斑马鱼尾鳍再生及小鼠大脑、心脏与棕色脂肪组织的节律性研究
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摘要
哺乳动物组织和器官的再生能力随着重复受伤或年龄的增长而逐渐减弱。近年来斑马鱼成为新的脊椎动物模式生物被用来研究组织和器官的再生。斑马鱼的许多组织都具有很强的再生能力，如视网膜、脊髓、肾、心脏和鳍。本研究检测了连续损伤尾鳍对再生的影响以及不同年龄斑马鱼尾鳍的再生情况。通过原位杂交和qRT-PCR比较不同再生阶段的标记基因(msxb,fgf20a,bmp2b)在新生组织中的表达，发现在重复多次切断尾鳍的斑马鱼中，基因表达量与第一次切断尾鳍相比没有显著性差异，表明重复损伤对尾鳍的再生能力没有影响。在再生初期（7days post-amputation, dpa），不同年龄组斑马鱼断尾后组织外长和相关再生标记基因（bmp2b）的表达无显著差异，表明尾鳍再生与年龄无关。总之，通过形态学和基因表达的分析，表明斑马鱼尾鳍具有无限再生能力。
正常条件下，哺乳动物的许多生理进程显示出每日的变化。当这些日变化模式在相同条件下恒定持续存在，即定义为昼夜节律（~24hours）。昼夜节律由内源性生物钟驱动。研究发现糖皮质激素（Glucocortcoids，GCs）对外周组织生物钟的调节在体内和体外均具有重要影响。昼夜节律在心脏组织无论是分子水平还是功能层面上都明显存在。先前的研究显示在一天的某个特定时间糖皮质激素受体激动剂地塞米松（DEX）会影响心脏生物钟基因的表达。鉴于生物钟基因对GCs的敏感性研究，本实验研究了DEX对心房组织的影响。通过对Per1和Per2-luciferase转基因小鼠体外心房组织培养，检测了DEX处理对心房生物钟的影响。据报道DEX对视交叉上核（SCN）中per1表达节律没有影响，但会改变肝脏中per1表达的节律相位，因此本研究对SCN和肝脏也进行了研究。结果发现不同时间点培养会影响心房生物钟的相位变化。结合培养液处理后观察到的相位转移，表明心房生物钟对操作处理存在时间依赖的敏感性，这与组织每天的机械活动变化如心脏的伸展和收缩运动高度相关。DEX也引起心房组织生物钟的相位转移，这种相位转移与肝脏处的显著不同，二者显示了距处理前最后一峰值的24h处理中除6-12h外所有时间点组织的特定反应。此外，心房组织中PER1-LUC的表达没有节律性，但DEX却能诱导出节律，而PER2-LUC有节律性并且在生理周期的特定时间有很强的振幅反应。这些数据表明心房生物钟在体外具有生物钟特点并对糖皮质激素存在明确的敏感度。
正电子发射断层扫描技术（Positron Emission Tomography, PET）是一种非侵入的定量原子能成像技术，被广泛应用于临床诊断和临床前的实验。PET通常利用~(18F)-氟代脱氧葡萄糖（FDG）作为示踪剂成像。这种成像技术提供了检测对葡萄糖高需求组织如心脏、大脑和各种癌症组织等的功能信息。本实验利用PET成像技术检测了C57BL/6小鼠活体内大脑、心脏和肩胛间棕色脂肪组织（iBAT）在24h光照-黑暗（LD）周期内对FDG的摄取变化。数据显示整个大脑对FDG的摄取具有显著的高振幅节律性，在LD周期的黑暗中间阶段达到高峰，此时正是夜行性小鼠活跃的阶段。此外，所检测的大部分大脑区域对FDG的摄取也均表现出24h的变化模式，包括在先前研究中报道的个别对葡萄糖利用没有差别的区域。相同条件下，相同小鼠的心脏对FDG的摄取在一天中不随时间改变，但存在生物学的波动。本研究表明在临床和临床前必需控制对大脑摄取FDG的扫描时刻，同时表明在一天的不同时间测量的FDG波形需标准化。iBAT的神经和内分泌调控具有明显的昼夜变化。本实验结果显示iBAT对FDG的摄取具有很强的24h变化分布，峰值大约在12h光照阶段的第9h。对iBAT摄取FDG的昼夜节律观察使得该组织成为代谢和节律系统之间相互作用的候选位点。此外，通过对个体内和个体间在一天的不同时间摄取FDG的检测，显示了24h内小鼠对FDG摄取模式的生物学和技术方面的可重复性。
The regenerative capacities of vertebrate tissues/organs tend to decrease afterrepeated injury or when the animals become older. The zebrafish (Danio rerio) haverecently emerged as a new vertebrate model for genetic studies of tissue/organregeneration. Zebrafish exhibit an enhanced capability of regenerating adult tissues,which include retina, spinal cord, kidney, heart and fin. In this study, we examinedthe effect of repeated injuries (fin amputation) in zebrafish on caudal fin regenerationand the capability of regeneration in zebrafish with different ages. In zebrafish thatreceived repeated injuries, the potential for caudal fin regeneration, such as tissuegrowth and the expression of regeneration marker genes (msxb, fgf20a, bmp2b), wasnot significant difference in comparison to zebrafish that received only oneamputation surgery. It shows that repeated injury has no effect on caudal finregeneration. During the process of initial fin regeneration (7days post-amputation,dpa), there were not significant differences in tissue outgrowth and the expression ofregeneration marker gene (bmp2b) between different aged zebrafish. This suggeststhat caudal fin regeneration did not seem to correlate with age. In summary, by usingmorphological and gene expression analyses, the data suggest that zebrafish hasunlimited regenerative potential in the injured caudal fin.
