發(fā)布時間：2018-05-24 06:24

  本文選題：微波輻射 + 雄性大鼠　； 參考：《蘇州大學》2014年博士論文

【摘要】：目的：微波輻射(Microwave Radiation, MWR)是一種重要的環(huán)境電磁污染物（EEPs），因無線通訊技術(shù)的快速發(fā)展，特別是全世界手機用戶的急劇增加而備受關(guān)注。由于微波輻射可對雄性生殖系統(tǒng)產(chǎn)生不良的影響，明確其作用途徑和損傷機制已成為迫切任務(wù)。已知哺乳動物的生理功能和行為存在晝夜節(jié)律，機體在24小時中不同時相對毒物及微波輻射敏感性不同。本課題采用時間生物學和生殖內(nèi)分泌毒理學相結(jié)合的策略，在建立微波輻射動物模型的基礎(chǔ)上，研究微波輻射對雄性生殖系統(tǒng)和褪黑素（Melatonin，MEL）分泌的晝夜時間毒性，以及褪黑素通過GATA-4/SF-1信號通路抑制睪酮合成的作用機制，為微波輻射對生殖功能損害的防治提供新的思路。 方法：(1)微波輻射對雄性大鼠生殖系統(tǒng)的晝夜時間毒性。以褪黑素日節(jié)律篩選具有晝夜(L:D,12h:12h)節(jié)律的SD大鼠，分別在授時時間（zeitgeber time，ZT,以ZT0為光照開始）ZT0, ZT4, ZT8, ZT12, ZT16和ZT20進行微波輻射（1800MHz，205μw/cm2），每次2h，每天一次，連續(xù)32天。對應(yīng)的假輻射組（Sham）同樣處理但不給予微波信號。輻射后24h內(nèi)每隔4h尾靜脈采血一次，ELISA法測定睪酮含量；同時Sham和MWR各時點組在輻射結(jié)束時點采集睪丸和附睪組織。測定附睪精子活動度；制作睪丸HE病理切片，測定睪丸每日精子生成量、睪丸標志酶ACP和γ-GT活性，Real-time PCR檢測睪酮合成基因StAR和p450cc mRNA表達。利用余弦節(jié)律分析軟件，根據(jù)方程式F(t)=M+Acos(ωt+Φ)將各指標數(shù)據(jù)擬合為余弦曲線，分析節(jié)律參數(shù)。(2)微波輻射改變大鼠褪黑素分泌的晝夜節(jié)律。微波輻射方法同上，輻射32天后，24h內(nèi)每隔4h尾靜脈采血一次，ELISA法檢測血漿褪黑素濃度，同時Sham和MWR各時點組在輻射結(jié)束時點采集松果體，Real-time PCR測定褪黑素合成酶NAT mRNA的表達。節(jié)律分析同上。(3)褪黑素抑制睪酮分泌的信號通路及微波輻射對睪丸間質(zhì)細胞睪酮分泌的影響。以小鼠睪丸間質(zhì)細胞系（TM3）細胞作為研究對象，添加不同濃度(0,10-6,10-8,10-10和10-12mol/L)的褪黑素，或先添加褪黑素受體阻斷劑（Luzindole，1μmol/L）再加褪黑素，MTT法檢測細胞增殖、流式細胞儀檢測線粒體膜電位、ELISA法檢測睪酮和cAMP，Real-time PCR和Western blotting測定褪黑素受體和睪酮相關(guān)基因GATA-4，SF-1，StAR，P450cc和3β-HSD的mRNA和蛋白的表達。通過睪丸注射褪黑素，利用免疫組化和免疫熒光方法以整體動物實驗驗證褪黑素抑制睪酮合成的信號通路。采用TM3細胞進行體外培養(yǎng)，在1800MHz微波輻射（205μw/cm2）2h后，流式細胞儀檢測線粒體膜電位，ELISA法測定睪酮和cAMP含量，Real-time PCR測定睪酮代謝調(diào)節(jié)因子相關(guān)基因SF-1、StAR和P450cc mRNA表達的改變。 結(jié)果：(1)微波輻射可使雄性大鼠睪丸組織發(fā)生病理改變，降低睪丸每日精子生成量、精子活動度、睪丸標志酶ACP和γ-GT活性，其中ACP和γ-GT活性以及每日精子生成量的下降在ZT0時輻射的MWR0組最為明顯。微波輻射還可改變血漿睪酮的晝夜節(jié)律，其中MWR0、MWR8、MWR12和MWR20組的晝夜節(jié)律消失，同時MWR0組大鼠血漿中睪酮的日均值水平下降最大。睪酮合成調(diào)控因子StAR和P450cc的mRNA表達在微波輻射后失去晝夜節(jié)律，且MWR0變化最大，提示大鼠雄性生殖系統(tǒng)對微波輻射最敏感的時點為ZT0:00。(2)微波輻射降低褪黑素的日均分泌量，其中以MWR16組最為明顯。