HCN4在微波辐射致大鼠窦房结损伤中的作用研究
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摘要
目的和意义：随着微波技术的飞速发展和信息时代的来临，人们在享受便捷生活的同时，也面临着微波污染的危害。此外，微波技术在军事上的应用发展迅速，它不仅能严重破坏敌方电子设备，同时也能造成机体损伤。心脏传导系统是微波辐射敏感的靶标之一，SAN是心脏传导系统最重要的组成部分。然微波辐射致SAN损伤规律及量效关系未明，其致伤机制尚未见报道。HCN4可调控P细胞的起搏电流及维持其电生理功能。SAN组织中HCN4的异常表达可能是各种SAN病变发生的分子基础之一，而HCN4及其调控可能在微波辐射致大鼠SAN损伤中发挥重要作用。因此，研究微波辐射致SAN损伤中HCN4的改变、调控及其意义，将为深入研究微波辐射致心脏损伤的分子机制和防治措施提供新靶标和思路，为寻找敏感诊断指标和制定防护标准提供实验依据。
材料和方法：（1）大鼠SAN定位及组织结构观察：采用二级Wistar成年大鼠20只，对右心房及与之相连的近段上腔静脉做水平连续切片，对切片行HE、Masson染色，光镜下观察连续切片，定位SAN并观察组织结构。（2）大鼠SAN超微结构观察：采用二级雄性Wistar成年大鼠10只，依据SAN位置、组织结构特点分两步取材，经树脂定向包埋，半薄切片甲苯胺蓝预染，光镜下定位后，再行超薄切片，透射电镜观察SAN超微结构。（3）微波辐射对大鼠SAN功能和结构的影响研究：采用平均功率密度为0、5、10、50mW/cm~2的脉冲微波辐射160只二级雄性Wistar大鼠，辐射时间为6min，于辐射前、辐射后即刻、7d、14d、28d、3m、6m和9m，采用多道生理记录仪检测大鼠ECG的变化；于辐射后1d、7d、14d、28d、3m、6m、9m、12m，采用光镜和电镜观察大鼠SAN组织结构和超微结构变化；采用Masson染色、天狼猩红染色和图像分析技术，观察SAN组织胶原纤维含量的动态变化规律。（4）微波辐射后大鼠SAN组织HCN4改变及其调控机制研究：采用ISH、IHC和图像分析等方法，检测50mW/m2微波辐射后大鼠SAN组织中HCN4及其上游分子β1-AR、M2-AchR基因和/或蛋白的表达变化。（5）微波辐射对原代培养乳鼠SAN细胞的损伤效应及机制研究：以体外原代培养的SAN细胞为研究对象，采用倒置显微镜、AFM、LSCM、IF等技术，观察微波辐射对细胞形态结构、搏动特征、细胞膜结构、细胞内[Ca~(2+)]及HCN4表达的影响。
结果：（1）正常成年大鼠SAN位置和组织学特点：正常成年大鼠SAN位于上腔静脉与右心耳交界区及其以上的上腔静脉壁内，呈马蹄形/C形；大鼠SAN组织结构疏松，主要含有P细胞、T细胞和少量心房肌细胞，间质胶原纤维含量丰富。（2）正常成年大鼠SAN超微结构观察：甲苯胺蓝预染半薄切片的方法简便迅速，着色效果好，可有效缩短定位时间。电镜观察显示，大鼠SAN内主要含有两种细胞。①P细胞：胞体小，胞浆丰富，细胞器含量少；肌原纤维含量少，杂乱分布于细胞膜附近，几乎不含肌节；②T细胞：胞体较P细胞大，细胞器含量较多，肌原纤维可见明显肌节，常沿细胞纵轴排列，分布于细胞膜附近。（3）微波辐射对大鼠SAN功能的影响：5mW/cm~2组未见明显异常；10和50mW/cm~2微波辐射后大鼠SAN功能受损，主要表现为：心率呈先加快后减慢趋势；P波振幅降低；ECG出现窦性心律不齐、SAN内游走性心律和房性心律失常等。（4）微波辐射后大鼠SAN组织结构变化：5mW/cm~2组未见明显异常；10和50mW/cm~2微波辐射后1d，表现为P、T细胞水肿；T细胞排列呈波浪状改变；14～28d损伤最重，表现为细胞排列紊乱，部分细胞胞浆嗜酸性染色增强，核固缩深染；3m～6m损伤减轻呈恢复趋势，仍有部分细胞呈水肿状态；9m～12m表现为实质细胞减少，间质胶原纤维增多及脂肪浸润。以上改变尤以50mW/cm~2组最为显著。（5）微波辐射后大鼠SAN超微结构变化：5mW/cm~2组未见明显异常；10和50mW/cm~2微波辐射后1d～28d表现为P、T细胞线粒体最早受累，出现肿胀，嵴断裂，甚至空化；肌原纤维局灶性溶解、断裂；可见P、T细胞核染色质浓缩、边集；细胞膜小凹减少或消失；血管周围间隙增宽、水肿；6m～12m表现为实质细胞退行性变，间质胶原原纤维增多、脂肪浸润。以上改变尤以50mW/cm~2组最为显著。（6）微波辐射致大鼠SAN损伤后HCN4、β1-AR、M2-AchR变化：50mW/cm~2微波辐射后1d～28d，大鼠SAN组织中HCN4mRNA表达上调（P<0.05或P<0.01），3m表达下调（P<0.05）；HCN4蛋白于辐射后1d～28d表达增加（P<0.05或P<0.01），3m～6m表达降低（P<0.05）；β1-AR mRNA于辐射后1d～3m表达上调（P<0.05或P<0.01）；β1-AR蛋白于辐射后1d～3m表达增加（P<0.05或P<0.01）；M2-AchR蛋白于辐射后1d～6m表达增加（P<0.05或P<0.01）。