【作者】 王玥；

【导师】 李扬；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2015， 硕士

【摘要】 [目的]应用L-02细胞建立长期低剂量镉转化细胞模型。观察转化细胞形态学变化，检测转化细胞增殖，凋亡和甲基化的改变，探讨长期低剂量镉导致细胞转化的机制。[方法]1）镉转化细胞模型的建立与鉴定。MTT法确定镉培养的浓度，形态学分析和WesternBlot确定镉培养的时间；台盼蓝染色计数法和细胞克隆形成检测转化细胞的增殖率，倍增时间和克隆形成率；Transwell检测转化细胞的迁移侵袭能力；流式细胞术检测转化细胞的周期，ROS水平和凋亡率的变化。2）转化细胞相关实验。实验分组：对照组（正常L-02细胞），CDT-L-02组（镉转化细胞），5-aza组（正常L-02细胞加5-aza处理），CDT-L-02+5-aza组（镉转化细胞加5-aza处理）。台盼蓝染色计数法检测各组细胞的增殖率；流式细胞术检测各组细胞凋亡率的变化；WesternBlot检测各组细胞DNMTs，凋亡和周期相关蛋白以及PCNA表达的变化；限制性内切酶酶切电泳和MSP检测各组细胞基因组DNA甲基化和caspase-8基因启动子甲基化的水平。3）癌细胞相关实验。实验分组：对照组（正常L-02细胞），CDT-L-02组（镉转化细胞），HepG-2组（肝癌细胞），HepG-2+5-aza组（肝癌细胞加5-aza处理）。台盼蓝染色计数法检测甲基化抑制剂5-aza处理后肝癌细胞HepG-2活性；MSP检测5-aza处理后肝癌细胞HepG-2caspase-8启动子甲基化的水平；WesternBlot检测5-aza处理后肝癌细胞HepG-2DNMT1，caspase-8的表达。[结果]1）1μM氯化镉培养L-02细胞第2周时，细胞形态开始发生变化，细胞长轴逐渐变长；10周时，细胞从方形变成长梭形，与正常对照组相比，转化细胞DNMT1表达稳定升高，Caspase-8表达稳定下降，p＜0.05。与正常对照组L-02细胞相比，转化细胞的增殖率，克隆形成率和迁移侵袭能力明显升高，p＜0.05，但是ROS的产生，倍增时间和凋亡率明显减少，p＜0.05。2）转化细胞在甲基化抑制剂5-aza的作用下，与正常对照组相比，升高的增殖率明显下降，减少的凋亡率明显增加，p＜0.05；DNMTs，Caspase-8与其他凋亡和周期相关蛋白的表达在5-aza的作用下恢复到正常对照组的水平，p＜0.05；基因组和caspase-8基因启动子甲基化的水平也有所恢复。3）5-aza作用48h，与HepG-2组相比HepG-2+5-aza组的细胞活性明显下降，p＜0.05； DNMT1表达减少，Caspase-8表达增加，p＜0.05；与正常L-02细胞相比，肝癌细胞HepG-2caspase-8基因启动子甲基化水平较高，5-aza的作用使肝癌细胞HepG-2caspase-8基因启动子甲基化水平下降。[结论]1.长期低剂量镉能促进正常肝细胞L-02增殖增加，凋亡减少，并改变细胞的形态，导致正常细胞发生转化。2.长期低剂量镉导致细胞转化的机制是其提高了基因组DNA和caspase-8基因启动子甲基化的水平；肝癌细胞的恶性增殖也与caspase-8基因启动子异常甲基化有关。更多还原

【Abstract】 ObjectiveTo establish and identify a transformed cell model of low dosechronic cadmium in L-02cell (human normal hepatocyte). Discussing thecarcinogenic mechanism of low dose chronic cadmium by analyzing theaberration of morphology, proliferation, apoptosis ratio and methylationstatus of transformed cell.Methods1) Establish and identify the transformed cell model. MTT assay formeasurement the culture concentration of cadmium, morphology andWesternblot for ensuring the culture length of cadmium; Trypan bluestaining method and Cell clone formation method were used for countingRelative growth rate, Doubling time and Colony-forming efficiency; Theinvasiveness and migration capability of transformed cell was tested byTranswell; Cell cycle, ROS level and cell apoptosis rate were detected byFCM.2) Experiment of transformed cells. The cells were divided into fourgroups, the control group (L-02cells), CDT-L-02group，5-aza group，CDT-L-02+5-aza group. Trypan blue staining method were used forcounting Relative growth rate；cell apoptosis rate were detected by FCM；The expression of DNMTs, apoptosis and cycle related proteinand PCNA were detected by Westernblot; HpaII/MspI enzymeelectrophoresis and MSP analysis to detect methylation status of genomicDNA and caspase-8gene promoter.3) Experiment of tumor cells. The cells were divided into fourgroups, the control group (L-02cells), CDT-L-02group, HepG-2group，HepG-2+5-aza group. To assay the cell viability of5-aza treated HepG-2by Trypan blue staining method; Methylation status of caspase-8genepromoter of5-aza treated HepG-2was detected by MSP analysis; Theexpression of DNMT1and Caspase-8protein of5-aza treated HepG-2were detected by Westernblot.Results1) After L-02cells were cultured with1μM CdCl2for2weeks, themacroaxis of the cells became longer; for10weeks, the shape of cellstransformed from polygon to long spindle, and compared with normalL-02(Control group) the cadmium transformed L-02(CDT-L-02group)expressed significantly and stably up-regulation DNMT1associated withdown-regulation Caspase-8.Compared with normal L-02, the transformedcells showed significantly increased proliferation, colony-formingefficiency and invasiveness and migration capability, however,demonstrated significantly reduced ROS level, doubling time andapoptosis rate.2) Methylation inhibitor5-aza treated transformed cells (CDT-L-02+5-aza group) demonstrated reduced proliferation and increasedapoptosis rate compared with transformed cells;5-aza restored the expression of DNMTs, caspase-8and other apoptosis and cycle relatedprotein significantly compared with normal L-02;5-aza changed theaberration of methylation status (of genomic DNA and caspase-8genepromoter) of transformed cells.3) After48hours treatment of5-aza HepG-2cells showedsignificantly reduced cell viability; compared with normal L-02, HepG-2cells demonstrated higher methlation status of caspase-8gene promoter,but the treatment of5-aza down-regulated the methylation status.Conclusion1.Low dose chronic cadmium promoted L-02cells demonstratingincreased proliferation and reduced apoptosis rate, and altered the shapeof L-02cells, leading to the transform of L-02cells.2.Correlated mechanism of cadmium carcinogenic is that cadmiumaltered the methylation status of genomic DNA and caspase-8genepromoter. The methylation of caspase-8may be concerned with the lowapoptosis ratio and malignant proliferation of tumor cell HepG-2.更多还原