微波輻射擾亂褪黑素合成的晝夜節(jié)律，其中MWR8，MWR16和MWR20組的血漿褪黑素晝夜節(jié)律被破壞，而振幅變化在MWR16組最為明顯，表明在ZT16:00時微波輻射對大鼠血漿褪黑素節(jié)律的影響最大。MWR后松果體NAT基因表達的晝夜節(jié)律改變，以ZT16時下調(diào)最為明顯，同時表達的中值和振幅明顯改變，峰值相位明顯滯后。(3)褪黑素在體外可抑制TM3細胞增殖，降低睪酮含量，減少細胞內(nèi)cAMP濃度，改變線粒體膜電位，下調(diào)核轉(zhuǎn)錄因子GATA-4及其目標基因SF-1，，StAR，Cyp11A，HSD3β的基因和蛋白表達。褪黑素的這些抑制作用可被褪黑素受體阻斷劑Luzindole所拮抗，特別是在褪黑素高水平作用時。大鼠睪丸組織注射褪黑素后0.5h、1h、2h，血漿中睪酮含量降低，睪丸間質(zhì)細胞膜受體Mel1a，GATA-4和SF-1表達減弱，4h后上述效應(yīng)消失。微波輻射后TM3細胞上清睪酮含量降低，細胞內(nèi)cAMP濃度和線粒體膜電位水平下降，睪酮合成調(diào)節(jié)因子SF-1，StAR，P450cc基因表達下調(diào)。 結(jié)論： 1、建立了微波輻射大鼠的動物模型，發(fā)現(xiàn)微波輻射對雄性大鼠的生殖功能和睪酮內(nèi)分泌具有晝夜時間毒性。 2、微波輻射可改變褪黑素分泌和褪黑素合成酶NAT mRNA的晝夜節(jié)律，并通過褪黑素介導(dǎo)對雄性大鼠生殖功能的時間毒性。 3、體內(nèi)外實驗證實褪黑素通過GATA-4/SF-1信號通路抑制睪酮分泌，其中GATA-4因子起著關(guān)鍵性的作用。
[Abstract]:Objective: Microwave Radiation (MWR) is an important environmental electromagnetic pollutant (EEPs). It has attracted much attention due to the rapid development of wireless communication technology, especially the rapid increase of mobile phone users all over the world. The effect of microwave radiation on the male reproductive system is undesirable, and its mechanisms of action and damage mechanism have been made clear. For the urgent task. The physiological function and behavior of mammals are known to be circadian rhythms, and the body has different sensitivity to toxic substances and microwave radiation at the same time in 24 hours. This subject uses a strategy of combining time biology with reproductive endocrine toxicology to study microwave radiation on males based on the establishment of a microwave radiation model. The diurnal time toxicity of the reproductive system and melatonin (Melatonin, MEL), as well as the mechanism of the inhibition of testosterone synthesis by melatonin through the GATA-4/SF-1 signaling pathway, provides a new idea for the prevention and control of reproductive function damage by microwave radiation.
