氟的遗传毒性及维生素C、E的干预研究

发布时间：2018-04-13 19:18

本文选题：氟 + 肿瘤　；参考：《广西医科大学》2013年硕士论文

【摘要】：目的探索氟的遗传毒性及维生素C(VC)、维生素E(VE)、维生素C联合维生素E (VC+VE)对染氟淋巴细胞损伤的干预作用效果。方法1.职业环境氟污染调查：以铝电解废气净化车间为污染源中心,在不同半径距离的工作位点设采样点检测空气中的氟,用氟离子选择电极法测定其含量。2.职业性氟接触与肿瘤发生的关系调查：通过问卷调查和医院病历收集该厂职工从1995～2009年底肿瘤发生情况,进行统计分析。3.氟的遗传损伤动物实验研究：(1)检测经低、中、高浓度(2mg·kg-1·d-18mg·kg-1·d-1、32mg·kg-1·d-1)氟化钠染毒14d后小鼠骨髓细胞微核率,(2)用衰减全反射傅立叶变换红外光谱技术(ATR-FTIR)在包括RNA/DNA标识波长(1020/1121cm-1)在内的波段进行扫描,检测小鼠肝细胞核化学结构的变化。4.氟的遗传损伤及VC、VE干预作用的体外细胞培养实验研究：提取正常人外周血淋巴细胞,培养48h后分组及处理：(1)用0.00、0.01、0.04、0.16、0.64mg/ml NaF(对照组、F1、F2、F3、F4)分别作用2h、4h、8h、16h后, MTT法测定淋巴细胞存活率。(2)淋巴细胞分为对照组、F1、F2、F3、F4,低、中、高VC剂量干预(VC1+F4、VC2+F4、 VC3+F4)组,低、中、高VE剂量干预(VE1+F4、VE2+F4、VE3+F4)组,低、中、高VC+VE剂量干预(VC1+VE1+F4、VC2+VE2+F4、 VC3+VE3+F4)组。对照组给予培养液28h, F1、F2、F3、F4组给予培养液24h后分别暴露于含0.01、0.04.0.16、0.64mg/ml NaF培养液4h, VC干预组分别于含4.16、64μmol/LVC培养液作用24h后暴露于0.64mg/mlNaF培养液4h, VE干预组分别于含2、10、50μmol/L VE培养液作用24h后暴露于0.64mg/ml NaF培养液4h, VC+VE干预组分别于含4+2、16+10、64+50μmol/L VC+VE培养液作用24h后暴露于0.64mg/ml NaF培养液4h,离心收集细胞,分别用MTT法测淋巴细胞存活率,Annexin V-FITC细胞凋亡试剂盒检测细胞凋亡率,单细胞凝胶电泳技术(SCGE)测定DNA的损伤,Elisa实验法检测细胞端粒酶含量。结果 1.职业环境氟污染调查结果：以氟污染源铝电解废气净化车间为中心,在不同半径距离的工作位点空气中的氟浓度依次降低,呈现氟污染扩散浓度与距离的负向趋势,但均没有超过国家卫生标准(0.50mg/m3)。 2.职业性氟接触与肿瘤发生的关系调查：厂区工人肿瘤粗发病率为117.95/10万(标化率为58.81/10万),标化发病率女性比男性高(男：女=1：2.64),肿瘤发病年龄高峰为40-49岁,前2位肿瘤男性为肝癌和肺癌,女性为乳腺癌和肺癌。与该地区非暴露人群比较,该铝厂工人的氟暴露可能使女性肿瘤发病率升高,为该市的2.14倍,肿瘤发病年龄提前,肿瘤构成基本相同。 3.氟的遗传损伤动物实验结果： 3.1动物实验小鼠骨髓细胞微核率随染氟浓度的增高而升高。 3.2肝细胞核ATR光谱图呈1650cm-1、1550cm-1、1380cm-1、1260cm-1、1225cm-1、1155cm-1、1080cm-1、1030cm-1、970cm-1、930cm-1峰降低；光谱数据用统计软件进行主成分分析和3-D图形处理后,可以观察到更细微的化学结构变化,与对照组比较,氟中、高浓度组的差异以第1主成分因子为主,而氟低浓度组的差异以第2主成分因子为主,显示不同氟浓度对小鼠肝细胞核的化学结构影响存在明显差异。 4.氟的遗传损伤及VC、VE干预作用的体外细胞培养实验研究： 4.1细胞存活率随染氟浓度与时间的增加而下降。与对照组相比,F4组细胞存活率显著下降(P0.05)。16h各处理组细胞存活率均低于2h处理组(P0.05)。 4.2淋巴细胞染氟4h后,(1)总凋亡率随染氟浓度增加而升高,与对照组相比,总凋亡率分别增加了2.47%、9.80%、13.52%、25.74%,除F1浓度组外,其余各组增加均有统计学意义(P0.05)；(2)与对照组相比,随着NaF染毒浓度的升高,拖尾细胞数增加,彗星头部变小,亮度增加,彗尾变长变圆,荧光强度增强,其尾部DNA百分率、尾长、Olive尾矩升高呈剂量依赖性(P0.01)；(3)端粒酶含量随染氟浓度增加而升高。与对照组相比,F3、F4浓度组端粒酶含量升高有统计学意义(P0.05),且F4浓度组端粒酶含量明显高于其余各组(P0.01)。 4.3经预防性干预后的各组与F4组比较,(1) VC、VE、VC+VE各干预组淋巴细胞存活率均有升高的趋势。VC各组淋巴细胞存活率随着浓度的增加而升高,其中VC3+F4组细胞存活率升高有统计学意义(P0.05),联合作用VC2+VE2+F4组细胞存率升高最显著(P0.05)；(2)VC、VE、VC+VE各干预组淋巴细胞总凋亡率均有降低的趋势。VC、VE各干预组淋巴细胞总凋亡率随着浓度的增加而降低,VC2+F4、VC3+F4、 VE3+F4组淋巴细胞总凋亡率降低有统计学意义(P0.05)。单纯VC、VE各总凋亡率低于VC+VE各干预组,但无统计学差异(P0.05)；(3)VC、VE、VC+VE各干预组彗星头部变大,彗尾变短,尾部DNA百分率、尾长、Olive尾矩均显著降低(P0.01),VE1组的Olive尾矩最短。VC+VE干预组均高于VC、VE各组尾部DNA百分率、尾长和Olive尾矩(P0.05)；(4)VC、VE、VC+VE各干预组淋巴细胞端粒酶含量均有降低。VC、VE各组淋巴细胞端粒酶含量随着浓度的增加而降低,VC3+F4、VE3+F4浓度组淋巴细胞端粒酶含量降低有统计学意义(P0.05)。 VC+VE各干预组以VC2+VE2+F4、VC3+VE3+F4组淋巴细胞端粒酶含量降低有统计学意义(P0.05),且VC2+VE2+F4浓度组降低更显著(P0.01)。结论在本研究条件下, 1.职业性氟接触组女性肿瘤发病率升高,肿瘤发病年龄提前,氟可能是一种潜在的致癌物。 2.氟可致实验动物小鼠染色体畸变、RNA/DNA结构的改变和体外培养人淋巴细胞细胞凋亡、DNA损伤、端粒酶含量升高,具有明显的遗传损伤效应,可能与肿瘤发生有关。 3.在一定剂量范围内,VC、VE对氟致淋巴细胞损伤有明显的干预作用。 4. ATR-FTIR可敏感地检测环境污染物引起的RNA、DNA.蛋白质等物质的改变,可作为早期遗传损伤监测的方法之一。