（7）微波辐射后体外培养SAN细胞形态结构、搏动特征变化：正常SAN细胞呈长梭形，伸出伪足或突起与周围细胞连接成片，呈单个细胞搏动，搏动速度快，节律规则。10和50mW/cm~2微波辐射后，SAN细胞搏动明显减慢且节律不规则，细胞肿胀变圆，伪足和突起减少。50mW/cm~2微波辐射后即刻SAN细胞内[Ca~(2+)]升高（P<0.05），细胞膜表面可见穿孔现象。（8）微波辐射致体外培养SAN细胞损伤后HCN4表达改变：50mW/cm~2微波辐射后即刻，SAN细胞中HCN4蛋白表达显著减弱（P＜0.01），12h表达显著增强（P＜0.05）。
结论：（1）成年大鼠SAN特殊的位置提示：大鼠SAN取材时一定要保留上腔静脉近心段；两步法前固定取材有助于大鼠SAN电镜标本的制作。（2）10、50mW/cm~2微波辐射导致大鼠SAN功能障碍、组织结构和超微结构损伤，上述改变与微波辐射剂量呈正相关。（3）10、50mW/cm~2微波辐射可导致培养的SAN细胞形态结构损伤和搏动下降，且与微波辐射剂量呈正相关。（4）HCN4异常表达可能是微波辐射致SAN损伤的重要致伤分子之一。（5）SAN组织β1-AR高表达可能通过促进HCN4表达加重微波辐射后SAN组织损伤过程；M2-AchR高表达可能通过抑制HCN4表达促进微波辐射致SAN损伤后的修复过程。（6）细胞膜穿孔、细胞内钙超载、HCN4的高表达是微波辐射致SAN损伤的重要机制。
Objective and Significance: With the advent of information era and the high speeddevelopment of microwave technology, people are accessible to the hazard of microwavepollution as well as the conveniences it brings about. Besides, microwave technology also saw itsincreasing development and application in military fields. It can not only severely damage theenemy’s electronic devices, but also do harm to the body. Conducting system of heart is one of thetargets sensitive to microwave radiation. However, as the essential component of heart conductingsystem, SAN’s pattern of damage from microwave radiation and their dose-effect relationshipremained unclear, and the damaging mechanism was uncovered. HCN4can regulate pacemakercells’ pacing current and maintains their physiologic functions. The abnormal expression of HCN4in the SAN tissues may be a molecular basis of a variety of SAN pathological changes, whileHCN4and its regulation may play an important role in rats’ SAN damage after microwaveradiation. It is followed that the study of the HCN4change, its regulation and significance in theSAN’s damage from microwave radiation will provide a new thought thread and target for thefurther study of molecular mechanism and protective measures of microwave radiation damage toheart. Meanwhile it can provide experimental evidence for the identification of sensitive diagnosisindex and the formulation of protective criteria.
