时钟基因PER2在喉癌中的表达水平及其预后意义

目的 探讨PER2基因在喉癌组织和癌旁非肿瘤组织中的表达水平及其与临床预后之间的相关性。方法 综合利用肿瘤基因组图谱（The Cancer Genome Atlas, TCGA）数据库和基因表达数据库（Gene Expression Omnibus,GEO）中的GSE84957数据集,分析PER2基因在喉癌患者癌组织及癌旁组织中的表达水平,并利用实时荧光定量PCR法在64例喉癌患者组织中验证PER2基因的表达。同时利用COX回归分析探讨PER2基因表达与喉癌患者临床预后和病理特征的相关性,进一步分析PER2基因的蛋白互作网络及功能富集,初步探索其在喉癌发生发展中的作用机制。结果 基于TCGA和GEO数据库分析结果显示PER2基因在喉癌组织中显著低表达（P<0.01）,PCR结果证实PER2基因在喉癌组织的表达量显著低于癌旁正常组织（P<0.01）。同时PER2基因高表达与喉癌患者的总生存期和无进展生存时间呈现显著正相关,且与喉癌患者的临床分期、分化程度和淋巴结转移状态等临床病理特征显著相关;蛋白互作及功能富集分析显示PER2基因主要通过与时钟相关基因相互作用参与昼夜节律等生...     

焦虑抑郁障碍与血压变异性研究

高血压与焦虑抑郁障碍之间密切相关。焦虑、抑郁可以引起血压昼夜节律变化,促发并加重高血压,降低降压药物的疗效,并可能导致难治性高血压。血压变异性作为新的血压监测参数,能更好地反应血压全天的总体水平、变化规律、昼夜的波动状况,能更好地评估降压治疗的疗效。随着血焦虑抑郁障碍引起异常的血压波动也越来越受重视,现就焦虑障碍与血压变异性之间关系的进行综述。     

PER2对人口腔鳞癌SCC15细胞增殖、凋亡以及生物钟基因表达的影响

目的探讨人口腔鳞癌SCC15细胞中生物钟基因PER2的表达改变对细胞增殖、凋亡以及其他生物钟基因的影响。方法用RNA干扰技术沉默人口腔鳞癌SCC15细胞中PER2基因;流式细胞术检测细胞增殖和凋亡水平;实时荧光定量PCR检测生物钟基因CLOCK、BMAL1、PER1、PER3、DEC1、DEC2、CRY1、CRY2、TIM、CKIε、RORα、NPAS2和REV-ERBαmRNA表达。结果沉默PER2后,SCC15细胞的增殖水平显著增加,凋亡显著下降(P0.05);SCC15细胞中生物钟基因PER3、BMAL1、DEC1、DEC2、CRY2、TIM、RORα和NPAS2 mRNA的表达水平显著降低,PER1和REV-ERBαmRNA的表达显著升高(P0.05)。结论在癌细胞中,生物钟基因PER2对生物钟基因网络中其他生物钟基因具有重要调控作用,PER2表达降低导致细胞增殖增加和细胞凋亡水平下降。     

老年非胰岛素依赖型糖尿病并高血压时血压昼夜节律改变

对比了22例健胀老华人和62例非胰岛素依赖型糖尿病(NIDDM)及合并高血压(NIDDM-HT)时的老年患者24小时动态血压昼夜节律,结果显示:健康老年人,老年NIDDM、老年NIDDM-HT者昼夜血压波动曲线均呈双峰、双谷状,老年人血压昼夜节律是存在的,但减弱;老年NIDDM血压昼夜节律消失;NIDDM-HT时夜间血压下降幅度进一步减小,提示:高龄、糖尿病、高血压均不同程度影响血压昼夜节律,而糖尿病起着主要作用,血压昼夜节律消失提示器官缺血,积极控制糖尿病及合并症以改善组织器官的缺血,是缓解血压昼夜节律消失的关键。     

