关于氟暴露早期生物学效应指标的研究

发布时间：2018-03-03 07:40

本文选题：氟化钠　切入点：人体骨髓瘤细胞　出处：《中央民族大学》2015年硕士论文　论文类型：学位论文

【摘要】：氟是人体必需的微量元素之一,对人体健康具有两面性。适量的氟摄入,有利于牙齿和骨骼生长,维持体内钙磷平衡,同时能有效预防龋齿；但长期过量氟暴露时,氟逐渐在体液和组织中蓄积,对机体产生毒性作用,引起严重的健康问题,典型临床症状为氟骨症和氟斑牙。目前,地氟病仍是我国许多地区最严重的公共卫生问题之一,除上海市和海南省外,其余各省、区几乎都有不同程度的发生,尤其在广大农村地区和边远山区。长期以来,人们对于氟暴露产生的骨相损伤进行了大量研究,逐渐以氟斑牙、氟骨症作为氟暴露效应终端,但后来发现具有一定局限性。氟斑牙和氟骨症均属于分类、跳跃型变量,不具备连续性。以此类指标和暴露浓度进行拟合时,由于评估指标的钝化和间断性,将直接影响推导结果的准确性。因此,为了探索氟暴露剂量-效应关系,寻求早期效应标志,本研究开展了细胞和动物氟化钠染毒实验,具体内容如下：(1)以人体骨髓瘤细胞RPMI8226为研究对象,用氟化钠进行染毒,建立细胞氟暴露模型。染毒浓度分别为0、10、20、40、80、160、320μM,染毒时间为48 h。染毒结束后,提取细胞RNA,运用RT-QPCR技术测定基因表达变化(上调或下调)。结果显示,与转录有关、与蛋白合成降解有关以及与细胞增殖、凋亡和周期有关的三类基因的表达受到氟暴露不同程度影响。其中,与转录有关的基因ANKRD1、KLF2、SBN02和ZNF649,与空白对照组相比,在高浓度染毒组(320 μM), mRNA相对表达量显著上升(P0.01)。与蛋白合成降解有关的基因FANCM的mRNA相对表达量在80、320μM染毒组明显上升。而与细胞增殖、凋亡和周期有关的基因PDGFA. RNF152、CDK10和CETN2,当染毒浓度大于160μM时,mRNA相对表达量相对于空白对照组显著上升(P0.01)。(2)以SD大鼠为研究对象,通过灌胃方式进行氟化钠染毒,建立动物慢性氟中毒模型。染毒剂量分别为0、2、4、8、16、32 mg/kg. b. w,染毒时间为3个月。染毒期间,隔天对大鼠进行称重,并观察氟斑牙发生情况。染毒结束后,测定动物常规指标、生化指标以及特异性蛋白指标变化情况。实验结果显示,氟化钠暴露对大鼠体重有一定影响。与空白对照组相比,随着氟化钠染毒剂量的增加,大鼠体重增长率逐渐下降。对于大鼠脏器系数,随着氟化钠染毒剂量的增大,心脏系数和胸腺系数逐渐减小,而肝脏系数逐渐增大。当染毒剂量为16、32mg/kg. b. w时,与空白对照组相比,大鼠肝脏系数显著增大(P0.05),表明两个高氟剂量组大鼠肝脏受到一定程度损伤。再有,肝功能生化指标检测显示,AST活力随染毒浓度的增加有降低趋势,当染毒剂量大于4 mg/kg. b. w,大鼠AST活力明显降低,差异具有统计学意义。最后,大鼠血液中特异性蛋白活性测定结果显示,随着氟暴露浓度的增大,蛋白GLRa2和RYR1含量没有显著变化；而蛋白ATF4的含量随着暴露浓度的增大,出现显著下降的趋势,并且蛋白含量和暴露浓度之间呈现显著负相关(R=-0.586,P0.01)。因此,可考虑将血液中ATF4含量作为流行病学效应指标,用于预测早期高氟暴露。本论文在成功建立细胞和动物氟暴露实验模型的基础上,测定了生物效应指标的变化,研究探讨了氟暴露剂量-效应关系,为氟暴露早期健康效应研究提供了建议,同时为地方性氟病的预防和治疗提供了科学依据。
[Abstract]:Fluorine is one of essential trace elements in human body, has two sides to human health. The amount of fluoride intake, is conducive to the growth of teeth and bones, to maintain the balance of calcium and phosphor, and can effectively prevent dental caries; but the long-term exposure to excessive fluoride, fluoride gradually accumulate in body fluids and tissues, produce toxic effects on the body, causing health the problem is serious, the typical clinical symptoms of skeletal fluorosis and dental fluorosis. At present, one of the most serious public health problems in many areas of endemic fluorosis in China is still in the provinces of Shanghai and Hainan provinces, area almost all have occurred in different degree, especially in the vast rural areas and remote mountainous areas. For a long time, the fluoride exposure caused bone damage has been studied, and gradually to dental fluorosis, skeletal fluorosis as fluoride exposure effect of terminal, but later found that it has certain limitations. Dental fluorosis and skeletal fluorosis are classified, jump Step type variable does not have continuity. By fitting to such indicators and exposure concentration, because the passivation evaluation index and discontinuity, the accuracy will directly affect the results. Therefore, in order to explore the dose-response relationship between the fluoride exposure, seeking early effect markers, this study developed the cell and animal experiment of sodium fluoride exposure and the specific contents are as follows: (1) in human myeloma cell RPMI8226 as the research object, were treated with sodium fluoride, fluoride exposure to establish cell model. Exposure concentration was 0,10,20,40,80160320 M, exposure time of 48 h. exposure after the extraction of RNA cells, determination