Materials and Methods:(1) Positioning of rats’ SANs and observation of their organizationalstructure: Twenty second-class adult Wistar rats were used to conduct the horizontal serial sectionsof the right atrium and the associated proximal segment of the superior vena cava. Serial sectionswere stained by HE and Masson staining and subsequently observed by light microscopy to locatethe position of SAN and to observe its organizational structure.(2) Observation of rats’ SAN’sultrastructure: Based on the SAN location and organizational characteristics,10second-class adultmale Wistar rats were taken to get the materials in two steps, the semi-thin sections werepre-stained by toluidine blue after the resin directed embedding and positioned through lightmicroscope. Following ultrathin sectioning, SAN ultrastructure was observed by the transmissionelectron microscopy.(3) Study of the effect of microwave radiation on rats’ SAN function andstructure:160Wistar rats were exposed to a pulse microwave field (0,5,10and50mW/cm~2) for6minutes. Multi-channel polygraph was applied to detect the change of ECG in rats beforemicrowave exposure and immediately,7,14,28days and3,6,9months after microwave radiation.Light microscopy and electron microscopy were used to observe the organizational structure andultrastructure of rats’ SAN at1d,7d,14d,28d,3m,6m,9m,12m after microwave exposure.Masson staining, Sirius red staining and image analysis techniques were applied to observe thedynamic development of the content of collagen fiber in rats’ sinoatrial nodes.(4) Research on thechange of HCN4in rats’ SAN tissue and the regulation mechanism after microwave radiation: ISH, IHC and image analysis were used to detect the changes in the gene or protein expression ofHCN4, β1-AR, M2-AchR in rat’s SAN tissues after50mW/m2microwave radiation.(5) Researchon the damaging effect and mechanism of microwave radiation on primary culture SAN cells:SAN cells primarily cultured in vitro were taken to do the research. Inverted microscope, AFM,LSCM and IF were used to observe the effect of microwave radiation on cells’ morphosis,pulsation features, membrane’s structure, the content of Ca~(2+)inside the cells and the expression ofHCN4.
