5-HT受体基因多态性与电磁辐射致认知功能改变易感性的相关性研究

发布时间：2018-02-25 04:08

本文关键词： 电磁辐射微波辐射学习记忆 5-HT受体单核苷酸多态性　出处：《中国人民解放军军事医学科学院》2015年博士论文　论文类型：学位论文

【摘要】：目的意义:随着现代科技应用领域的不断扩展,电磁辐射日益成为威胁人类健康的环境因素。中枢神经系统是电磁辐射的敏感靶器官之一,但电磁辐射对中枢神经系统的影响及机制研究目前尚无统一定论。5-HT系统在大脑发育、认知功能及情绪调节中发挥着重要作用,基因多态性是导致不同个体间生物性状差异的遗传学基础,5-HT受体基因多态性是否在电磁辐射生物效应的易感性中发挥作用尚不明确。本研究从认知功能着手,以SNP为遗传标记,着眼于5-HT_(1A/2A/2C)R基因,展开5-HT受体基因多态性与电磁辐射致认知功能改变易感性的相关性研究,以期筛选出对电磁辐射易感的相关基因SNP位点,为电磁暴露人群早期的防诊治和职业电磁操作人员的遴选提供候选指标。材料方法 :(1)辐射方法:2.856 GHz微波辐射,在体实验:以Wistar大鼠为研究对象,平均功率密度5、10、20、30 m W/cm_2,3次/周,持续6周;离体实验:以PC12及293T细胞系为研究对象,平均功率密度30 m W/cm_2,5 min×3次,每次间隔5 min;(2)MWM检测大鼠空间学习记忆功能;(3)多导生理记录及分析系统记录分析大鼠脑电图;(4)HE染色观察大鼠海马组织结构;(5)HPLC检测大鼠海马和脑脊液中氨基酸及单胺类神经递质含量;(6)MAO活性检测试剂盒检测大鼠海马MAO活性;(7)WB检测大鼠海马TPH1含量及5-HT_(1A/2A/2C)R表达量;(8)免疫组化检测大鼠大脑皮层和海马5-HT_(1A/2A/2C)R表达量;(9)全血提取大鼠DNA,PCR扩增5-HT_(1A/2A/2C)R基因启动子区序列,测序法筛查SNP位点;(10)报告基因及EMSA实验检测候选SNP不同等位基因转录活性及微波辐射对不同等位基因转录活性的影响;(11)Real-Time PCR检测各基因型大鼠5-HT_1AR m RNA水平;(12)问卷调查和量表收集电厂职工基本资料并评估其精神状态;(13)采集电厂职工血液标本,提取DNA后采用RFLP方法针对5-HT_(1A/2A/2C)R的SNP位点rs6311、rs6313、rs6314、rs6318、rs6295对人群进行基因分型;检测血常规、血清生化、BNP、LENK及NPY,并记录ECG及ERPs等。结果:1.5-HT系统在微波辐射致认知功能改变中的作用:(1)空间学习记忆功能:长期微波辐射(5、10、20、30 m W/cm_2)可导致大鼠空间学习记忆功能降低。(2)脑电图:辐射后14 d,各剂量组大鼠脑电图与假辐射组相比波幅增加、频率降低、δ频段功率百分比增加。(3)海马组织结构:辐射后14 d、28 d及2m各剂量组大鼠海马组织部分神经元固缩深染,以齿状回和CA3区损伤明显,辐射后28 d,30 m W/cm_2剂量组大鼠损伤程度最为严重。(4)神经递质:微波辐射后氨基酸及单胺类神经递质紊乱,其中5-HT改变主要为远期效应:辐射后28 d,10、20及30 m W/cm_2剂量组海马组织5-HT增加;辐射后2m,各辐射剂量组5-HT均明显增加,而其代谢产物5-HIAA明显降低。(5)5-HT合成及代谢中的限速酶TPH1及MAO:30 m W/cm_2微波辐射后大鼠海马TPH1表达未发生明显改变,辐射后28 d及2 m,MAO活性明显降低。(6)5-HT_(1A/2A/2C)R表达:WB结果表明,海马及大脑皮层5-HT2AR表达量无明显改变。5-HT_1AR及5-HT2CR在海马组织的表达量于辐射后14 d及2 m明显增加,28 d时明显降低;免疫组化结果表明,辐射后14 d,5-HT_1AR在海马CA1、CA2、CA3区表达明显增加,辐射后28 d,5-HT_1AR在海马CA1、CA2区表达明显增加,辐射后2m,5-HT_1AR在海马CA3区表达量增加。辐射后14 d及2 m,5-HT2CR表达量在CA3区显著增加,辐射后28 d,其在CA2区表达量明显降低。WB及免疫组化结果均表明5-HT_1AR及5-HT2CR在大鼠大脑皮层的表达于辐射后28d明显增加。2.大鼠5-HT受体基因启动子区SNP位点与微波辐射致认知功能改变易感性的相关性:(1)5-HTR基因启动子区SNP位点筛查:在不同动物中心的Wistar大鼠5-HT_(1A/2A/2C)R基因启动子区筛查到两个SNP位点:5-HT_1AR-215 TC(rs198585630)及5-HT2AR 74_75 ins G,Wistar大鼠rs198585630基因分型为TT、TC、CC基因型。(2)5-HT_1AR rs198585630功能研究:①包含rs198585630-C等位基因的序列结构启动荧光素酶报告基因的表达功能强于包含T等位的序列结构,30 m W/cm_2微波辐射后含rs198585630-C的序列结构启动报告基因的表达功能明显增强;②C等位基因与PC12细胞核蛋白的结合能力明显高于T等位基因,30 m W/cm_2微波辐射PC12细胞后,T、C等位基因与核蛋白的结合能力均明显增强,C等位与辐射后细胞核蛋白的结合能力明显强于T等位。(3)5-HT_1AR rs198585630与微波辐射后大鼠认知功能改变易感性的相关性研究:①空间学习能力:TT型大鼠空间学习能力明显低于TC及CC型大鼠,30 m W/cm_2微波辐射后TT型大鼠空间学习能力增强,TC、CC型大鼠空间学习、记忆能力降低;②脑电图:辐射后14 d大鼠脑电图波幅增加、频率降低、δ频段功率百分比明显升高,其中TC、CC型大鼠δ频段功率百分比的增加幅度高于TT型大鼠;③神经递质:TT型大鼠辐射后5-HIAA含量有降低趋势而5-HT含量有升高趋势,但未见统计学差异,5-HIAA/5-HT比值明显降低;④5-HT_1AR表达:TC及CC型大鼠海马组织5-HT_1AR m RNA及蛋白水平于微波辐射后明显上调,辐射组中TC及CC型大鼠5-HT_1AR m RNA及蛋白水平较TT型明显增加。3.5-HTR基因多态性与人群电磁辐射易感性关联的初步探讨:(1)心脑功能ECG、ERPs等与电磁辐射暴露剂量相关:操作组及巡检组工人较对照组心电图异常发生率增加(主要为心率不齐、心动过缓、T波改变等);巡检组电力工人N2潜伏期与对照组及操作组比较,显著增加;操作组较对照组BNP明显增加;巡检组较操作组LENK含量显著上升。(2)5-HT_1AR rs6295、5-HT2AR rs6313与辐射暴露剂量的交互作用对心脑功能的影响具有统计学意义:rs6313 TT型随暴露剂量增加,N2、P3潜伏期显著延长,与CC型比较有统计学差异;rs6295 GG型随暴露剂量增加,LENK含量增加,N2潜伏期显著延长,与CG型比较有统计学差异。rs6313 TT型、rs6295 GG型对电磁辐射易感性高。结论:(1)长期微波(2.856 GHz,平均功率密度5、10、20及30 m W/cm_2)辐射可致大鼠认知功能损伤。(2)5-HT系统参与微波辐射所致认知功能受损。(3)大鼠5-HT_1AR基因启动子区SNP-215 TC为功能性位点,包含C等位的启动子区转录活性高于T等位,且C等位对微波辐射更为易感。(4)5-HT_1AR-215 TC的TC、CC型大鼠于微波辐射后5-HT_1AR高表达,促使认知功能损伤。(5)长期电力作业可对工人心脑功能产生一定的影响。(6)5-HT_1AR rs6295及5-HT2AR rs6313可能作为电磁辐射作业人员筛选的候选靶点。
