氟的细胞损伤作用及硒和维生素E的干预效果研究

发布时间：2018-08-28 14:29

【摘要】：目的探讨氟的细胞损伤作用及硒(Se)、维生素E(VE)的干预效果。方法1.氟的细胞损伤作用研究 (1)动物实验：选取SPF纯种昆明小鼠,随机分为：低、中、高氟组32mg/kg.d)和对照组(相应体积生理盐水),连续14天。末次注射后24h内用梯度离心方法分离小鼠骨髓细胞作微核率检测；分离肝细胞核、线粒体、微粒体三种亚细胞结构并制作玻片,用傅里叶红外光谱仪对各片进行扫描检测,比较各组之间的差异。 (2)体外细胞培养实验：抽取正常人外周静脉血,用Ficoll密度梯度离心法提取淋巴细胞培养48h后,分组及处理：淋巴细胞分组为F1、F2、F3、F4和对照组,分别加入氟化钠使终浓度为0.01mg/L0.04mg/L、0.16mg/L、0.64mg/L,对照组加等容培养液,加氟培养4h后收集细胞,分别检测各孔淋巴细胞存活率(MTT法)、DNA损伤(单细胞凝胶电泳试验SCGE)、凋亡率(Annexin V-FITC/PI法)、端粒酶含量(酶联免疫法)。 2.Se、Se+VE对氟致细胞损伤作用的干预效果实验研究抽取正常人外周静脉血,用Ficoll密度梯度离心法提取淋巴细胞培养48h后,分组及处理：0.64mg/ml氟组(F4)(下同)、Se10.005mg/L Se20.05mg/L、Se30.5mg/L干预组(Sel+F4、Se2+F4、Se3+F4(下同))、Se1+VE50umol/L、Se2+VE50umol/L、Se3+VE50umol/L干预组(Se1+VE+F4、Se2+VE+F4、Se3+VE+F4)(下同)。体外常规培养4h后收集细胞,分别检测各孔淋巴细胞的存活率(MTT法)、凋亡率(Annexin V-FITC/PI法)、DNA损伤(单细胞凝胶电泳试验SCGE)、端粒酶含量(酶联免疫法)。结果 1.氟的细胞损伤作用研究 (1)动物实验与对照组相比,中氟和高氟组小鼠骨髓细胞微核率升高有统计意义(P0.01)。红外光谱分析(ATR-FTIR)显示：染氟使实验小鼠肝细胞核、线粒体、微粒体的化学成分发生改变,这些改变与细胞遗传功能、微粒体代谢酶活力、线粒体代谢酶活力密切相关。 (2)体外细胞培养实验与对照组相比,各染氟组淋巴细胞活性均降低,F3、F4组细胞活性降低有统计学意义(P0.05)；各染氟组与对照组相比,细胞凋亡率随着氟浓度的增加而逐渐升高,F2、F3、F4组细胞凋亡率升高有统计学意义(P0.05或P0.01)。氟对DNA损伤的影响：表3-3显示,经氟作用4h后,与对照组相比,F1、F2、F3、F4组的彗星细胞率均升高,经卡方检验,差异均有统计学意义(P0.01)。而实验组的细胞彗星尾长(Tail Length)、尾部DNA%、尾距(TailMoment)、Olive尾距(Olive Tail Moment)均大于对照组,其中0.04～0.64mg/mL (F2、F3、F4)氟组与对照组比较,差异均有统计学意义。氟对端粒酶含量的影响：加氟培养4h后,染氟组淋巴细胞端粒酶含量逐渐升高,其中F3组端粒酶含量与对照相比,差异有统计学意义(P0.05),F4组端粒酶含量明显高于其他各组。 2.Se、Se+VE对氟致细胞损伤的干预效果研究 2.1Se、Se+VE对氟致淋巴细胞活性下降的干预效果：Se1+F4、Se2+F4、Se3+F4干预组与F4组相比,淋巴细胞活性有所升高,且随着Se的浓度增加而逐渐升高,Se2+F4、Se3+F4干预组细胞活性升高有统计学意义(P0.05,P0.01)。Se+VE干预组中,只有Se3+VE+F4组细胞活性明显高于F4组(P0.01)。Se+VE各干预组与对应的Se单独干预组相比,细胞活性差异无统计学意义(P0.05)。 2.2Se、Se+VE对氟致淋巴细胞凋亡率升高的干预效果：与F4组比较,Se1+F4、Se2+F4、Se3+F4、Se1+VE+F4、Se2+VE+F、Se3+VE+F4干预组细胞凋亡率均有下降趋势,除了Se2+VE+F4组外,其他干预组凋亡率下降均有统计学意义(P0.05或P0.01)；Se干预组与相应的Se+VE干预组相比,组间差异无统计学意义(P0.05)。 2.3Se、Se+VE对氟损伤淋巴细胞DNA的干预效果：Se、Se+VE干预组与F4比较,细胞彗星尾长(Tail Length)、尾部DNA%、尾距(Tail Moment)、Olive尾距(Olive Tail Moment)均降低,差异均有统计学意义(P0.05,或P0.01)；Se干预组与相应的Se+VE干预组相比,组间差异无统计学意义(P0.05)。 2.4Se、Se+VE对氟致淋巴细胞端粒酶含量升高的干预效果：Se、Se+VE干预组与F4比较,端粒酶含量随Se浓度的升高而降低,各组端粒酶含量差异有统计学意义(P0.05或P0.01)；Se干预组与相应的Se+VE干预组相比,组间差异无统计学意义(P0.05)。结论 1.染氟可致小鼠骨髓细胞微核率升高； 2.染氟使肝细胞核、线粒体、微粒体的化学成分发生改变,这些改变与细胞遗传功能、微粒体和线粒体代谢酶活力密切相关； 3.氟对体外培养淋巴细胞也具有明显的遗传损伤效应,可致细胞凋亡率升高、DNA损伤增加、端粒酶含量升高,这些改变可能与肿瘤发生有关； 4.在本实验剂量范围内,Se、Se+VE对氟致淋巴细胞损伤有明显的干预作用； 5. ATR-FTIR可敏感地检测氟引起RNA、DNA、蛋白质等物质的化学结构改变,可望作为早期遗传损伤监测的方法之一。