Results:(1) SAN’s position and histological feature of normal adult rat: Adult rats’ SANswere located in the junctional zone of superior vena cava and right atricle and on the inner-wall ofsuperior vena cava above the junctional zone. The adult rats’ SANs were shaped like the horseshoeor the letter ‘C’. The rats’ SANs, being loose in structure and rich in interstitial collagen fiber,mainly contain P cells, T cells and a small amount of atrial muscle cells.(2) Observation ofSAN’s ultrastructure of adult rats: The toluidine blue prestained semi-thin sections is a rapidand efficient method, which can effectively shorten the positioning time. The electron microscopyshowed that two types of cells were present in the rat SAN: P cells and T cells. With a small cellbody, P cell was rich in cytoplasm but poor in organelle. There were a small number of myofibrilswhich were messily distributed nearby the cell membrane and almost free of sarcomeres. T cellhad a bigger cell body and more organelles than P cell and the myofibrils. With visible sarcomeres,were often arranged along the cell longitudinal axis and close to the cell membrane.(3) Effect ofmicrowave radiation on SAN’s function. There were no significant change in the5mW/cm~2group but appeared some damage to SAN’s functions in the10and50mW/cm~2groups, whichwere mainly as follows: the heart rate tended to accelerate and then slow down; the amplitude of Pwave was decreased; the ECG showed sinus arrhythmia, sinoatrial node wandering heart rhythmand atrial arrhythmia, etc.(4) Structural changes in rats’ SAN tissues after microwaveradiation: There was no significant change in the group of5mW/cm~2. But in the10and50mW/cm~2groups, changes could be observed like edema in P and T cells, wavy changes in T cells,increasing of acidophilia staining in the cytoplasm of P and T cells, and karyopyknosis andanachromasis from1to28days after microwave exposure. From3to6months, the damagestended to lessen and recover but there were still some cells in the edematous state, and from9to12months, parenchyma cells began to decrease and interstitial collagen fibers were increased, andthere appeared fatty infiltration. Changes above were remarkably observed in the group of50mW/cm~2.(5) SAN’s ultrastructural changes after exposure to microwave radiation. Therewas no significant change in the group of5mW/cm~2. In the period of1to28days, mitochondriaof T cells and P cells in the10and50mW/cm~2groups were the earliest to be effected to appearswelling, crista breakage, and even cavitation; then came the focal dissolution and even breakageof myofibrils, and concentration of nuclear chromatin of P and T cells to the edge, followed by thewidening and edema of perivascular canal, and the reduction or disappearance of foveolas ofmembrane. In the period of6to12months, there came degeneration in parenchyma cells, increase of interstitial collagen fibrils and fatty infiltration.(6) Changes in HCN4, β1-AR, and M2-AchRin rats’ SAN tissues injured by50mW/cm~2microwave radiation. The expression of HCN4mRNA began to be up-regulated at1to28days (P<0.05or P<0.01) and down-regulated at3months (P<0.05).The expression of HCN4protein was increased at1to28days (P<0.05orP<0.01) and was decreased at3to6months (P<0.05). The expression of β1-AR mRNA wasup-regulated at1d to3m (P<0.05or P<0.01) while the expression of β1-AR protein was increasedat1d to3m (P<0.05or P<0.01). The expression of M2-AchR protein was increased after1d~6m(P<0.05or P<0.01).(7) Changes in cellular morphosis and pulsation feature of SAN cellscultured in vitro after microwave exposure. The normal cell of SAN was shaped like longspindle, sticking out pseudopodia or apophysis and connected with the cells around, and beatrhythmically in single with a high speed. But immediately after the exposure to10and50mW/cm~2microwave, the cells of SAN began to beat significantly slower and irregular and tendedto become swollen and rounding, with pseudopodia or apophysis reducing. Besides, the contentsof Ca~(2+)inside the cells of SAN rose immediately after50mW/cm~2microwave exposure, andperforation could be observed in the surface of membrane.(8) Changes in the expression ofHCN4in the vitro cultured SAN cells after microwave radiation. The expression of HCN4protein was weakened significantly immediately after50mW/cm~2microwave radiation (P＜0.01)and was strengthened significantly after12hours (P＜0.05).
Conclusion:(1) The special location of SANs in adult rats indicated that the proximal segmentof superior vena cava must be reserved when taking SANs from rats and the two-step methodbefore positioning was helpful for the making of electron microscope specimens of rats’ SANs.(2)Exposure to10and50mW/cm~2microwave radiation can lead to dysfunction and damage toorganizational structure and ultrastructure of rats’ SANs. The above changes are positivelycorrelated to microwave radiation dose.(3) Exposure to10and50mW/cm~2microwave can lead todamage to morphosis and reduction of pulsation ability of cultured SAN cells, which arepositively correlated to microwave radiation dose.(4) The abnormal expression of HCN4is likelyto be one of the important vulnerating molecules in the damaging process of microwave radiationto SANs.(5) In the tissue of SAN, the high expression of β1-AR is likely to aggratate SAN’sinjuries resulted from microwave radiation by stimulating the expression of HCN4while the highexpression of M2-AchR is likely to promote the recovery of SAN’s injuries from microwaveradiation by inhibiting the expression of HCN4.(6) The perforation of membrane, the overloadingof intracellular Ca~(2+), and the high expression of HCN4are important mechanisms leading to SANinjury resulted from microwave radiation.
