【作者】 赵黎；

【导师】 彭瑞云；

【作者基本信息】 中国人民解放军军事医学科学院 ， 病理学与病理生理学， 2010， 博士

【摘要】 目的和意义:随着微波技术的广泛应用和信息时代的到来,人们暴露于微波辐射的机会与日俱增,各种通讯设施和医疗设备的影响尤为突出。微波的生物效应及其机制研究是目前生物电磁学的前沿课题。微波辐射具有明显的中枢神经系统损伤效应,海马是最敏感的靶区,但其致伤机制未明。线粒体结构和功能与能量代谢密切相关,也是微波辐射最早累及的靶点之一,而HIF-1α/ERK信号通路可能在微波辐射致海马神经元线粒体损伤过程中发挥重要作用。因此,研究微波辐射致海马线粒体损伤中HIF-1α/ERK信号通路的改变及意义,将为深入研究微波辐射损伤机制和防治措施提供新靶标和思路,对于平时作业人员的卫生防护和现代高科技战争中作战人员的安全保障具有重要的理论和现实意义。材料和方法:(1)采用2.5、5和10mW/cm~2的微波辐射192只二级雄性Wistar大鼠,辐射时间为6min/次,5次/w,连续辐射1m。大鼠于辐射后6h、7d、14d、1m、2m和6m,采用Morris水迷宫检测其学习和记忆能力;通过HE、尼氏体染色和电镜观察海马组织结构和线粒体超微结构的变化;采用高效液相色谱法检测海马组织中氨基酸类神经递质含量的变化;采用Real-time PCR检测海马组织中hif-1αmRNA表达;通过Western Blot、免疫组织化学和图像分析等手段检测海马组织中HIF-1α、ERK1/2和p-ERK1/2表达。(2)采用30mW/cm~2微波辐射经NGF诱导的PC12细胞5min,通过电镜、Luminometer、多功能酶标仪、荧光显微镜等技术,观察PC12细胞线粒体结构和功能变化;采用Real-time PCR、Western Blot、免疫细胞化学和图像分析等技术,检测PC12细胞中hif-1αmRNA、HIF-1α、ERK通路分子(ERK1/2、p-ERK1/2、Raf、p-c-Raf和p-MEK1/2)、HIF-1靶基因(VEGF)、能量代谢相关基因(COXⅠ和COXⅣ)的表达变化;通过U0126和HIF-1α高表达质粒的干预等手段,研究微波辐射后ERK通路对HIF-1α的调控作用以及HIF-1α对PC12细胞线粒体功能的影响。实验结果:(1)大鼠学习记忆能力和氨基酸类神经递质变化:辐射后7d,10mW/cm~2组大鼠平均逃避潜伏期显著长于假辐射组(P<0.05);辐射后14d,5和10mW/cm~2组大鼠平均逃避潜伏期明显延长(P<0.05);辐射后1m,各辐射组与假辐射组相比,平均逃避潜伏期均延长(P<0.05或P<0.01)。2.5和5mW/cm~2微波辐射后6h、14d和2m,海马4种氨基酸类神经递质含量均明显增加(P<0.05或P<0.01);10 mW/cm~2微波辐射后6h,GABA含量降低(P<0.05);辐射后14d和2m,Asp和Glu含量均降低(P<0.05或P<0.01)。(2)大鼠海马组织结构和线粒体超微结构变化:2.5、5和10mW/cm~2微波辐射后6m内,大鼠海马组织见水肿、疏松,神经元呈不同程度的变性、坏死,血管周间隙增宽。2.5和5mW/cm~2辐射后7d见海马损伤,1m损伤最为严重,2m呈恢复趋势,2.5 mW/cm~2辐射后6m恢复,而5mW/cm~2组仍见部分组织损伤;10mW/cm~2组海马结构损伤发生早(辐射后6h),7d和14d损伤最重,6m仍未恢复。各辐射组尼氏体数量均呈不同程度减少,以10mW/cm~2组最重,其变化规律与HE染色结果基本一致。5mW/cm~2组辐射后6m内,大鼠海马线粒体肿胀、嵴断裂,可见线粒体空化和畸形的线粒体;辐射后6h即见损伤,1m最重,6m呈恢复趋势。(3)大鼠海马组织中hif-1αmRNA和HIF-1α蛋白变化:大鼠海马hif-1αmRNA于2.5mW/cm~2组未见明显改变;5mW/cm~2组于辐射后14d明显增加(P<0.01),辐射后1m恢复;10mW/cm~2组均低于假辐射组,以6h(P<0.01)、1m(P<0.05)和2m(P<0.05)减少显著。大鼠海马组织中HIF-1α蛋白于2.5mW/cm~2辐射后6h和1m增加(P<0.05或P<0.01),5mW/cm~2辐射后1m增加(P<0.01),10mW/cm~2辐射后14d~2m降低(P<0.05或P<0.01)。(4)大鼠海马组织中ERK1/2和p-ERK1/2表达变化:2.5、5和10mW/cm~2微波辐射后2m内,大鼠海马ERK1/2未见明显改变。假辐射组p-ERK1/2于海马神经元胞浆中呈弱阳性;2.5mW/cm~2辐射后2m内,p-ERK1/2表达无明显变化;5mW/cm~2辐射后7d~1m,p-ERK1/2于海马神经元胞浆和胞核中呈阳性或强阳性(P<0.05或P<0.01);10mW/cm~2辐射后7d~2m,p-ERK1/2于海马神经元胞浆和胞核中呈阳性或强阳性(P<0.05或P<0.01)。(5)PC12细胞线粒体结构和功能变化:30mW/cm~2微波辐射后6h,PC12细胞线粒体分布不均,大小、形状不规则,多数线粒体肿胀、嵴断裂和空化。辐射后1h~3d,ATP含量均低于假辐射组(P<0.01或P<0.05);辐射后1h,SDH和COX活性显著降低(P<0.05);辐射后6h,ΔΨm降低显著(P<0.05);辐射后1~24h,ROS均显著高于假辐射组(P<0.01)。(6)PC12细胞hif-1αmRNA和HIF-1α蛋白变化:hif-1αmRNA于30mW/cm~2微波辐射后6h显著增加(P<0.05);HIF-1α蛋白于30mW/cm~2微波辐射后1h和12h增加(P<0.05),辐射后6h降低(P<0.05)。(7)PC12细胞ERK通路信号分子ERK1/2、p-ERK1/2、Raf、p-c-Raf和p-MEK1/2表达变化:30mW/cm~2微波辐射后1h,PC12细胞中p-ERK1/2蛋白显著增加(P<0.05),30mW/cm~2微波辐射后1~24h,PC12细胞中ERK1/2、Raf、p-c-Raf和p-MEK1/2蛋白均无明显改变。(8)PC12细胞HIF-1靶基因VEGF和能量代谢基因COXⅠ和Ⅳ表达变化:VEGF、COXⅠ和Ⅳ蛋白变化与上述HIF-1α变化呈现一致性,即30mW/cm~2微波辐射后6h,PC12细胞VEGF、COXⅠ和Ⅳ蛋白显著减少(P<0.05),辐射后12h明显增加(P<0.05)。