本文關(guān)鍵詞： 電磁輻射 微波輻射 學(xué)習(xí)記憶 5-HT受體 單核苷酸多態(tài)性　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2015年博士論文　論文類型：學(xué)位論文

【摘要】：目的意義:隨著現(xiàn)代科技應(yīng)用領(lǐng)域的不斷擴展,電磁輻射日益成為威脅人類健康的環(huán)境因素。中樞神經(jīng)系統(tǒng)是電磁輻射的敏感靶器官之一,但電磁輻射對中樞神經(jīng)系統(tǒng)的影響及機制研究目前尚無統(tǒng)一定論。5-HT系統(tǒng)在大腦發(fā)育、認知功能及情緒調(diào)節(jié)中發(fā)揮著重要作用,基因多態(tài)性是導(dǎo)致不同個體間生物性狀差異的遺傳學(xué)基礎(chǔ),5-HT受體基因多態(tài)性是否在電磁輻射生物效應(yīng)的易感性中發(fā)揮作用尚不明確。本研究從認知功能著手,以SNP為遺傳標(biāo)記,著眼于5-HT_(1A/2A/2C)R基因,展開5-HT受體基因多態(tài)性與電磁輻射致認知功能改變易感性的相關(guān)性研究,以期篩選出對電磁輻射易感的相關(guān)基因SNP位點,為電磁暴露人群早期的防診治和職業(yè)電磁操作人員的遴選提供候選指標(biāo)。材料方法 :(1)輻射方法:2.856 GHz微波輻射,在體實驗:以Wistar大鼠為研究對象,平均功率密度5、10、20、30 m W/cm_2,3次/周,持續(xù)6周;離體實驗:以PC12及293T細胞系為研究對象,平均功率密度30 m W/cm_2,5 min×3次,每次間隔5 min;(2)MWM檢測大鼠空間學(xué)習(xí)記憶功能;(3)多導(dǎo)生理記錄及分析系統(tǒng)記錄分析大鼠腦電圖;(4)HE染色觀察大鼠海馬組織結(jié)構(gòu);(5)HPLC檢測大鼠海馬和腦脊液中氨基酸及單胺類神經(jīng)遞質(zhì)含量;(6)MAO活性檢測試劑盒檢測大鼠海馬MAO活性;(7)WB檢測大鼠海馬TPH1含量及5-HT_(1A/2A/2C)R表達量;(8)免疫組化檢測大鼠大腦皮層和海馬5-HT_(1A/2A/2C)R表達量;(9)全血提取大鼠DNA,PCR擴增5-HT_(1A/2A/2C)R基因啟動子區(qū)序列,測序法篩查SNP位點;(10)報告基因及EMSA實驗檢測候選SNP不同等位基因轉(zhuǎn)錄活性及微波輻射對不同等位基因轉(zhuǎn)錄活性的影響;(11)Real-Time PCR檢測各基因型大鼠5-HT_1AR m RNA水平;(12)問卷調(diào)查和量表收集電廠職工基本資料并評估其精神狀態(tài);(13)采集電廠職工血液標(biāo)本,提取DNA后采用RFLP方法針對5-HT_(1A/2A/2C)R的SNP位點rs6311、rs6313、rs6314、rs6318、rs6295對人群進行基因分型;檢測血常規(guī)、血清生化、BNP、LENK及NPY,并記錄ECG及ERPs等。結(jié)果:1.5-HT系統(tǒng)在微波輻射致認知功能改變中的作用:(1)空間學(xué)習(xí)記憶功能:長期微波輻射(5、10、20、30 m W/cm_2)可導(dǎo)致大鼠空間學(xué)習(xí)記憶功能降低。(2)腦電圖:輻射后14 d,各劑量組大鼠腦電圖與假輻射組相比波幅增加、頻率降低、δ頻段功率百分比增加。(3)海馬組織結(jié)構(gòu):輻射后14 d、28 d及2m各劑量組大鼠海馬組織部分神經(jīng)元固縮深染,以齒狀回和CA3區(qū)損傷明顯,輻射后28 d,30 m W/cm_2劑量組大鼠損傷程度最為嚴重。(4)神經(jīng)遞質(zhì):微波輻射后氨基酸及單胺類神經(jīng)遞質(zhì)紊亂,其中5-HT改變主要為遠期效應(yīng):輻射后28 d,10、20及30 m W/cm_2劑量組海馬組織5-HT增加;輻射后2m,各輻射劑量組5-HT均明顯增加,而其代謝產(chǎn)物5-HIAA明顯降低。(5)5-HT合成及代謝中的限速酶TPH1及MAO:30 m W/cm_2微波輻射后大鼠海馬TPH1表達未發(fā)生明顯改變,輻射后28 d及2 m,MAO活性明顯降低。(6)5-HT_(1A/2A/2C)R表達:WB結(jié)果表明,海馬及大腦皮層5-HT2AR表達量無明顯改變。5-HT_1AR及5-HT2CR在海馬組織的表達量于輻射后14 d及2 m明顯增加,28 d時明顯降低;免疫組化結(jié)果表明,輻射后14 d,5-HT_1AR在海馬CA1、CA2、CA3區(qū)表達明顯增加,輻射后28 d,5-HT_1AR在海馬CA1、CA2區(qū)表達明顯增加,輻射后2m,5-HT_1AR在海馬CA3區(qū)表達量增加。