材料和方法：（1）大鼠SAN定位及组织结构观察：采用二级Wistar成年大鼠20只，对右心房及与之相连的近段上腔静脉做水平连续切片，对切片行HE、Masson染色，光镜下观察连续切片，定位SAN并观察组织结构。（2）大鼠SAN超微结构观察：采用二级雄性Wistar成年大鼠10只，依据SAN位置、组织结构特点分两步取材，经树脂定向包埋，半薄切片甲苯胺蓝预染，光镜下定位后，再行超薄切片，透射电镜观察SAN超微结构。（3）微波辐射对大鼠SAN功能和结构的影响研究：采用平均功率密度为0、5、10、50mW/cm~2的脉冲微波辐射160只二级雄性Wistar大鼠，辐射时间为6min，于辐射前、辐射后即刻、7d、14d、28d、3m、6m和9m，采用多道生理记录仪检测大鼠ECG的变化；于辐射后1d、7d、14d、28d、3m、6m、9m、12m，采用光镜和电镜观察大鼠SAN组织结构和超微结构变化；采用Masson染色、天狼猩红染色和图像分析技术，观察SAN组织胶原纤维含量的动态变化规律。（4）微波辐射后大鼠SAN组织HCN4改变及其调控机制研究：采用ISH、IHC和图像分析等方法，检测50mW/m2微波辐射后大鼠SAN组织中HCN4及其上游分子β1-AR、M2-AchR基因和/或蛋白的表达变化。（5）微波辐射对原代培养乳鼠SAN细胞的损伤效应及机制研究：以体外原代培养的SAN细胞为研究对象，采用倒置显微镜、AFM、LSCM、IF等技术，观察微波辐射对细胞形态结构、搏动特征、细胞膜结构、细胞内[Ca~(2+)]及HCN4表达的影响。
结果：（1）正常成年大鼠SAN位置和组织学特点：正常成年大鼠SAN位于上腔静脉与右心耳交界区及其以上的上腔静脉壁内，呈马蹄形/C形；大鼠SAN组织结构疏松，主要含有P细胞、T细胞和少量心房肌细胞，间质胶原纤维含量丰富。（2）正常成年大鼠SAN超微结构观察：甲苯胺蓝预染半薄切片的方法简便迅速，着色效果好，可有效缩短定位时间。电镜观察显示，大鼠SAN内主要含有两种细胞。①P细胞：胞体小，胞浆丰富，细胞器含量少；肌原纤维含量少，杂乱分布于细胞膜附近，几乎不含肌节；②T细胞：胞体较P细胞大，细胞器含量较多，肌原纤维可见明显肌节，常沿细胞纵轴排列，分布于细胞膜附近。（3）微波辐射对大鼠SAN功能的影响：5mW/cm~2组未见明显异常；10和50mW/cm~2微波辐射后大鼠SAN功能受损，主要表现为：心率呈先加快后减慢趋势；P波振幅降低；ECG出现窦性心律不齐、SAN内游走性心律和房性心律失常等。（4）微波辐射后大鼠SAN组织结构变化：5mW/cm~2组未见明显异常；10和50mW/cm~2微波辐射后1d，表现为P、T细胞水肿；T细胞排列呈波浪状改变；14～28d损伤最重，表现为细胞排列紊乱，部分细胞胞浆嗜酸性染色增强，核固缩深染；3m～6m损伤减轻呈恢复趋势，仍有部分细胞呈水肿状态；9m～12m表现为实质细胞减少，间质胶原纤维增多及脂肪浸润。以上改变尤以50mW/cm~2组最为显著。（5）微波辐射后大鼠SAN超微结构变化：5mW/cm~2组未见明显异常；10和50mW/cm~2微波辐射后1d～28d表现为P、T细胞线粒体最早受累，出现肿胀，嵴断裂，甚至空化；肌原纤维局灶性溶解、断裂；可见P、T细胞核染色质浓缩、边集；细胞膜小凹减少或消失；血管周围间隙增宽、水肿；6m～12m表现为实质细胞退行性变，间质胶原原纤维增多、脂肪浸润。以上改变尤以50mW/cm~2组最为显著。（6）微波辐射致大鼠SAN损伤后HCN4、β1-AR、M2-AchR变化：50mW/cm~2微波辐射后1d～28d，大鼠SAN组织中HCN4mRNA表达上调（P<0.05或P<0.01），3m表达下调（P<0.05）；HCN4蛋白于辐射后1d～28d表达增加（P<0.05或P<0.01），3m～6m表达降低（P<0.05）；β1-AR mRNA于辐射后1d～3m表达上调（P<0.05或P<0.01）；β1-AR蛋白于辐射后1d～3m表达增加（P<0.05或P<0.01）；M2-AchR蛋白于辐射后1d～6m表达增加（P<0.05或P<0.01）。（7）微波辐射后体外培养SAN细胞形态结构、搏动特征变化：正常SAN细胞呈长梭形，伸出伪足或突起与周围细胞连接成片，呈单个细胞搏动，搏动速度快，节律规则。10和50mW/cm~2微波辐射后，SAN细胞搏动明显减慢且节律不规则，细胞肿胀变圆，伪足和突起减少。50mW/cm~2微波辐射后即刻SAN细胞内[Ca~(2+)]升高（P<0.05），细胞膜表面可见穿孔现象。（8）微波辐射致体外培养SAN细胞损伤后HCN4表达改变：50mW/cm~2微波辐射后即刻，SAN细胞中HCN4蛋白表达显著减弱（P＜0.01），12h表达显著增强（P＜0.05）。
