氯乙烯对大鼠肝细胞周期相关蛋白表达的影响

发布时间：2018-01-26 19:00

本文关键词： 氯乙烯单体细胞周期 G1/S关卡细胞周期蛋白依赖性激酶　出处：《山西医科大学》2013年硕士论文　论文类型：学位论文

【摘要】：目的通过研究氯乙烯单体(vinyl chloride monomer, VCM)对大鼠肝细胞P53介导的细胞周期G1/S关卡的影响和相关蛋白表达的变化,以期探索VCM可能的致癌机制。材料与方法将64只SD大鼠随机分成4组,分别为5、25、125mg/kg体重3个染毒组和对照组,对照组为同中剂量染毒组相同体积的清洁空气,采用腹腔注射染毒,每周3次,持续12周。实验过程中记录大鼠的一般情况。染毒6周和12周分别随机处死32只大鼠(雌雄各半),流式细胞术检测大鼠肝细胞周期和凋亡,Western blot检测肝细胞周期相关蛋白(正性调控cyclinD1和负性调控P53、P21)的表达,免疫组织化学法检测P16蛋白表达的变化。所有数据均采用SPSS16.0统计软件进行分析。结果 1.大鼠的一般状况在染毒过程中大鼠未发现明显中毒症状,体重正常增加,4组间差异没有统计学意义(P0.05)。 2.大鼠染毒6周后,大鼠肝细胞G0/G1期分布差异P=0.055,各染毒组的G0/G1期分布百分比均高于对照组。染毒12周时,S期细胞百分比随着染毒剂量的增加而增加(P=0.016)。 3.VCM对大鼠染毒6周后,大鼠肝细胞P21蛋白表达量随染毒剂量的增加而降低,差异有统计学意义(P0.05),P53蛋白表达量随染毒剂量的增加而增加,差异有统计学意义(P0.05), cyclinA和cyclinD1蛋白表达量均随染毒剂量的增加而增加,差异均有统计学意义(P0.001)。 4.VCM对大鼠染毒12周后,P21蛋白的表达量随染毒剂量的增加而增加,差异有统计学意义(P0.05),中、高剂量组P21蛋白表达量高于对照组,高剂量组高于低剂量组,差异均有统计学意义(P0.05)；P53蛋白的表达量随染毒剂量的增加呈上升趋势,高剂量组高于对照组,差异有统计学意义(P0.05),但总体差异没有统计学意义(P0.05)。cyclinA和cyclinD1蛋白表达量随染毒剂量增加而增加,差异均有统计学意义(P0.05),其中高剂量组表达量高于对照组,差异有统计学意义(P0.001)。 5.免疫组化法检测VCM染毒12周,P16蛋白表达量各组间差异没有统计学意义(P0.05),CDK4蛋白表达量随染毒剂量的增加呈降低趋势,染毒组的蛋白表达量均较对照组降低,差异有统计学意义(P0.05)。 6.组织病理学检查发现VCM染毒后,对照组和低剂量组肝细胞形态基本无变化,中剂量和高剂量组肝细胞有气球样变,汇管区变大,纤维化等病理改变。结论 1.VCM在低剂量早期暴露时,能够影响细胞周期分布,引起G0/G1期阻滞,随着染毒剂量的增加和染毒时间的延长G1期阻滞消失,发生S期阻滞。 2.VCM染毒对大鼠肝细胞早期凋亡有影响,对晚期凋亡的影响不大。 3. cyclin A、cyclinD1和P21蛋白的过表达可能与氯乙烯导致的细胞增殖失控有关。
[Abstract]:Purpose Vinyl chloride monomer (VCM) vinyl chloride monomer was studied. The effect of VCM on p53 mediated G1 / S level and the expression of related proteins in rat hepatocytes were studied in order to explore the possible carcinogenic mechanism of VCM. Materials and methods 64 Sprague-Dawley rats were randomly divided into 4 groups: the control group and the control group. The rats in the control group were given the same volume of clean air at the same dose. The rats were injected intraperitoneally three times a week for 12 weeks. During the experiment, 32 rats (half of male and half female) were randomly killed at 6 and 12 weeks of exposure. Flow cytometry was used to detect hepatocyte cycle and apoptosis. Western blot was used to detect hepatocyte cycle associated protein (positive regulation of cyclinD1 and negative regulation of p53). The expression of P21) and the expression of P16 protein were detected by immunohistochemistry. All the data were analyzed by SPSS16.0 software. Results 1. During the course of exposure, no obvious toxic symptoms were found in rats, and there was no significant difference among the four groups in normal weight gain (P 0.05). 2. After 6 weeks of exposure, the difference of G0 / G1 phase distribution in rat hepatocytes was 0.055. The percentage of G0 / G1 phase distribution in each exposure group was higher than that in the control group. At 12 weeks of exposure, the percentage of G0 / G1 phase distribution was higher than that of the control group. The percentage of S phase cells increased with the increase of exposure dose. 3. After 6 weeks of VCM exposure, the expression of P21 protein decreased with the increase of the dose of VCM, and the difference was statistically significant (P 0.05). The expression of p53 protein increased with the increase of exposure dose, the difference was statistically significant (P0.05). The expression of cyclinA and cyclinD1 protein increased with the increase of exposure dose, and the difference was statistically significant (P 0.001). 4. After 12 weeks of VCM exposure, the expression of P21 protein increased with the increase of the dose of VCM, and the difference was statistically significant (P 0.05). The expression of P21 protein in the high dose group was higher than that in the control group, and in the high dose group was higher than that in the low dose group (P 0.05). The expression of p53 protein in high dose group was higher than that in control group, the difference was statistically significant (P 0.05). But the total difference was not statistically significant (P0.05A. Cyclin A and cyclinD1 protein expression increased with the increase of exposure dose, the difference was statistically significant (P0.05). The expression level of high dose group was higher than that of control group, the difference was statistically significant (P 0.001). 5. There was no significant difference in the expression of P16 protein between the groups exposed to VCM for 12 weeks by immunohistochemical method. The expression of CDK4 protein decreased with the increase of the dose of VCM. The expression of protein in the exposed group was lower than that in the control group, and the difference was statistically significant (P 0.05). 6. Histopathological examination showed that the morphology of hepatocytes in the control group and the low dose group was almost unchanged after VCM exposure, and the hepatic cells in the middle and high dose groups had balloon changes and the catchment area became larger. Fibrosis and other pathological changes. Conclusion 1. VCM could affect the cell cycle distribution and induce G _ 0 / G _ 1 phase arrest during early exposure at low dose, which disappeared with the increase of exposure dose and the prolongation of exposure time. Stage S block occurred. 2. VCM exposure had an effect on early apoptosis of rat hepatocytes, but had little effect on late apoptosis. 3. Overexpression of cyclin cyclin D1 and P21 proteins may be related to uncontrolled cell proliferation induced by vinyl chloride.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

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