In mammals, many processes in physiology show daily variation under normalconditions. When these daily patterns persist under constant conditions, they aredescribed as circadian (~24hours) rhythms, and are driven by an endogenous clock.Glucocortcoids (GCs) have been implicated as being important in synchronizingclocks in peripheral tissues in vivo and in vitro. Circadian rhythms are apparent incardiac tissue at the molecular and functional level. It has been shown previously thatdexamethasone (DEX) which is a glucocortcoid receptor agonist can affect clockgene expression in the heart, although this has been tested only at one particular timeof day. Because of the reported susceptibility of clock genes to GCs, we set out to study the response of atrial tissue to dexamethasone (DEX) in a time informedmanner. Using in vitro atrial tissue cultures of mice carrying bioluminescencereporters for PER1and PER2activity, the circadian clock in this tissue and itsresponse to DEX treatment was monitored. Because DEX reportedly does not shiftthe rhythm of per1expression in the suprachiasmatic nucleus (SCN), but has a knownresponse in liver, the SCN and liver of the same individuals were also cultured. Theresults show that the clock in atrial tissue shows a strong phase shifting effect inresponse to time of culturing. Together with the phase shift observed after mediumtreatment, this could suggest a time-dependent sensitivity of the atrial clock tomechanical treatment, which is highly relevant for a tissue that shows a dailyvariation in mechanical (stretch and contractile) activity. Atrial tissue also showsphase shifting responses to DEX, which is markedly different from that of the liver.DEX treated liver and atria show tissue specific response for all time points exceptbetween6-12hours after the last peak in bioluminescence. Moreover, in atrial tissuePER1LUCexpression is not rhythmic, but can be induced by DEX, while PER2LUCisrhythmic and shows a strong amplitude response at certain times within the circadiancycle. These data indicate that the clock in the atrium has defined glucocorticoidsensitivity and circadian clock characteristics in vitro.
Positron Emission Tomography (PET) is a non-invasive and quantitative nuclearimaging modality used for a range of clinical diagnostic and pre-clinical experimentalapplications. PET often employs~(18F)-fluorodeoxyglucose (FDG) as the nuclear probefor imaging. This imaging technique provides functional information in detectingtissues with high glucose demands such as the heart, the brain, and many types ofcancers. In this study, we measured in vivo uptake of FDG in the brain, heart andinterscapular brown adipose tissue (iBAT) of C57BL/6mice at intervals across a24-hour light-dark cycle by PET. Our data describe a significant, high amplituderhythm in FDG uptake throughout the whole brain, peaking at the mid-dark phase ofthe light-dark cycle, which is the active phase for nocturnal mice. In addition, ourdata show24-hour patterns in glucose uptake in most of the brain regions examined,including several regions that do not show a difference in glucose utilization in theprevious studies. Under these conditions, heart FDG uptake did not vary with time-of-day, but did show biological variation throughout the24-hour period formeasurements within the same mice. Our data also emphasizes a methodologicalrequirement of controlling for the time-of-day of scanning FDG uptake in the brain inboth clinical and pre-clinical settings, and suggests waveform normalization of FDGmeasurements at different times of the day. Nervous and endocrine control of iBATpotentially exhibit circadian variation. Our data reveals a strong24-hour profile ofglucose uptake of iBAT, peaking at approximately9hours into the light phase of the12hour light,12hour dark day. The observation of a24-hour rhythm in glucoseuptake in iBAT makes this tissue a candidate site of interaction between metabolicand circadian systems. Moreover, FDG uptake was scanned at different times-of-daywithin an individual mouse, and also compared to different times-of-day betweenindividuals, showing both biological and technical reproducibility of the24-hourpattern in FDG uptake.