[Abstract]:Objective to explore the genetic toxicity of fluoride and vitamin C (VC), vitamin E (VE), vitamin C and vitamin E (VC+VE) on the intervention effect of fluoride on lymphocytes.
Investigation of 1. occupation of environmental fluoride pollution method: aluminum electrolytic gas purification workshop for pollution source, sampling points were set in the air fluoride working positions at different radii, investigate the relationship to determine the content of.2. occupation exposed to fluorine and tumor by fluoride ion selective electrode method: through the questionnaire survey and the hospital medical records factory workers from the end of 1995~2009 the tumor incidence of genetic damage in animal experimental study of statistical analysis: (1).3. fluoride detection by low, high concentration (2mg - kg-1 - d-18mg - kg-1 - d-1,32mg - kg-1 - D-1) mice bone marrow cell micronucleus rate of sodium fluoride exposure after 14d (2) with attenuation total reflection Fu Liye transform infrared spectroscopy (ATR-FTIR) in the RNA/DNA logo (1020/1121cm-1), wavelength band scanning, genetic damage and VC fluoride.4. to detect changes in the chemical structure of mouse liver nuclei, VE intervention Experimental study of cultured cells in vitro: peripheral blood lymphocytes from normal people, grouping and treatment after 48h culture: (1) 0.00,0.01,0.04,0.16,0.64mg/ml NaF (control group, F1, F2, F3, F4) respectively. 2h, 4h, 8h, 16h, MTT lymphocyte survival rate determination method. (2) lymphocytes into the control group, F1, F2, F3, F4, low, high dose of VC (VC1+F4, VC2+F4, VC3+F4 intervention group), low, high dose of VE (VE1+F4, VE2+F4, VE3+F4 intervention group), low, high dose of VC+VE (VC1+VE1+F4, VC2+VE2+F4, VC3+ intervention VE3+F4) control group. 28h group received medium, F1, F2, F3, F4 group received medium 24h respectively after exposure to NaF medium containing 0.01,0.04.0.16,0.64mg/ml 4H and VC 4.16,64 respectively in the intervention group containing mol/LVC medium 24h after exposure to 0.64mg/mlNaF medium 4h, VE group respectively with 2,10,50 mol/L VE medium 24h after exposure to 0.64mg/ml NaF culture Liquid 4h, VC+VE group respectively with 4+2,16+10,64+50 mol/L VC+VE cultured 24h after exposure to 0.64mg/ml NaF medium 4h, cells were collected, respectively. MTT method was used to test lymphocyte survival, apoptotic cells were detected by Annexin V-FITC apoptosis kit, single cell gel electrophoresis (SCGE) determination of DNA damage. Determination of telomerase Elisa assay.