[Abstract]:Purpose: with the continuous expansion of the application fields of modern science and technology, electromagnetic radiation has become a threat to human health and environmental factors. The central nervous system is one of the electromagnetic radiation sensitive organs, but the effects of electromagnetic radiation on the central nervous system and mechanism at present there is no unified conclusion of.5-HT system in brain development, cognitive function and play an important role emotion regulation, gene polymorphism is the result of the genetic basis for differences in biological traits among different individuals, whether 5-HT receptor gene polymorphism and susceptibility to radiation in the electromagnetic biological effects play a role is not clear. This study from the cognitive function in hand, with SNP as the genetic marker, which focuses on 5-HT_ (1A/2A/2C) R gene, correlation study 5-HT receptor gene polymorphism and susceptibility to electromagnetic radiation caused by changes in cognitive function, in order to screen the electromagnetic radiation susceptible genes related to SNP Point selection, early diagnosis and prevention in occupation operations personnel to provide a candidate target electromagnetic electromagnetic exposure. Materials and methods: (1): 2.856 GHz microwave radiation method, the in vivo experiment: Wistar rats as the research object, the average power density of 5,10,20,30 m W/cm_2,3 times / week, for 6 weeks in vitro; in PC12 and 293T cell lines as the research object, the average power density of 30 m W/cm_2,5 min * 3 times, each time interval of 5 min; (2) the learning and memory function of MWM rats was detected; (3) multi channel physiological analysis and rat EEG recording analysis system; (4) to observe the hippocampal structure in rats HE staining; (5) the content of amino acid and monoamine neurotransmitters in rat hippocampus and cerebrospinal fluid HPLC detection; (6) MAO kit to detect the activity of rat hippocampal MAO activity; (7) WB rats were detected TPH1 content in hippocampus and the expression of R 5-HT_ (1A/2A/2C); (8) immunohistochemical detection of cerebral cortex rat 5-HT_ (1A/2A/2C) in hippocampus and the expression of R; (9) blood from the rat DNA, PCR amplification of 5-HT_ (1A/2A/2C) R gene promoter sequence, sequencing SNP loci; (10) effects of candidate gene and EMSA test SNP test report of transcription activity of different alleles and microwave radiation on isoallele transcriptional activity; (11) to detect the level of RNA of each genotype Real-Time PCR rats 5-HT_1AR m; (12) questionnaire and questionnaire to collect basic data and evaluate the mental state of the workers of power plants; (13) collecting power plant workers blood samples, DNA was extracted using RFLP method for 5-HT_ (1A/2A/2C) R SNP rs6311 site rs6313, rs6314, rs6318, rs6295, on the population were genotyped; blood routine examination, serum biochemistry, BNP, LENK and NPY, ECG and ERPs were recorded. Results: 1.5-HT radiation induced changes of cognitive function in the role of microwave: (1) spatial learning and memory function: long term microwave radiation (5,10,20,30 m W/cm_2) can lead to rat spatial learning and memory function decreased. (2) EEG: 14 d after irradiation, the dose group rat EEG amplitude increased compared with the sham radiation group, reduce the frequency of delta band power percentage increased. (3) the organizational structure of the hippocampus: after radiation of 14 D, 28 d and 2m each dose group of rat hippocampus neurons pycnosised damage to dentate gyrus and CA3 areas, after exposure to 28 d, 30 m W/cm_2 dose group rat damage was the most severe. (4) after microwave radiation: neurotransmitter amino acid and monoamine neurotransmitter disorder, which is the main feature of 5-HT long term effect radiation: after 28 d 10,20 and 30 m W/cm_2 group in hippocampus 5-HT increased; 2m after radiation, the radiation dose of 5-HT group was significantly increased, and its metabolite 5-HIAA decreased obviously. (5) in hippocampus of rats with TP TPH1 and MAO:30 m enzyme W/cm_2 microwave radiation synthesis and metabolism of 5-HT The expression of H1 was not significantly changed after radiation, 28 d and 2 m, MAO activity decreased significantly. (6) 5-HT_ (1A/2A/2C) R expression: WB results showed that the expression of 5-HT2AR in hippocampus and cerebral cortex expression had no obvious change in.5-HT_1AR and 5-HT2CR in the hippocampus after radiation of 14 d and 2 m increased significantly. 28 d decreased significantly; immunohistochemistry results showed that 14 d after radiation, 5-HT_1AR in the hippocampus of CA1, CA2, CA3 expression increased significantly after irradiation of 28 d, 5-HT_1AR CA1 in the hippocampus, CA2 expression increased significantly after 2m radiation, the expression of 5-HT_1AR in hippocampus CA3 region increased after radiation and 14 D. 2 m, 5-HT2CR expression increased significantly in the CA3 region, 28 d after irradiation, the expression decreased.WB and immunohistochemical results showed that the expression of 5-HT_1AR and 5-HT2CR in the cerebral cortex of rats after radiation 28d significantly increased 5-HT receptor gene of.2. rat SNP promoter start sites and microwave radiation induced cognitive in CA2 area The correlation function change of susceptibility: (1) 5-HTR gene promoter SNP loci screening in different animal center Wistar rat 5-HT_ (1A/2A/2C) R gene promoter screening to two SNP loci: 5-HT_1AR-215 TC (rs198585630) and 5-HT2AR 74_75 ins G, Wistar of rat rs198585630 gene type was TT, TC CC, gene type. (2) study the function of rs198585630 5-HT_1AR: sequence structure sequence structure containing rs198585630-C allele promoter luciferase reporter gene expression function to contain T alleles, sequence structure of rs198585630-C containing 30 m W/cm_2 after microwave radiation to initiate the expression of reporter gene function was enhanced; the binding capacity of C PC12 gene and nuclear protein was significantly higher than that of T allele 30 m microwave radiation W/cm_2 PC12 cells, T binding ability of C allele and nuclear protein were significantly enhanced, C and nuclear radiation The protein binding ability was stronger than the T allele (3). The