氟的細胞損傷作用及硒和維生素E的干預(yù)效果研究
發(fā)布時間：2018-08-28 14:29
【摘要】：目的探討氟的細胞損傷作用及硒(Se)、維生素E(VE)的干預(yù)效果。 方法1.氟的細胞損傷作用研究 (1)動物實驗：選取SPF純種昆明小鼠,隨機分為：低、中、高氟組32mg/kg.d)和對照組(相應(yīng)體積生理鹽水),連續(xù)14天。末次注射后24h內(nèi)用梯度離心方法分離小鼠骨髓細胞作微核率檢測；分離肝細胞核、線粒體、微粒體三種亞細胞結(jié)構(gòu)并制作玻片,用傅里葉紅外光譜儀對各片進行掃描檢測,比較各組之間的差異。 (2)體外細胞培養(yǎng)實驗：抽取正常人外周靜脈血,用Ficoll密度梯度離心法提取淋巴細胞培養(yǎng)48h后,分組及處理：淋巴細胞分組為F1、F2、F3、F4和對照組,分別加入氟化鈉使終濃度為0.01mg/L0.04mg/L、0.16mg/L、0.64mg/L,對照組加等容培養(yǎng)液,加氟培養(yǎng)4h后收集細胞,分別檢測各孔淋巴細胞存活率(MTT法)、DNA損傷(單細胞凝膠電泳試驗SCGE)、凋亡率(Annexin V-FITC/PI法)、端粒酶含量(酶聯(lián)免疫法)。 2.Se、Se+VE對氟致細胞損傷作用的干預(yù)效果實驗研究 抽取正常人外周靜脈血,用Ficoll密度梯度離心法提取淋巴細胞培養(yǎng)48h后,分組及處理：0.64mg/ml氟組(F4)(下同)、Se10.005mg/L Se20.05mg/L、Se30.5mg/L干預(yù)組(Sel+F4、Se2+F4、Se3+F4(下同))、Se1+VE50umol/L、Se2+VE50umol/L、Se3+VE50umol/L干預(yù)組(Se1+VE+F4、Se2+VE+F4、Se3+VE+F4)(下同)。體外常規(guī)培養(yǎng)4h后收集細胞,分別檢測各孔淋巴細胞的存活率(MTT法)、凋亡率(Annexin V-FITC/PI法)、DNA損傷(單細胞凝膠電泳試驗SCGE)、端粒酶含量(酶聯(lián)免疫法)。 結(jié)果 1.氟的細胞損傷作用研究 (1)動物實驗 與對照組相比,中氟和高氟組小鼠骨髓細胞微核率升高有統(tǒng)計意義(P0.01)。紅外光譜分析(ATR-FTIR)顯示：染氟使實驗小鼠肝細胞核、線粒體、微粒體的化學成分發(fā)生改變,這些改變與細胞遺傳功能、微粒體代謝酶活力、線粒體代謝酶活力密切相關(guān)。 (2)體外細胞培養(yǎng)實驗 與對照組相比,各染氟組淋巴細胞活性均降低,F3、F4組細胞活性降低有統(tǒng)計學意義(P0.05)；各染氟組與對照組相比,細胞凋亡率隨著氟濃度的增加而逐漸升高,F2、F3、F4組細胞凋亡率升高有統(tǒng)計學意義(P0.05或P0.01)。 氟對DNA損傷的影響：表3-3顯示,經(jīng)氟作用4h后,與對照組相比,F1、F2、F3、F4組的彗星細胞率均升高,經(jīng)卡方檢驗,差異均有統(tǒng)計學意義(P0.01)。而實驗組的細胞彗星尾長(Tail Length)、尾部DNA%、尾距(TailMoment)、Olive尾距(Olive Tail Moment)均大于對照組,其中0.04～0.64mg/mL (F2、F3、F4)氟組與對照組比較,差異均有統(tǒng)計學意義。 氟對端粒酶含量的影響：加氟培養(yǎng)4h后,染氟組淋巴細胞端粒酶含量逐漸升高,其中F3組端粒酶含量與對照相比,差異有統(tǒng)計學意義(P0.05),F4組端粒酶含量明顯高于其他各組。 2.Se、Se+VE對氟致細胞損傷的干預(yù)效果研究 2.1Se、Se+VE對氟致淋巴細胞活性下降的干預(yù)效果：Se1+F4、Se2+F4、Se3+F4干預(yù)組與F4組相比,淋巴細胞活性有所升高,且隨著Se的濃度增加而逐漸升高,Se2+F4、Se3+F4干預(yù)組細胞活性升高有統(tǒng)計學意義(P0.05,P0.01)。Se+VE干預(yù)組中,只有Se3+VE+F4組細胞活性明顯高于F4組(P0.01)。Se+VE各干預(yù)組與對應(yīng)的Se單獨干預(yù)組相比,細胞活性差異無統(tǒng)計學意義(P0.05)。 2.2Se、Se+VE對氟致淋巴細胞凋亡率升高的干預(yù)效果：與F4組比較,Se1+F4、Se2+F4、Se3+F4、Se1+VE+F4、Se2+VE+F、Se3+VE+F4干預(yù)組細胞凋亡率均有下降趨勢,除了Se2+VE+F4組外,其他干預(yù)組凋亡率下降均有統(tǒng)計學意義(P0.05或P0.01)；Se干預(yù)組與相應(yīng)的Se+VE干預(yù)組相比,組間差異無統(tǒng)計學意義(P0.05)。 2.3Se、Se+VE對氟損傷淋巴細胞DNA的干預(yù)效果：Se、Se+VE干預(yù)組與F4比較,細胞彗星尾長(Tail Length)、尾部DNA%、尾距(Tail Moment)、Olive尾距(Olive Tail Moment)均降低,差異均有統(tǒng)計學意義(P0.05,或P0.01)；Se干預(yù)組與相應(yīng)的Se+VE干預(yù)組相比,組間差異無統(tǒng)計學意義(P0.05)。 2.4Se、Se+VE對氟致淋巴細胞端粒酶含量升高的干預(yù)效果：Se、Se+VE干預(yù)組與F4比較,端粒酶含量隨Se濃度的升高而降低,各組端粒酶含量差異有統(tǒng)計學意義(P0.05或P0.01)；Se干預(yù)組與相應(yīng)的Se+VE干預(yù)組相比,組間差異無統(tǒng)計學意義(P0.05)。 結(jié)論 1.染氟可致小鼠骨髓細胞微核率升高； 2.染氟使肝細胞核、線粒體、微粒體的化學成分發(fā)生改變,這些改變與細胞遺傳功能、微粒體和線粒體代謝酶活力密切相關(guān)； 3.氟對體外培養(yǎng)淋巴細胞也具有明顯的遺傳損傷效應(yīng),可致細胞凋亡率升高、DNA損傷增加、端粒酶含量升高,這些改變可能與腫瘤發(fā)生有關(guān)； 4.在本實驗劑量范圍內(nèi),Se、Se+VE對氟致淋巴細胞損傷有明顯的干預(yù)作用； 5. ATR-FTIR可敏感地檢測氟引起RNA、DNA、蛋白質(zhì)等物質(zhì)的化學結(jié)構(gòu)改變,可望作為早期遺傳損傷監(jiān)測的方法之一。
[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.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R114
本文編號：2209660
[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.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R114
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