(9)U0126干预后ERK1/2、p-ERK1/2和HIF-1α表达以及线粒体功能变化:10μmol/l U0126干预2h经30mW/cm~2微波辐射后1h,PC12细胞中ERK1/2无明显改变,p-ERK1/2和HIF-1α明显降低(P<0.05);10μmol/l U0126干预2h经30mW/cm~2微波辐射后1~24h,PC12细胞线粒体ATP含量和ΔΨm降低。(10)HIF-1α高表达质粒及U0126干预对30mW/cm~2微波辐射后PC12细胞线粒体功能的影响:经G418筛选后,对照质粒pEGFP-N1(N)和目的质粒pEGFP-N1-HIF-1α(H)转染PC12细胞效率大于90%;H组HIF-1α表达显著高于N组(P<0.01);30mW/cm~2微波辐射后1~24h,H组线粒体ΔΨm显著升高(P<0.01),SDH活性无明显改变(P>0.05);10μmol/l U0126干预2h后经30mW/cm~2微波辐射1~24h,H组ATP含量抑制率低于N组,ΔΨm于辐射后1h和6h无明显变化(P>0.05),辐射后12h和24h明显升高(P<0.01)。结论:(1)2.5~10mW/cm~2微波长期辐射可使大鼠海马组织结构尤其是线粒体结构损伤,学习记忆能力下降,氨基酸类神经递质紊乱;上述改变与微波辐射剂量呈正相关。(2)2.5和5mW/cm~2微波长期辐射引起大鼠海马组织中HIF-1α表达增加,10mW/cm~2微波辐射则使HIF-1α表达降低;2.5~10mW/cm~2微波长期辐射使p-ERK1/2表达增加,表明HIF-1α和ERK通路参与微波辐射致海马线粒体损伤的病理生理过程;(3)30mW/cm~2微波辐射使PC12细胞线粒体结构损伤,ATP含量、SDH和COX活性及ΔΨm下降,ROS堆积;(4)30mW/cm~2微波辐射可使PC12细胞HIF-1α表达呈升高、降低再升高的波动性改变,其mRNA呈短暂升高,p-ERK1/2表达升高。表明微波辐射可激活HIF-1α和通过活化p-ERK1/2激活ERK通路。HIF-1的靶基因VEGF和能量代谢相关基因COXⅠ和Ⅳ与HIF-1α的表达变化呈现一致性,表明上述基因表达参与微波辐射致线粒体损伤过程;(5)U0126抑制ERK通路,可引起30mW/cm~2微波辐射后PC12细胞p-ERK1/2和HIF-1α表达下降, ATP含量下降和ΔΨm降低,表明微波辐射后ERK信号通路正向调控HIF-1α,ERK信号通路在微波辐射致线粒体损伤中发挥保护作用;(6)HIF-1α高表达可使30mW/cm~2微波辐射后PC12细胞ATP含量和ΔΨm明显升高,U0126抑制ERK通路后可降低HIF-1α高表达对PC12细胞ATP含量和ΔΨm的作用,表明HIF-1α激活在微波辐射致PC12细胞线粒体损伤中起保护作用,ERK通路对线粒体功能的保护作用部分通过HIF-1α的作用实现。更多还原

【Abstract】 Objective: With the wide application of microwave technology and information era, the opportunities of people exposed to microwave are increasing particularly all communication facilities and medical equipment. The biological effects and mechanisms of microwave are the subject of bioelectromagnetics forward. Microwave has a significant effect on central nervous system while hippocampus is the most sensitive target, but the injury mechanism is unknown. Mitochondrial structure and function are closely related to energy metabolism, which is one of the first involving targets of microwave exposure, while the HIF-1α/ERK signaling pathway may play an important role in the process of injury of hippocampal neurons in mitochondria induced by microwave. Therefore, the study of HIF-1α/ERK signaling pathway and its significance in the damage of the hippocampus induced by microwave radiation provides new targets for in-depth study of microwave injury mechanisms and prevention and control measures and approaches, and has important theoretical and practical significance for the normal operation of the health protection staff and modern technology war combatant’s security.Materials and methods: (1) 192 male Wistar rats were exposed to microwave at 2.5, 5 and 10mW/cm~2. Exposure time was 6min/times, 5 times/week, and continuouing exposure for 1m. The abilities of learning and memory were detected by Morris water maze at 6h, 7d, 14d, 1m, 2m and 6m after exposure. The structure of rats’hippocampus and mitochondria ultrastructure were observed by HE and tigroid body