正常条件下，哺乳动物的许多生理进程显示出每日的变化。当这些日变化模式在相同条件下恒定持续存在，即定义为昼夜节律（~24hours）。昼夜节律由内源性生物钟驱动。研究发现糖皮质激素（Glucocortcoids，GCs）对外周组织生物钟的调节在体内和体外均具有重要影响。昼夜节律在心脏组织无论是分子水平还是功能层面上都明显存在。先前的研究显示在一天的某个特定时间糖皮质激素受体激动剂地塞米松（DEX）会影响心脏生物钟基因的表达。鉴于生物钟基因对GCs的敏感性研究，本实验研究了DEX对心房组织的影响。通过对Per1和Per2-luciferase转基因小鼠体外心房组织培养，检测了DEX处理对心房生物钟的影响。据报道DEX对视交叉上核（SCN）中per1表达节律没有影响，但会改变肝脏中per1表达的节律相位，因此本研究对SCN和肝脏也进行了研究。结果发现不同时间点培养会影响心房生物钟的相位变化。结合培养液处理后观察到的相位转移，表明心房生物钟对操作处理存在时间依赖的敏感性，这与组织每天的机械活动变化如心脏的伸展和收缩运动高度相关。DEX也引起心房组织生物钟的相位转移，这种相位转移与肝脏处的显著不同，二者显示了距处理前最后一峰值的24h处理中除6-12h外所有时间点组织的特定反应。此外，心房组织中PER1-LUC的表达没有节律性，但DEX却能诱导出节律，而PER2-LUC有节律性并且在生理周期的特定时间有很强的振幅反应。这些数据表明心房生物钟在体外具有生物钟特点并对糖皮质激素存在明确的敏感度。
正电子发射断层扫描技术（Positron Emission Tomography, PET）是一种非侵入的定量原子能成像技术，被广泛应用于临床诊断和临床前的实验。PET通常利用~(18F)-氟代脱氧葡萄糖（FDG）作为示踪剂成像。这种成像技术提供了检测对葡萄糖高需求组织如心脏、大脑和各种癌症组织等的功能信息。本实验利用PET成像技术检测了C57BL/6小鼠活体内大脑、心脏和肩胛间棕色脂肪组织（iBAT）在24h光照-黑暗（LD）周期内对FDG的摄取变化。数据显示整个大脑对FDG的摄取具有显著的高振幅节律性，在LD周期的黑暗中间阶段达到高峰，此时正是夜行性小鼠活跃的阶段。此外，所检测的大部分大脑区域对FDG的摄取也均表现出24h的变化模式，包括在先前研究中报道的个别对葡萄糖利用没有差别的区域。相同条件下，相同小鼠的心脏对FDG的摄取在一天中不随时间改变，但存在生物学的波动。本研究表明在临床和临床前必需控制对大脑摄取FDG的扫描时刻，同时表明在一天的不同时间测量的FDG波形需标准化。iBAT的神经和内分泌调控具有明显的昼夜变化。本实验结果显示iBAT对FDG的摄取具有很强的24h变化分布，峰值大约在12h光照阶段的第9h。对iBAT摄取FDG的昼夜节律观察使得该组织成为代谢和节律系统之间相互作用的候选位点。此外，通过对个体内和个体间在一天的不同时间摄取FDG的检测，显示了24h内小鼠对FDG摄取模式的生物学和技术方面的可重复性。
The regenerative capacities of vertebrate tissues/organs tend to decrease afterrepeated injury or when the animals become older. The zebrafish (Danio rerio) haverecently emerged as a new vertebrate model for genetic studies of tissue/organregeneration. Zebrafish exhibit an enhanced capability of regenerating adult tissues,which include retina, spinal cord, kidney, heart and fin. In this study, we examinedthe effect of repeated injuries (fin amputation) in zebrafish on caudal fin regenerationand the capability of regeneration in zebrafish with different ages. In zebrafish thatreceived repeated injuries, the potential for caudal fin regeneration, such as tissuegrowth and the expression of regeneration marker genes (msxb, fgf20a, bmp2b), wasnot significant difference in comparison to zebrafish that received only oneamputation surgery. It shows that repeated injury has no effect on caudal finregeneration. During the process of initial fin regeneration (7days post-amputation,dpa), there were not significant differences in tissue outgrowth and the expression ofregeneration marker gene (bmp2b) between different aged zebrafish. This suggeststhat caudal fin regeneration did not seem to correlate with age. In summary, by usingmorphological and gene expression analyses, the data suggest that zebrafish hasunlimited regenerative potential in the injured caudal fin.