结论：（1）成年大鼠SAN特殊的位置提示：大鼠SAN取材时一定要保留上腔静脉近心段；两步法前固定取材有助于大鼠SAN电镜标本的制作。（2）10、50mW/cm~2微波辐射导致大鼠SAN功能障碍、组织结构和超微结构损伤，上述改变与微波辐射剂量呈正相关。（3）10、50mW/cm~2微波辐射可导致培养的SAN细胞形态结构损伤和搏动下降，且与微波辐射剂量呈正相关。（4）HCN4异常表达可能是微波辐射致SAN损伤的重要致伤分子之一。（5）SAN组织β1-AR高表达可能通过促进HCN4表达加重微波辐射后SAN组织损伤过程；M2-AchR高表达可能通过抑制HCN4表达促进微波辐射致SAN损伤后的修复过程。（6）细胞膜穿孔、细胞内钙超载、HCN4的高表达是微波辐射致SAN损伤的重要机制。
Objective and Significance: With the advent of information era and the high speeddevelopment of microwave technology, people are accessible to the hazard of microwavepollution as well as the conveniences it brings about. Besides, microwave technology also saw itsincreasing development and application in military fields. It can not only severely damage theenemy’s electronic devices, but also do harm to the body. Conducting system of heart is one of thetargets sensitive to microwave radiation. However, as the essential component of heart conductingsystem, SAN’s pattern of damage from microwave radiation and their dose-effect relationshipremained unclear, and the damaging mechanism was uncovered. HCN4can regulate pacemakercells’ pacing current and maintains their physiologic functions. The abnormal expression of HCN4in the SAN tissues may be a molecular basis of a variety of SAN pathological changes, whileHCN4and its regulation may play an important role in rats’ SAN damage after microwaveradiation. It is followed that the study of the HCN4change, its regulation and significance in theSAN’s damage from microwave radiation will provide a new thought thread and target for thefurther study of molecular mechanism and protective measures of microwave radiation damage toheart. Meanwhile it can provide experimental evidence for the identification of sensitive diagnosisindex and the formulation of protective criteria.
