和0.5μM NaAsO2组明显低于未用siRNA转染组(P<0.05),而与其自身对照组无显著性差别(P>0.05);结果说明JNK/c-Jun信号通路参与了低浓度NaAsO2所致HELF细胞周期紊乱过程。五、JNK/c-Jun信号通路在低浓度NaAsO2诱导cyclin D1蛋白表达升高中的作用(一)JNK信号通路阻滞对低浓度NaAsO2诱导c-Jun激活和cyclin D1蛋白表达升高的影响HELF细胞磷酸化JNK表达水平在用SP600125预处理时0.1和0.5μM NaAsO2组均明显低于同一浓度无抑制剂组,磷酸化c-Jun表达水平和cyclin D1蛋白表达水平也明显降低;类似地,HELF细胞JNK蛋白表达水平在用JNK siRNA转染时0.1和0.5μM NaAsO2组均明显低于同一浓度未用siRNA转染组,磷酸化c-Jun表达水平和cyclin D1蛋白表达水平也均明显降低。结果说明低浓度NaAsO2可能通过激活JNK/c-Jun信号通路,从而引起cyclin D1蛋白表达水平升高。(二)C-Jun蛋白下调对低浓度NaAsO2诱导cyclin D1蛋白表达升高的影响HELF细胞c-Jun蛋白表达水平在用c-Jun siRNA转染时0.1和0.5μM NaAsO2组均明显低于同一浓度未用siRNA转染组,cyclin D1蛋白表达水平也均随之明显降低,而磷酸化JNK表达水平未受c-Jun siRNA的影响。结果说明低浓度NaAsO2通过激活JNK而激活c-Jun,然后再诱导cyclin D1蛋白表达升高。结论1.低浓度NaAsO2可引起HELF细胞增殖升高,而高浓度NaAsO2可引起HELF细胞增殖降低。2.低浓度NaAsO2可促进HELF细胞从G1期跃迁到S期而引起细胞周期紊乱。3.低浓度NaAsO2可激活JNK/c-Jun信号通路而引起细胞周期素cyclin D1蛋白表达升高。4. JNK/c-Jun信号通路和细胞周期素cyclin D1在低浓度NaAsO2所致HELF细胞增殖异常和细胞周期紊乱中发挥重要作用。更多还原

【Abstract】 Arsenic is a metalloid element that is widely distributed in the environment. Sodium arsenite is the main inorganic form of arsenic in the environment. Available epidemiological data have shown that long-term exposure to high concentration of arsenide may induce various cancers including skin cancer, bladder cancer and liver cancer. Importantly, occupational inhalation of arsenic, which exists in nonferrous metal ore smelters and insecticide manufacturers, is associated with increased risks of human lung cancer. Arsenic is an identified human carcinogen. Sodium arsenite is demonstrated to induce cellular proliferation and cell cycle disorder both in cell culture model and animal model; however, its mechanisms are not well known.Cancer is one of diseases caused by environmental factors and genetic factors. Cell exposure to carcinogenic agents can lead to a series of events, such as oncogene reinforcement and anti-oncogene inhibition caused by gene mutation, dysfunction of cell signal transduction, deregulation of cell cycle and uncontrolled cellular proliferation, which cause eventually cancer. Of these, the abnormal cellular proliferation and cell cycle disorders are the bases of cancer.Some studies indicate that arsenite may induce biphase effects of cellular proliferation and cell apoptosis in human cells. Furthermore, ROS and MAPKs signal pathway may play crucial roles in cellular proliferation and cell apoptosis induced by difference concentration of sodium arsenite. However, molecular mechanisms underlying this phenomenon are not well understood.In the present study, we investigated the effects of different concentration of sodium arsenite on cellular proliferation, cell cycle, JNK activation, c-Jun activation and cycline D1 express and their relationship in human embryo lung fibroblast (HELF) cells. Furthermore, we studied the effects of blocking JNK/c-Jun signal pathway, which were caused by JNK inhibitor (SP600125), JNK siRNA, and c-Jun siRNA, on cellular proliferation abnormilty and cell cycle disorder induced by low concentration of sodium arsenite in HELF cells. By these, we demonstrated the roles of JNK/c-Jun signal pathway in cellular proliferation abnormity and cell cycle disorder induced by low concerntration of sodium arsenite in HELF cells.Methods1. The effects of different concentrantion of sodium arsenite on cellular proliferation and cell cycle in HELF cells To evaluate the effects of sodium arsenite on cellular proliferation and cell cycle in HELF cells, the HELF cells were exposed to 0.0, 0.1, 0.5 or 5.0μM of sodium arsenite for 12, 24 or 48 h, respectively. Cellular proliferation was evaluated by Cell Counting Kit-8 assay, and the proportion of cell cycle was analyzed by Flow Cytometry with propidium iodide staining.2. The effects of different concentrantion of sodium arsenite on cyclin D1 expression in HELF cellsTo examine cyclin D1 level induced by sodium arsenite in HELF cells, After HELF cells were treated with 0.0, 0.1, 0.5, or 5.0μM of sodium