staining and electron microscope. Meanwhile, amino acid neurotransmitters contents in hippocampus were detected by HPLC. The expression of hif-1αmRNA in hippocampus was detected by Real-time PCR. HIF-1α, ERK1/2, p-ERK1/2 expression were detected by Western Blot, IHC and image analysis. (2) PC12 cells which were induced by NGF were exposed to 30mW/cm~2 microwave. The structure and function of mitochondria were observed by electron microscope, luminometer, fluorospectrophotometer and fluoromicroscope. Real-time PCR, Western Blot, IHC and image analysis were used to detect hif-1αmRNA, HIF-1α, molecular of ERK pathway (ERK1/2, p-ERK1/2, Raf, p-c-Raf, p-MEK1/2), target gene of HIF-1α(VEGF) and energy metabolism related gene (COXⅠand COXⅣ) expression. The regulation of ERK pathway on HIF-1αand the effect of HIF-1αto mitochondria function of PC12 cells were invested by intervention means of U0126 and hypso-expression plasmid of HIF-1α.Results: (1) The changes of rats’learning and memory abilities and amino acids neurotransmitter: The AELs of 10mW/cm~2 group were longer than sham group (P<0.05) at 7d after exposure. AELs of 5 and 10mW/cm~2 group were longer than sham group at 14d (P<0.05 or P<0.01). AELs of exposed groups were all longer than sham group at 1m(P<0.05 or P<0.01). Four kinds of amino acid neurotransmitter contents in hippocampus were increased significantly at 6h, 14d and 2m in 2.5 and 5mW/cm~2 groups(P<0.05 or P<0.01). GABA contents were decreased at 6h after exposure (P<0.05), and Asp and Glu contents were decreased at 14d and 2m in 10mW/cm~2 group(P<0.05 or P<0.01). (2) The changes of hippocampus structure and ultrastructure of mitochondria: The hippocampus structure of exposed groups appeared tissue edema, osteoporosis, and different degrees of degeneration and necrosis of neurons, vascular congestion and hemorrhage and enlarged perivascular space within 6m after 2.5, 5 and 10mW/cm~2 exposure. At 2.5 and 5mW/cm~2 groups, the hippocampus injuries appeared at 7d, 1m at the most serious stage, and 2m on the recovery of the trend. In 2.5 mW/cm~2 group, the hippocampus structure recovered at 6m while in 5 mW/cm~2 group, the injuries still could be seen then. In 10mW/cm~2 group, the injury of hippocampus structure appeared earlier (6h after exposure), 7d and 14d at the most serious stage, and could not recovered at 6m. The numbers of tigroid body were decreased at different extend in exposure groups and were the most serious in 10 mW/cm~2 group. The change rule of it was agreed with HE staining. The mitochondria showed swell, cristae break, cavitation and malformed which were injured at 6h, 1m at the most serious stage, and 6m on the recovery of the trend during 6m in 5 mW/cm~2 group. (3) The mRNA and protein expression changes