Materials and Methods:(1) Positioning of rats’ SANs and observation of their organizationalstructure: Twenty second-class adult Wistar rats were used to conduct the horizontal serial sectionsof the right atrium and the associated proximal segment of the superior vena cava. Serial sectionswere stained by HE and Masson staining and subsequently observed by light microscopy to locatethe position of SAN and to observe its organizational structure.(2) Observation of rats’ SAN’sultrastructure: Based on the SAN location and organizational characteristics,10second-class adultmale Wistar rats were taken to get the materials in two steps, the semi-thin sections werepre-stained by toluidine blue after the resin directed embedding and positioned through lightmicroscope. Following ultrathin sectioning, SAN ultrastructure was observed by the transmissionelectron microscopy.(3) Study of the effect of microwave radiation on rats’ SAN function andstructure:160Wistar rats were exposed to a pulse microwave field (0,5,10and50mW/cm~2) for6minutes. Multi-channel polygraph was applied to detect the change of ECG in rats beforemicrowave exposure and immediately,7,14,28days and3,6,9months after microwave radiation.Light microscopy and electron microscopy were used to observe the organizational structure andultrastructure of rats’ SAN at1d,7d,14d,28d,3m,6m,9m,12m after microwave exposure.Masson staining, Sirius red staining and image analysis techniques were applied to observe thedynamic development of the content of collagen fiber in rats’ sinoatrial nodes.(4) Research on thechange of HCN4in rats’ SAN tissue and the regulation mechanism after microwave radiation: ISH, IHC and image analysis were used to detect the changes in the gene or protein expression ofHCN4, β1-AR, M2-AchR in rat’s SAN tissues after50mW/m2microwave radiation.(5) Researchon the damaging effect and mechanism of microwave radiation on primary culture SAN cells:SAN cells primarily cultured in vitro were taken to do the research. Inverted microscope, AFM,LSCM and IF were used to observe the effect of microwave radiation on cells’ morphosis,pulsation features, membrane’s structure, the content of Ca~(2+)inside the cells and the expression ofHCN4.
Results:(1) SAN’s position and histological feature of normal adult rat: Adult rats’ SANswere located in the junctional zone of superior vena cava and right atricle and on the inner-wall ofsuperior vena cava above the junctional zone. The adult rats’ SANs were shaped like the horseshoeor the letter ‘C’. The rats’ SANs, being loose in structure and rich in interstitial collagen fiber,mainly contain P cells, T cells and a small amount of atrial muscle cells.(2) Observation ofSAN’s ultrastructure of adult rats: The toluidine blue prestained semi-thin sections is a rapidand efficient method, which can effectively shorten the positioning time. The electron microscopyshowed that two types of cells were present in the rat SAN: P cells and T cells. With a small cellbody, P cell was rich in cytoplasm but poor in organelle. There were a small number of myofibrilswhich were messily distributed nearby the cell membrane and almost free of sarcomeres. T cellhad a bigger cell body and more organelles than P cell and the myofibrils. With visible sarcomeres,were often arranged along the cell longitudinal axis and close to the cell membrane.(3) Effect ofmicrowave radiation on SAN’s function. There were no significant change in the5mW/cm~2group but appeared some damage to SAN’s functions in the10and50mW/cm~2groups, whichwere mainly as follows: the heart rate tended to accelerate and then slow down; the amplitude of Pwave was decreased; the ECG showed sinus arrhythmia, sinoatrial node wandering heart rhythmand atrial arrhythmia, etc.