輻射后14 d及2 m,5-HT2CR表達量在CA3區(qū)顯著增加,輻射后28 d,其在CA2區(qū)表達量明顯降低。WB及免疫組化結(jié)果均表明5-HT_1AR及5-HT2CR在大鼠大腦皮層的表達于輻射后28d明顯增加。2.大鼠5-HT受體基因啟動子區(qū)SNP位點與微波輻射致認知功能改變易感性的相關(guān)性:(1)5-HTR基因啟動子區(qū)SNP位點篩查:在不同動物中心的Wistar大鼠5-HT_(1A/2A/2C)R基因啟動子區(qū)篩查到兩個SNP位點:5-HT_1AR-215 TC(rs198585630)及5-HT2AR 74_75 ins G,Wistar大鼠rs198585630基因分型為TT、TC、CC基因型。(2)5-HT_1AR rs198585630功能研究:①包含rs198585630-C等位基因的序列結(jié)構(gòu)啟動熒光素酶報告基因的表達功能強于包含T等位的序列結(jié)構(gòu),30 m W/cm_2微波輻射后含rs198585630-C的序列結(jié)構(gòu)啟動報告基因的表達功能明顯增強;②C等位基因與PC12細胞核蛋白的結(jié)合能力明顯高于T等位基因,30 m W/cm_2微波輻射PC12細胞后,T、C等位基因與核蛋白的結(jié)合能力均明顯增強,C等位與輻射后細胞核蛋白的結(jié)合能力明顯強于T等位。(3)5-HT_1AR rs198585630與微波輻射后大鼠認知功能改變易感性的相關(guān)性研究:①空間學(xué)習(xí)能力:TT型大鼠空間學(xué)習(xí)能力明顯低于TC及CC型大鼠,30 m W/cm_2微波輻射后TT型大鼠空間學(xué)習(xí)能力增強,TC、CC型大鼠空間學(xué)習(xí)、記憶能力降低;②腦電圖:輻射后14 d大鼠腦電圖波幅增加、頻率降低、δ頻段功率百分比明顯升高,其中TC、CC型大鼠δ頻段功率百分比的增加幅度高于TT型大鼠;③神經(jīng)遞質(zhì):TT型大鼠輻射后5-HIAA含量有降低趨勢而5-HT含量有升高趨勢,但未見統(tǒng)計學(xué)差異,5-HIAA/5-HT比值明顯降低;④5-HT_1AR表達:TC及CC型大鼠海馬組織5-HT_1AR m RNA及蛋白水平于微波輻射后明顯上調(diào),輻射組中TC及CC型大鼠5-HT_1AR m RNA及蛋白水平較TT型明顯增加。3.5-HTR基因多態(tài)性與人群電磁輻射易感性關(guān)聯(lián)的初步探討:(1)心腦功能ECG、ERPs等與電磁輻射暴露劑量相關(guān):操作組及巡檢組工人較對照組心電圖異常發(fā)生率增加(主要為心率不齊、心動過緩、T波改變等);巡檢組電力工人N2潛伏期與對照組及操作組比較,顯著增加;操作組較對照組BNP明顯增加;巡檢組較操作組LENK含量顯著上升。(2)5-HT_1AR rs6295、5-HT2AR rs6313與輻射暴露劑量的交互作用對心腦功能的影響具有統(tǒng)計學(xué)意義:rs6313 TT型隨暴露劑量增加,N2、P3潛伏期顯著延長,與CC型比較有統(tǒng)計學(xué)差異;rs6295 GG型隨暴露劑量增加,LENK含量增加,N2潛伏期顯著延長,與CG型比較有統(tǒng)計學(xué)差異。rs6313 TT型、rs6295 GG型對電磁輻射易感性高。結(jié)論:(1)長期微波(2.856 GHz,平均功率密度5、10、20及30 m W/cm_2)輻射可致大鼠認知功能損傷。(2)5-HT系統(tǒng)參與微波輻射所致認知功能受損。(3)大鼠5-HT_1AR基因啟動子區(qū)SNP-215 TC為功能性位點,包含C等位的啟動子區(qū)轉(zhuǎn)錄活性高于T等位,且C等位對微波輻射更為易感。(4)5-HT_1AR-215 TC的TC、CC型大鼠于微波輻射后5-HT_1AR高表達,促使認知功能損傷。(5)長期電力作業(yè)可對工人心腦功能產(chǎn)生一定的影響。(6)5-HT_1AR rs6295及5-HT2AR rs6313可能作為電磁輻射作業(yè)人員篩選的候選靶點。
[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.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R594.8

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