Result
1. occupation of environmental fluoride pollution survey results: the fluorine pollution source of aluminum electrolysis gas purification plant as the center, the fluoride concentration in different radial distance in the air in order to reduce the work site, the negative trend of fluorine pollution diffusion concentration and distance, but did not exceed the national hygienic standard (0.50mg/m3).
Investigation on the relationship between the 2. occupation exposed to fluorine and cancer: the workers of crude cancer incidence rate of 117.95/10 million (the standardized rate was 58.81/10 million), the standardized incidence rate was higher in females than in males (male: female =1:2.64), the peak age of onset of tumors was 40-49 years ago, 2 tumors were liver cancer and lung cancer in men, women for breast cancer and lung cancer. And the area of non exposed population, the factory workers exposed to fluoride may make women increased tumor incidence, is 2.14 times the city's tumor early age of onset, tumor structure is basically the same.
3. fluorine genetic damage in animal experiments:
3.1 animal experimental mouse bone marrow cell micronucleus rate increased with the increase of fluoride concentration increased.
3.2 liver nuclear ATR spectra was decreased 1650cm-11550cm-11380cm-11260cm-11225cm-11155cm-11080cm-11030cm-1970cm-1930cm-1 peak spectral data; principal component analysis and 3-D graphics processing with statistical software, can be observed that the chemical structure of the more subtle change, compared with control group, fluoride, the difference of the high concentration group with first principal component factors, and the difference of fluoride and low concentration group based on second principal component factors, the effects of different fluoride concentration and chemical structure of the mouse liver cell nuclei were significantly different.
The genetic damage and VC 4. fluorine, experimental study of effects of VE on the in vitro cell culture:
4.1 cell survival rate increased with the increase of fluoride concentration and time decreased. Compared with the control group, F4 group significantly decreased cell viability in each treatment group (P0.05).16h cell survival rate were lower than 2H treatment group (P0.05).
4.2 lymphocyte fluoride 4h, (1) the total apoptosis rate with fluoride concentration increased, compared with the control group, the apoptosis rate were increased by 2.47%, 9.80%, 13.52%, 25.74%, in addition to the concentration of F1 group, the other groups were statistically significant increased (P0.05); (2) compared with the control group. With the increase of NaF concentrations, the trailing cell number increased, the comet head becomes small, increased brightness, long tail round, fluorescence intensity, the percentage of tail DNA, tail length, Olive tail moment increased in a dose dependent manner (P0.01); (3) telomerase content increased with increasing concentration. Compared with fluoride with the control group, F3 group, F4 concentration increased telomerase content was statistically significant (P0.05), and the concentration of F4 group was significantly higher than that of other groups of telomerase (P0.01).
The 4.3 group compared with the F4 group intervention of prevention, (1) VC, VE, VC+VE lymphocyte survival rate in each intervention group increased.VC lymphocyte survival rate were increased with increasing concentration, the survival rate of cells in VC3+F4 group increased significantly (P0.05), the combined effect of VC2+VE2+F4 group cell survival rate the most significant increase (P0.05); (2) VC, VE.VC VC+VE, the trend in each intervention group were decreased the apoptosis rate of lymphocytes, VE lymphocytes apoptosis rate in each intervention group with the concentration increased, VC2+F4, VC3+F4, VE3+F4 group had a significant decrease in apoptosis rate of lymphocytes (P0.05). Only VC. VE the total apoptosis rate lower than the intervention group VC+VE, but no significant difference (P0.05); (3) VC, VE, VC+VE of the intervention group head of the comet tail becomes larger and shorter, tail DNA, tail length, Olive tail moment were significantly decreased (P0.01), VE1 group, Olive tail moment most Short.VC+VE intervention group were higher than VC, VE groups of tail DNA, tail length and Olive tail moment (P0.05); (4) VC, VE, VC+VE of the intervention group had decreased.VC lymphocyte telomerase, VE lymphocytes were telomerase content decreased with the increase of the concentration of VC3+F4, VE3+ concentration of F4 group decreased the content of lymphocyte telomerase there was statistical significance (P0.05). VC+VE VC2+VE2+F4 in the intervention group, VC3+VE3+F4 group decreased the content of lymphocyte telomerase was statistically significant (P0.05), and the concentration of VC2+VE2+F4 group decreased more significantly (P0.01).
conclusion
Under the condition of this study,
The group of women increased tumor incidence of 1. occupation of fluoride exposure, tumor early age of onset, fluoride is a potential carcinogen.
2. fluorine can experimental animal mouse chromosome aberration, lymphocyte apoptosis, and in vitro culture change the structure of RNA/DNA DNA injury, the telomerase content increased, with significant genetic damage, may be related to tumorigenesis.
3. in a certain dose range, VC, VE has a significant intervention effect on lymphocyte damage induced by fluoride.
4. ATR-FTIR can sensitively detect the environmental pollution caused by RNA, the changes of substance DNA. protein, can be used as one of the methods for monitoring genetic damage in early stage.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

【参考文献】