arsenite for 6, 12 or 24 h, cyclin D1 level was detected by western blot assay.3. The effects of different concentrantion of sodium arsenite on JNK/c-Jun signal pathway in HELF cellsWe investigated whether sodium arsenite induced JNK and c-Jun activation in HELF cells. After HELF cells were treated with 0.0, 0.1, 0.5, or 5.0μM of sodium arsenite for 6, 12 or 24 h, the levels of JNK, phospho-JNK, c-Jun and phospho-c-Jun were detected by western blot assay.4. The roles of JNK/c-Jun signal pathway in the cellular proliferation and cell cycle induced by low concentrantion of sodium arsenite in HELF cellsTo confirm the roles of JNK/c-Jun signal pathway in the cellular proliferation and the cell cycle induced by low concerntrantion of sodium arsenite, HELF cells were left untreated or pre-treated with 20μM of JNK inhibitor (SP600125) for 30 min or transfected with 20 nM of JNK siRNA or c-Jun siRNA for 24 h, then cells were exposed to 0.0, 0.1, 0.5 or 5.0μM of sodium arsenite for 24 h, respectively. Cellular proliferation and the proportion of cell cycle were measured with Cell Counting Kit-8 assay and Flow Cytometry, respectively.5. The roles of JNK/c-Jun signal pathway in the cyclin D1 expression induced by low concentrantion of sodium arseniteWe investigated whether JNK/c-Jun signal pathway was involved in sodium arsenite-induced cyclin D1 expression. HELF cells were left untreated or pre-treated with 20 ofμM JNK inhibitor (SP600125) for 30 min or transfected with 20 nM of JNK siRNA or c-Jun siRNA for 12 h, then cells were exposed to 0.0, 0.1, or 0.5μM of sodium arsenite for 24 h, respectively. The levels of JNK protein, phospho-JNK, c-Jun protein, phospho-c-Jun, and cyclin D1 were detected by western blot assay.Results1. The effects of different concentrantion of sodium arsenite on cellular proliferation and cell cycle in HELF cells(1) The effects of different concerntration of sodium arsenite on cellular proliferation in HELF cellsCellular proliferation was significantly increased by 0.1 or 0.5μM of sodium arseinte at 12, 24 or 48 h in HELF cells. The curve peak of cellular proliferation presented at 0.1μM of sodium arsenite and at exposure 24 h. In contrast, cellular proliferation was markedly decreased in 5.0μM of arsenite at each time. Data indicated that low concentarion of sodium arsenite increased cell proliferation but high concerntration of sodium arsenite inhibited cell growth at in HELF cells.(2) The effects of different concerntration of sodium arsenite on cell cycle in HELF cellsThe cell proportion in S phase was significantly elevated by 0.1 or 0.5μM of sodium arsenite at 24 h, as well as the cell proportion in G1 phase was markedly decreased. The curve peak was present at 0.1μM sodium arsenite group, 5.0μM of sodium arsenite induced significantly the increases of the cell proportion in G2/M phase at 24 h. Data indicated that low concentration of sodium arsenite promoted cell cycle transition from G1 to S phases. However, high concerntration of sodium arsenite induced cell cycle arrest at G2/M phase.2. The effects of different concerntration of sodium arsenite on cyclin D1 expression in HELF cellsCyclin D1 expression levels were markedly increased by 0.1 or 0.5μM of sodium arsenite at 6, 12, or 24 h in HELF cells. In contrast, 5.0μM of sodium arsenite showed no effect on cyclin D1 expression in HELF cells. Data indicated that cyclin D1 expression was inducted by low concentration of sodium arsenite in HELF cells.3. The effects of different concentrantion of sodium arsenite on JNK/c-Jun signal pathway in HELF cells(1) The effects of different concerntration of sodium arsenite on phosho-JNK levels in HELF cellsPhopho-JNK levels were dramatically increased by 0.1 or 0.5μM of arsenite at 6, 12, or 24 h in HELF cells, whereas 5.0μM of arsenite did not show any effect on phospo-JNK in HELF cells. Data suggested that JNK signal pathway could be activated by low concerntration of arsenite in HELF cells, furthermore, the change similar to the cyclin D1 expression induction by sodium arsenite.