Methods: (1) the diurnal time toxicity of microwave radiation on the reproductive system of male rats. The SD rats with circadian (L:D, 12h:12h) rhythm were screened with melatonin diurnal rhythm, and ZT0, ZT4, ZT8, ZT12, ZT16, and ZT12, each time, once every day, at the time of time (zeitgeber time, ZT, ZT0 as light), respectively. For 32 consecutive days, the corresponding false radiation group (Sham) was treated with the same treatment but no microwave signal was given. Blood samples were collected every 4H tail vein in 24h after radiation, ELISA method was used to determine testosterone content. At the same time, the testis and epididymis were collected at each time point of Sham and MWR at the end of radiation. The motility of epididymis spermatozoa was measured; testicular HE pathological section was made and testicle was determined for each testicular test. Diurnal sperm production, testicular marker enzyme ACP and gamma -GT activity, Real-time PCR to detect the expression of testosterone synthesis gene StAR and p450cc mRNA. Using cosine rhythm analysis software, according to the equation F (T) =M+Acos (omega t+), each index data is fitted to cosine curve, and rhythmic parameters are analyzed. (2) microwave radiation changes the circadian rhythm of melatonin secretion in rats. The method of wave radiation was the same. 32 days after radiation, blood was collected once every 4H tail vein in 24h. ELISA method was used to detect the concentration of melatonin in plasma. At the same time, the pineal body was collected at the end point of radiation and Sham and MWR. Real-time PCR was used to determine the expression of melatonin synthase NAT mRNA. Rhythmic analysis was same. (3) melatonin inhibits the signaling pathway and micro of testosterone secretion. The effect of wave radiation on testosterone secretion in Leydig cells of testis. The mouse Leydig cell line (TM3) cells were used as the research object, adding melatonin of different concentrations (0,10-6,10-8,10-10 and 10-12mol/L), or adding melatonin receptor blocker (Luzindole, 1 mu mol/L) with melatonin, MTT method to detect cell proliferation, and flow cytometry to detect the line particles Testosterone and cAMP, Real-time PCR and Western blotting were used to detect the expression of melatonin receptor and testosterone related genes, GATA-4, SF-1, StAR, P450cc and 3 beta -HSD mRNA and protein, by ELISA method. Melatonin was injected through the testis, and immunohistochemistry and immunofluorescence methods were used to test the inhibition of testosterone synthesis by melatonin in whole animal experiments. The signal pathway was cultured in vitro by TM3 cells. After 1800MHz microwave radiation (205 u w/cm2) 2h, the mitochondrial membrane potential was detected by flow cytometry. The content of testosterone and cAMP was measured by ELISA. Real-time PCR measured the changes in the expression of testosterone metabolic regulator related genes SF-1, StAR and P450cc mRNA.
Results: (1) microwave radiation can make the testicular tissue of male rats pathological changes, reduce the daily sperm production, sperm motility, testicular marker enzyme ACP and gamma -GT activity, in which the activity of ACP and gamma -GT and the decrease of daily sperm production at ZT0 are most obvious. Microwave radiation can also change the day and night of plasma testosterone. The circadian rhythm of MWR0, MWR8, MWR12 and MWR20 disappeared, while the daily average level of testosterone in the plasma of MWR0 rats decreased most. The mRNA expression of the testosterone synthesis regulator StAR and P450cc lost the circadian rhythm after microwave radiation, and the MWR0 changes were the most, suggesting that the most sensitive time point of the male reproductive system to microwave radiation was the time point for the male reproductive system of rats. ZT0:00. (2) microwave radiation reduced the daily average secretion of melatonin, which was the most obvious in the MWR16 group. Microwave radiation disrupted the circadian rhythm of melatonin synthesis, in which the plasma melatonin circadian rhythm was destroyed in MWR8, MWR16 and MWR20 groups, and the amplitude changes were most obvious in the MWR16 group, indicating that microwave radiation at ZT16:00 was used in the plasma melatonin node in rats. The circadian rhythm of the NAT gene expression in the pineal body after the maximum effect of.MWR was the most obvious, and the median and amplitude of the expression were obviously changed, and the peak phase was obviously lagged. (3) melatonin could inhibit the proliferation of TM3 cells, reduce the content of testosterone, reduce the cAMP concentration in the cells, change the mitochondrial membrane potential and down regulate the nuclear transcription. The gene and protein expression of factor GATA-4 and its target gene SF-1, StAR, Cyp11A, HSD3 beta. These inhibitory effects of melatonin can be antagonized by melatonin receptor blocker Luzindole, especially at the high level of melatonin. After injection of melatonin in rat testicular tissue, 0.5h, 1H, 2h, plasma testosterone levels are reduced, and the membrane receptors of Leydig cells The expression of Mel1a, GATA-4 and SF-1 decreased. After 4h, the above effect disappeared. After microwave radiation, the content of testosterone in the supernatant of TM3 cells decreased, the concentration of cAMP in the cells and the level of mitochondrial membrane potential decreased, and the expression of testosterone synthesis regulator SF-1, StAR, and P450cc genes were downregulated.
Conclusion:
1, a rat model of microwave radiation was established. It was found that microwave radiation had a diurnal toxicity on reproductive function and testosterone secretion in male rats.
2, microwave radiation can change the circadian rhythm of melatonin secretion and melatonin synthetase NAT mRNA, and the time toxicity of Guro Susuke conductance to reproductive function in male rats.
3, in vivo and in vitro tests confirmed that melatonin inhibited the secretion of testosterone through the GATA-4/SF-1 signaling pathway, of which GATA-4 factor played a key role.
【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R114

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