In mammals, many processes in physiology show daily variation under normalconditions. When these daily patterns persist under constant conditions, they aredescribed as circadian (~24hours) rhythms, and are driven by an endogenous clock.Glucocortcoids (GCs) have been implicated as being important in synchronizingclocks in peripheral tissues in vivo and in vitro. Circadian rhythms are apparent incardiac tissue at the molecular and functional level. It has been shown previously thatdexamethasone (DEX) which is a glucocortcoid receptor agonist can affect clockgene expression in the heart, although this has been tested only at one particular timeof day. Because of the reported susceptibility of clock genes to GCs, we set out to study the response of atrial tissue to dexamethasone (DEX) in a time informedmanner. Using in vitro atrial tissue cultures of mice carrying bioluminescencereporters for PER1and PER2activity, the circadian clock in this tissue and itsresponse to DEX treatment was monitored. Because DEX reportedly does not shiftthe rhythm of per1expression in the suprachiasmatic nucleus (SCN), but has a knownresponse in liver, the SCN and liver of the same individuals were also cultured. Theresults show that the clock in atrial tissue shows a strong phase shifting effect inresponse to time of culturing. Together with the phase shift observed after mediumtreatment, this could suggest a time-dependent sensitivity of the atrial clock tomechanical treatment, which is highly relevant for a tissue that shows a dailyvariation in mechanical (stretch and contractile) activity. Atrial tissue also showsphase shifting responses to DEX, which is markedly different from that of the liver.DEX treated liver and atria show tissue specific response for all time points exceptbetween6-12hours after the last peak in bioluminescence. Moreover, in atrial tissuePER1LUCexpression is not rhythmic, but can be induced by DEX, while PER2LUCisrhythmic and shows a strong amplitude response at certain times within the circadiancycle. These data indicate that the clock in the atrium has defined glucocorticoidsensitivity and circadian clock characteristics in vitro.
Positron Emission Tomography (PET) is a non-invasive and quantitative nuclearimaging modality used for a range of clinical diagnostic and pre-clinical experimentalapplications. PET often employs~(18F)-fluorodeoxyglucose (FDG) as the nuclear probefor imaging. This imaging technique provides functional information in detectingtissues with high glucose demands such as the heart, the brain, and many types ofcancers. In this study, we measured in vivo uptake of FDG in the brain, heart andinterscapular brown adipose tissue (iBAT) of C57BL/6mice at intervals across a24-hour light-dark cycle by PET. Our data describe a significant, high amplituderhythm in FDG uptake throughout the whole brain, peaking at the mid-dark phase ofthe light-dark cycle, which is the active phase for nocturnal mice. In addition, ourdata show24-hour patterns in glucose uptake in most of the brain regions examined,including several regions that do not show a difference in glucose utilization in theprevious studies. Under these conditions, heart FDG uptake did not vary with time-of-day, but did show biological variation throughout the24-hour period formeasurements within the same mice. Our data also emphasizes a methodologicalrequirement of controlling for the time-of-day of scanning FDG uptake in the brain inboth clinical and pre-clinical settings, and suggests waveform normalization of FDGmeasurements at different times of the day. Nervous and endocrine control of iBATpotentially exhibit circadian variation. Our data reveals a strong24-hour profile ofglucose uptake of iBAT, peaking at approximately9hours into the light phase of the12hour light,12hour dark day. The observation of a24-hour rhythm in glucoseuptake in iBAT makes this tissue a candidate site of interaction between metabolicand circadian systems. Moreover, FDG uptake was scanned at different times-of-daywithin an individual mouse, and also compared to different times-of-day betweenindividuals, showing both biological and technical reproducibility of the24-hourpattern in FDG uptake.
引文
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