(2) The effects of different concerntration of sodium arsenite on phosho-c-Jun levels in HELF cellsPhopho-c-Jun levels were dramatically increased after HELF cells incubated by 0.1 or 0.5μM of sodium arsenite at 6, 12, or 24 h, however, although 5.0μM of sodium arsenite may also increased phopho-c-Jun level, the effect was weakly. Data suggested c-Jun activation was induced by low concentration of sodium arsenite in HELF cells.4. The roles of JNK/c-Jun signal pathway in cellular proliferation and cell cycle induced by low concentrantion of sodium arsenite in HELF cells(1) The effects of blocking JNK/c-Jun signal pathway on cellular proliferation induced by low concerntration of sodium arseniteJNK inhibitor, SP600125, significantly blocked increases of cellular proliferation induced by 0.1 or 0.5μM of sodium arsenite in HELF cells. JNK siRNA and c-Jun siRNA also markedly suppressed the increases of cellular proliferation induced by 0.1 or 0.5μM of sodium arsenite in HELF cells. Data suggested that JNK/c-Jun signal pathway play a critical role on cellular proliferation induced by low concerntration of sodium arsenite in HELF cells.(2) The effects of JNK/c-Jun knockdown on cell cycle induced by low concerntration of sodium arseniteThe elevation of cell proportion in S phase induced by low concerntration of sodium arsenite was markedly inhibited by the JNK siRNA. Furthermore, the c-Jun siRNA inhibited dramatically the sodium arsenite-dependent transition of G1 to S phases in HELF. Data suggested that JNK/c-Jun signal pathway involvedin the cell cycle transition induced by low concerntration of sodium arsenite in HELF cells.5. The roles of JNK/c-Jun signal pathway in cyclin D1 expression induced by low concentrantion of sodium arsenite in HELF cells(1) The effects of blocking JNK signal pathway on c-Jun activation and cyclin D1 expression induced by low concerntration of sodium arsenitePretreatment of HELF cells with SP600125, resulted in a dramatic inhibition of the phosphorylation of JNK and c-Jun. Furthermore, inhibition of JNK signal pathway by SP600125 resulted in a significant decrease in sodium arsenite-induced cyclin D1 expression. Similarly, with JNK expression was dramatically knockdown by JNK siRNA, the phospho-c-Jun levels and cyclin D1 expression were significantly decreased as well. Data demonstrated that activation of JNK signal pathway may be implicated in c-Jun activation and cyclin D1 expression induction by low concerntrantion of sodium arsenite in HELF cells. (2) The effects of c-Jun kockdown on cyclin D1 expression induced by low concerntration of sodium arseniteKnockdown of c-Jun expression by transfection of HELF cells with c-Jun siRNA resulted in a significant decrease in cyclin D1 expression induced by low concerntration of sodium arsenite. Whereas, c-Jun siRNA did not show any inhibitory effect on phospho-JNK levels which induction by the same concerntration of sodium arsenite in HELF cells. Data suggested that induction of cyclin D1 expression by low concerntration of sodium arsenite in HELF cells requires JNK/c-Jun signal pathway.Conclusions:1. The low concerntration of sodium arsenite induces the increases of cellular proliferation; however, the high concerntration of sodium arsenite inhibites cellular proliferation in HELF cells.2. The low concerntration of sodium arsenite prometes cell cycle transition from G1 to S phases, which induces cell cycle disorder in HELF cells3. Activation of JNK/c-Jun pathway and the cyclin D1 protein expression are induced by low concerntration of sodium arsenite in HELF cells.4. JNK/c-Jun pathway and cyclin D1 play critical roles in cellular proliferation abnormity and cell cycle disorder induced by low concerntration of sodium arsenite in HELF cells.更多还原