of HIF-1αin rats’hippocampus: There was no change of hif-1αmRNA in 2.5mW/cm~2 group, while hif-1αmRNA was increased significantly at 14d (P<0.01) and recovered at 1m in 5mW/cm~2 group, then hif-1αmRNA was expressed in 10mW/cm~2 group lower than sham group, which was decreased significantly at 6h (P<0.01), 1m (P<0.05) and 2m (P<0.05). The protein of HIF-1αin rats’hippocampus was increased at 6h and 1m (P<0.01 or P<0.05) in 2.5mW/cm~2 group and at 1m in 5mW/cm~2 group (P<0.01), and was decreased at 14d~2m in 10mW/cm~2 group (P<0.01 or P<0.05). (4) The expression changes of ERK1/2 and p-ERK1/2 in rats’hippocampus: There was no significant change within 2m in exposure groups. In sham group, p-ERK1/2 was expressed weakly positive in neuron cytoplasm of hippocampus, while there was no changes within 2m in 2.5mW/cm~2 group, and p-ERK1/2 was expressed positive or strong positive at 7d~1m in 5mW/cm~2 group (P<0.01 or P<0.05) and at 7d~2m in 10mW/cm~2 group (P<0.01 or P<0.05). (5) The structure and function changes of mitochondria of PC12 cells: The mitochondria of PC12 cells showed unevenness distribution, irregular sizes and shapes, swelling, cristae break and cavitation at 6h after 30mW/cm~2 microwave exposure. ATP contents were lower than sham group (P<0.01 or P<0.05) at 1h~3d. The activities of SDH and COX were decreased significantly (P<0.05) at 1h.ΔΨm was decreased significantly at 6h (P<0.05). ROS was higher than sham group at 1~24h (P<0.01). (6) The mRNA and protein expression changes of HIF-1αin PC12 cells: hif-1αmRNA was increased at 6h after 30mW/cm~2 microwave exposure (P<0.05). HIF-1αwas increased at 1h and 12h after 30mW/cm~2 microwave exposure (P<0.05) and was decreased at 6h (P<0.05). (7) The expression changes of ERK pathway signal moleculars ERK1/2, p-ERK1/2, Raf, p-c-Raf and p-MEK1/2: The expression of p-ERK1/2 was increased significantly at 1h after 30mW/cm~2 microwave exposure (P<0.05). There were no changes of ERK1/2, Raf, p-c-Raf and p-MEK1/2 expression at 1~24h after 30mW/cm~2 microwave exposure. (8) The changes of VEGF, COXⅠand COXⅣ: The expressions of VEGF, COXⅠandⅣwere on concordance with HIF-1αabove, i.e. VEGF, COXⅠandⅣwere decreased at 6h after 30mW/cm~2 microwave exposure (P<0.05), and were increased at 12h (P<0.05). (9) ERK1/2, p-ERK1/2 and HIF-1αexpression and function of mitochondria changes after U0126 intervention: PC12 cells were exposed to 30mW/cm~2 microwave 2h after 10μmol/l U0126 intervention, and there was no change of ERK1/2 expression, while the expression of p-ERK1/2 and HIF-1αwere decreased significantly at 1h after (P<0.05). With the same conditions, the ATP contents andΔΨm were decreased at 1~24h after exposure. (10) The effect of high expression plasmid of HIF-1αand U0126 intervention on mitochondria