[Abstract]:Objective to investigate the cellular damage effect of fluoride and the intervention effect of selenium (Se) and vitamin E (VE).
Methods 1. cell damage induced by fluoride.
(1) Animal experiment: Kunming mice were randomly divided into two groups: low, medium and high fluoride group (32mg/kg.d) and control group (corresponding volume of normal saline) for 14 days. Fourier transform infrared spectrometer was used to scan the slices and compare the differences among the groups.
(2) In vitro cell culture experiment: normal peripheral venous blood was extracted and lymphocytes were cultured for 48 hours by Ficoll density gradient centrifugation. Lymphocytes were divided into F1, F2, F3, F4 and control groups. The final concentration of sodium fluoride was 0.01mg/L, 0.04mg/L, 0.16mg/L, 0.64mg/L, and the control group was cultured for 4 hours with fluoride. Cells were collected and the survival rate (MTT), DNA damage (SCGE), apoptosis rate (Annexin V-FITC/PI) and telomerase content (ELISA) of lymphocytes were measured.
Experimental study on intervention effect of 2.Se and Se+VE on fluoride induced cell injury
After 48 hours of lymphocyte culture, the normal PeripheralVenous blood was extracted and lymphocytes were cultured by Ficoll density gradientient centrifugation for 48 hours. Then the lymphocytes were divided into 0.64mg/ml fluoride group (F4) (the same below), Se10.005mg/L Se20.05mg/L, Se30.5mg/L intervention group (Sel + F4, Se2 + F4, Se3 + F4 (the same below), Se1 + VE50umol/L, Se2 + VE50umol/L intervention group (Se1 + VE + VE + F4, Se2 + F4, 2 + F4, Se3 + VE3 + VE3 + VE50 umol/L intervention group) (Se1 + VE1 + VE + VE + VE + VE + VE + VE + F4, Se2 + 2 + F4, Se3 + In the meantime, it is necessary to study the relationship between the two. Cells were collected after 4 hours of conventional culture in vitro. The survival rate (MTT), apoptosis rate (Annexin V-FITC/PI), DNA damage (SCGE) and telomerase content (ELISA) of lymphocytes were detected.