correlation between the susceptibility of cognitive function changes in rats 5-HT_1AR and rs198585630 after microwave radiation: the spatial learning ability: TT spatial learning ability of rats was significantly lower than that of TC and CC in rats, TT rats spatial learning ability enhancement, 30 m microwave W/cm_2 after the radiation of TC, CC type rats learning memory ability decreased; the EEG: after radiation of 14 d rats increased the amplitude of EEG frequency decreased, delta band power percentage increased significantly, including TC, CC rats increased the amplitude of the delta band power percentage is higher than that of the TT rats; the neurotransmitters: 5-HIAA the content of TT in rats after radiation decreased while the content of 5-HT increased, but no significant difference, 5-HIAA/5-HT ratio decreased significantly; the expression of 5-HT_1AR in hippocampus of TC and CC: 5-HT_1AR rats m RNA and protein levels in microwave radiation significantly TC increase, and CC type radiation group in the rat 5-HT_1AR m RNA and protein level was significantly increased compared with TT type of.3.5-HTR gene polymorphism and susceptibility of the population of electromagnetic radiation: (1) the cerebral function ECG, ERPs and other related electromagnetic radiation exposure dose: operation group and inspection group of people than in the control group of ECG abnormalities the rate of increase (mainly for arrhythmia, bradycardia, T wave changes etc.); inspection group significantly increased the latency of N2 electrical workers and control group and operation group, operation group than in the control group; BNP group was significantly increased; the inspection operation group LENK increased significantly. The content of rs6313 and 5-HT_1AR (2) rs6295,5-HT2AR radiation the exposure was statistically significant interaction effect of dosage on cerebral function: rs6313 TT with the exposure dose increased, N2, P3 latency was significantly prolonged, with significant difference compared with CC type; rs6295 type GG with exposure dose increasing, the content of LENK Increased N2 latency was significantly prolonged, there were significant differences in.Rs6313 TT type compared with CG type, rs6295 GG type of electromagnetic radiation of high susceptibility. Conclusion: (1) long term microwave (2.856 GHz, the average power density of 5,10,20 and 30 m W/cm_2) cognitive function injury in rats can cause radiation. (2) 5-HT system in microwave radiation impaired cognitive function. (3) the rat 5-HT_1AR gene promoter SNP-215 TC as functional sites, transcriptional activity of promoter containing C allele was higher than that of T allele and C allele of microwave radiation is more susceptible. (4) 5-HT_1AR-215 TC TC, CC in rats after microwave radiation the high expression of 5-HT_1AR, promote cognitive function damage. (5) long term work may have a certain impact on the cardiovascular function of workers. (6) 5-HT_1AR rs6295 and 5-HT2AR rs6313 may be used as electromagnetic radiation workers screening candidate targets.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R594.8

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