of gene expression using RT-QPCR Technology (up or down). The results showed that transcription, protein synthesis and degradation is related with cell proliferation, apoptosis and gene expression of three kinds of cycle related to the different influence of fluoride exposure. Among them, with relevant transcription The gene ANKRD1, KLF2, SBN02 and ZNF649, compared with the control group, the high concentration exposure group (320 M), the relative expression level of mRNA increased significantly (P0.01). FANCM gene related to protein synthesis and degradation of the relative expression of mRNA increased significantly in the 80320 M exposure group. The cell proliferation and, cycle and apoptosis related genes PDGFA., RNF152, CDK10 and CETN2, when the exposure concentration is greater than 160 M, the relative expression of mRNA compared with the control group increased significantly (P0.01). (2) with SD rats as the research object, were exposed by intragastric administration of sodium fluoride, establish the animal model of chronic fluorosis at doses of 0,2,4,8,16,32 mg/kg.. B. W, the exposure time was 3 months. During the exposure, the rats were weighed and observed the occurrence of dental fluorosis. After exposure, determination of conventional indicators of animal, biochemical indexes and specific protein index changes. Experimental results Results showed that sodium fluoride exposure has a certain effect on the body weight of rats. Compared with the control group, with the increase of sodium fluoride dose, rat body weight growth rate decreased gradually. The organ coefficient of rats, with the increase of sodium fluoride dose, cardiac index and thymus index was gradually decreased, and the liver coefficient increases gradually when the dose was 16,32mg/kg.. B. W, compared with the control group, the liver index of rats increased significantly (P0.05), two showed that the high fluoride dose group rat liver by a certain degree of damage. Furthermore, detection of biochemical indexes of liver function showed that AST activity decreased with exposure concentration decreased with the increase of exposure, when more than 4 mg/kg. B. dose of W, activity of AST rats decreased significantly, the difference was statistically significant. Finally, results show that the determination of specific protein activity in blood of rats exposed to fluoride, with the increasing concentration of egg white, GLRa2 and RYR1 were not There are significant changes; and the content of ATF4 protein increased with the exposure concentration, significantly decreased, and between protein content and exposure concentration showed a significant negative correlation (R=-0.586, P0.01). Therefore, the content of ATF4 in blood can be considered as epidemiological effect index for the prediction of early high fluoride exposure. This thesis established cell and the animal experiment model of fluoride in successful exposure, changes of biological effect index determination, study the dose effect relationship of fluoride exposure, provides suggestions on health effects of early exposure to fluoride, and provide scientific basis for prevention and treatment of endemic fluorosis.

【学位授予单位】：中央民族大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R599.1

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