Result
1. cell damage induced by fluoride
(1) animal experiments
Compared with the control group, the micronucleus rate of bone marrow cells in the medium fluoride and high fluoride groups increased significantly (P 0.01). Infrared spectrum analysis (ATR-FTIR) showed that fluoride could change the chemical composition of hepatocyte nucleus, mitochondria and microsome in the experimental mice. These changes were closely related to cell genetic function, microsomal metabolic enzyme activity and mitochondrial metabolic enzyme activity. Cut the correlation.
(2) in vitro cell culture experiment
Compared with the control group, the lymphocyte activity of fluoride-exposed groups decreased, and the cell activity of F3 and F4 groups decreased significantly (P 0.05); compared with the control group, the apoptosis rate of fluoride-exposed groups increased gradually with the increase of fluoride concentration, and the apoptosis rate of F2, F3 and F4 groups increased significantly (P 0.05 or P 0.01).
The effect of fluoride on DNA damage: Table 3-3 shows that the comet cell rates in F1, F2, F3 and F4 groups were significantly higher than those in the control group after 4 hours of fluoride exposure (P 0.01). The comet tail length (Tail Length), tail DNA, Tail Moment and Olive Tail Moment in the experimental group were all larger than those in the control group. The difference was statistically significant between the 0.04 ~ 0.64mg/mL (F2, F3, F4) fluorine group and the control group.
Effect of fluoride on telomerase content: Telomerase content of lymphocytes in fluoride-exposed group increased gradually after 4 hours of culture. Telomerase content in F3 group was significantly higher than that in control group (P 0.05). Telomerase content in F4 group was significantly higher than that in other groups.
Intervention effect of 2.Se and Se+VE on fluoride induced cell injury
Intervention effect of 2.1Se, Se+VE on fluoride-induced decrease of lymphocyte activity: Compared with F4 group, the activity of lymphocyte in Se1+F4, Se2+F4, Se3+F4 intervention group was higher, and gradually increased with the increase of Se concentration. The increase of cell activity in Se2+F4, Se3+F4 intervention group was statistically significant (P 0.05, P 0.01). Compared with F4 group (P 0.01). There was no significant difference in cell activity between the SE + VE intervention group and the corresponding SE alone intervention group (P 0.05).
2.2Se, Se+VE intervention effect on fluoride-induced lymphocyte apoptosis rate: compared with F4 group, the apoptosis rate of Se1+F4, Se2+F4, Se3+F4, Se1+VE+F4, Se2+VE+F, Se3+VE+F4 intervention group had a downward trend, except Se2+VE+F4 group, the other intervention group apoptosis rate decreased significantly (P 0.05 or P 0.01); There was no significant difference between the two groups (P0.05).
Intervention effect of 2.3Se and Se+VE on DNA of fluoride-injured lymphocytes: Compared with F4, tail Length, tail DNA, tail Moment and Olive Tail Moment were all decreased in Se and Se+VE intervention group, and there was no statistical difference between the two groups (P 0.05, or P 0.01). Academic meaning (P0.05).
Intervention effect of 2.4Se and Se+VE on the elevation of telomerase content in lymphocytes induced by fluoride: Telomerase content decreased with the elevation of Se concentration in the Se and Se+VE intervention group compared with F4, and the difference was statistically significant (P 0.05 or P 0.01); there was no significant difference between the Se intervention group and the corresponding Se+VE intervention group (P 0.05).
conclusion
1. fluoride exposure can increase the micronucleus rate of bone marrow cells in mice.
2. Fluoride exposure alters the chemical composition of hepatocyte nucleus, mitochondria and microsomes, which are closely related to cellular genetic function, microsome and mitochondrial metabolic enzyme activity.
3. Fluoride also has obvious genetic damage effect on cultured lymphocytes in vitro, which can increase the apoptosis rate, DNA damage and telomerase content. These changes may be related to tumorigenesis.
4. in the dose range of this experiment, Se and Se+VE have obvious intervention effect on lymphocyte damage induced by fluorine.
5. ATR-FTIR can sensitively detect the chemical structure changes of RNA, DNA, protein and other substances caused by fluoride. It is expected to be one of the early genetic damage monitoring methods.
【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

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