(4) Structural changes in rats’ SAN tissues after microwaveradiation: There was no significant change in the group of5mW/cm~2. But in the10and50mW/cm~2groups, changes could be observed like edema in P and T cells, wavy changes in T cells,increasing of acidophilia staining in the cytoplasm of P and T cells, and karyopyknosis andanachromasis from1to28days after microwave exposure. From3to6months, the damagestended to lessen and recover but there were still some cells in the edematous state, and from9to12months, parenchyma cells began to decrease and interstitial collagen fibers were increased, andthere appeared fatty infiltration. Changes above were remarkably observed in the group of50mW/cm~2.(5) SAN’s ultrastructural changes after exposure to microwave radiation. Therewas no significant change in the group of5mW/cm~2. In the period of1to28days, mitochondriaof T cells and P cells in the10and50mW/cm~2groups were the earliest to be effected to appearswelling, crista breakage, and even cavitation; then came the focal dissolution and even breakageof myofibrils, and concentration of nuclear chromatin of P and T cells to the edge, followed by thewidening and edema of perivascular canal, and the reduction or disappearance of foveolas ofmembrane. In the period of6to12months, there came degeneration in parenchyma cells, increase of interstitial collagen fibrils and fatty infiltration.(6) Changes in HCN4, β1-AR, and M2-AchRin rats’ SAN tissues injured by50mW/cm~2microwave radiation. The expression of HCN4mRNA began to be up-regulated at1to28days (P<0.05or P<0.01) and down-regulated at3months (P<0.05).The expression of HCN4protein was increased at1to28days (P<0.05orP<0.01) and was decreased at3to6months (P<0.05). The expression of β1-AR mRNA wasup-regulated at1d to3m (P<0.05or P<0.01) while the expression of β1-AR protein was increasedat1d to3m (P<0.05or P<0.01). The expression of M2-AchR protein was increased after1d~6m(P<0.05or P<0.01).(7) Changes in cellular morphosis and pulsation feature of SAN cellscultured in vitro after microwave exposure. The normal cell of SAN was shaped like longspindle, sticking out pseudopodia or apophysis and connected with the cells around, and beatrhythmically in single with a high speed. But immediately after the exposure to10and50mW/cm~2microwave, the cells of SAN began to beat significantly slower and irregular and tendedto become swollen and rounding, with pseudopodia or apophysis reducing. Besides, the contentsof Ca~(2+)inside the cells of SAN rose immediately after50mW/cm~2microwave exposure, andperforation could be observed in the surface of membrane.(8) Changes in the expression ofHCN4in the vitro cultured SAN cells after microwave radiation. The expression of HCN4protein was weakened significantly immediately after50mW/cm~2microwave radiation (P＜0.01)and was strengthened significantly after12hours (P＜0.05).
Conclusion:(1) The special location of SANs in adult rats indicated that the proximal segmentof superior vena cava must be reserved when taking SANs from rats and the two-step methodbefore positioning was helpful for the making of electron microscope specimens of rats’ SANs.(2)Exposure to10and50mW/cm~2microwave radiation can lead to dysfunction and damage toorganizational structure and ultrastructure of rats’ SANs. The above changes are positivelycorrelated to microwave radiation dose.(3) Exposure to10and50mW/cm~2microwave can lead todamage to morphosis and reduction of pulsation ability of cultured SAN cells, which arepositively correlated to microwave radiation dose.(4) The abnormal expression of HCN4is likelyto be one of the important vulnerating molecules in the damaging process of microwave radiationto SANs.(5) In the tissue of SAN, the high expression of β1-AR is likely to aggratate SAN’sinjuries resulted from microwave radiation by stimulating the expression of HCN4while the highexpression of M2-AchR is likely to promote the recovery of SAN’s injuries from microwaveradiation by inhibiting the expression of HCN4.(6) The perforation of membrane, the overloadingof intracellular Ca~(2+), and the high expression of HCN4are important mechanisms leading to SANinjury resulted from microwave radiation.
引文
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