function of PC12 cells after 30mW/cm~2 microwave exposure: After G418 screening, the transfection efficiency of sham plasmid pEGFP-N1 (N) and objective plasmid pEGFP-N1-HIF-1α(H) to PC12 cells exceeded 90%. The expressions of HIF-1αin H group were higher than N group (P<0.01). After 30mW/cm~2 microwave exposure at 1~24h, theΔΨm step up significantly in H group (P<0.01), but there was no change of SDH activity (P>0.05). The ATP inhibition ratio in H group was lower than N group 2h after 10μmol/l U0126 intervention and 30mW/cm~2 microwave exposure, and there was no change ofΔΨm at 1h and 6h after exposure (P>0.05), but was increased at 12h and 24h (P<0.01).Conclusions: (1) Long-term microwave exposure at 2.5, 5 and 10mW/cm~2 may cause structural damage in rats’hippocampus especially in mitochondria, with the abilities of learning and memory declining and amino acid neurotransmitter disorders. These changes above were positively correlated with exposure dose. (2) HIF-1αexpression in rats’hippocampus was increased in 2.5 and 5mW/cm~2 groups, while was decreased in 10 mW/cm~2 groups. The p-ERK1/2 expression was increased in exposure groups, which indicated that HIF-1αand ERK pathway participated in the processes of hippocampus mitochondria damage induced by microwave. (3) The mitochondria structure of PC12 cells were injuried by microwave at 30mW/cm~2, and the contents of ATP, the activities of SDH and COX,ΔΨm were decreased, while ROS accumulated. (4) HIF-1αexpression in PC12 cells was fluctuated which showed step up first, then step down and up. The hif-1αmRNA showed increase temporary and the expression of p-ERK1/2 increased. It indicated that microwave exposure may activate HIF-1αand activate ERK pathway by p-ERK1/2. The changes of expression of VEGF which was the target gene of HIF-1α, COXⅠandⅣwhich were energy metabolism genes appeared the same rule of HIF-1α, which indicated that genes above participated in the processes of mitochondria damage induced by microwave exposure. (5) ERK pathway inhibited by U0126 can cause the down expression of p-ERK1/2 and HIF-1α, and the decreasing of ATP contents andΔΨm, which manifested that ERK pathway participated in the positive regulation of HIF-1αafter microwave exposure and ERK pathway played a protection role in the mitochondria damage induced by microwave exposure. (6) High expression of HIF-1αcan increase the ATP contents andΔΨm and ERK pathway inhibited by U0126 can decrease the effects of high expression of HIF-1αon ATP contents andΔΨm, which indicated that the activation of HIF-1αprotected mitochondria in the processes of mitochondria damages of PC12 cells induced by microwave exposure and the protection effect of ERK pathway on mitochondria function was implemented by HIF-1α.更多还原