小鼠外周组织时钟基因启动子区甲基化分析

 摘要：目的 建立一种简单快速经济的检测八个主要时钟基因BMAL1、BMAL2、CLOCK、NPAS2、PER1、PER2、CRY1和CRY2启动子区甲基化状态的方法。同时检测小鼠外周组织肝、心、肾、胸腺、睾丸时钟基因启动子区甲基化状态。方法 用偏重亚硫酸氢钠和对苯二酚对基因组DNA进行脱氨基修饰。修饰后的DNA为模板,两套不同的引物对：甲基化特异性引物对和非甲基化特异性引物扩增小鼠肝、心、肾、胸腺、睾丸组织时钟基因启动子区。PCR产物进行电泳和测序。结果 扩增产物与预期片段大小相符合。PCR产物经过直接测序得到进一步证实。结论 成功建立了检测小鼠时钟基因启动子区甲基化的方法,为检测小鼠周围组织时钟基因启动子区甲基化提供了新的方法。同时,成年小鼠所有的八个时钟基因均为非甲基化状态。     

Suvorexant通过调控PER1蛋白表达改善APP/PS1小鼠昼夜节律紊乱行为

目的:探讨orexin双受体拮抗剂suvorexant对9月龄APPswe/PS1dE9(APP/PS1)双转基因小鼠行为昼夜节律紊乱的影响及机制。方法:8月龄APP/PS1小鼠和具有同样遗传背景的野生型(WT)小鼠随机分为4组,在ZT0口服灌胃suvorexant或等体积溶剂28 d,采用跑轮行为学观察小鼠昼夜节律变化,免疫组织化学和Western Blot分别检测SCN脑区Aβ斑块和PER1蛋白的表达。结果:(1)在光暗交替环境中,给予suvorexant后APP/PS1小鼠相对振幅(P 0. 01)和鲁棒值(P 0. 01)明显升高,昼夜变异性明显降低(P 0. 01)。(2)在持续黑暗环境中给予suvorexant后,APP/PS1小鼠的自由运转周期明显缩短(P 0. 05),活动持续时间缩短(P 0. 01),相对振幅增大(P 0. 01),昼夜变异性减小(P 0. 01)。(3)免疫组化结果显示各组动物的SCN脑区未检测到Aβ阳性斑块。(4) APP/PS1小鼠给予Suvorexant后部分恢复了PER1蛋白表达的昼夜节律性。结论:suvorexant通过恢复SCN脑区PER1的昼夜节律性表达改善APPswe/PS1dE9小鼠的昼夜节律紊乱,增强其昼夜节律稳定性。     

山羊Goldblatt高血压昼夜节律及硝苯啶对其影响的研究

7只山羊经颈动脉插管直接测量动脉血压,计算机自动记录与贮存血压数据。动态观察山羊的正常血压范围。用Goldplatt双肾双夹法建立了山羊慢性高血压模型,用时间生物学群体余弦法显示山羊血压的昼夜节律。观察硝苯啶常规治疗和择时治疗对山羊Goldplatt高血压昼夜节律的影响。     

原发性高血压合并微量白蛋白尿患者动态血压观察

目的了解原发性高血压患者微量白蛋白尿(micro-albuminuria,MAU)与动态血压的特征性改变,包括血压变异指数(ABPVs、ABPVd)、血压负荷(BPload)及血压昼夜节律的相关性。方法确诊的141例原发性高血压患者进行24h动态血压和24h尿微量白蛋白检查,分为微量白蛋白尿(MAU)组及尿微量白蛋白正常组(NAU)。结果MAU组血压变异指数及血压负荷较NAU组增大(P<0.05),合并MAU的患者血压昼夜节律不明显。结论微量白蛋白尿的发生与血压变异指数、血压负荷和血压昼夜节律密切相关,有无微量白蛋白尿可一定程度上反映血压控制状况。     

高血压病血压昼夜节律与心肌缺血的关系

目的 探讨高血压病患者血压昼夜节律改变与心肌缺血之间的关系及临床意义。方法对90例高血压病患者,根据动态心电图上有无心肌缺血(MI)分为MI组(40例)和无MI组(50例),并与20例健康体检者进行对照,观察3组的动态血压变化,同时分析血压昼夜节律正常组和异常组的心肌缺血的发生情况。结果 高血压病伴有MI的患者中,除dSBP、24hDBPL、dSBPL、dDBPL与无MI组比较无统计学差异外,其余各项动态血压指标均明显高于无MI组(P<0.01);高血压病血压昼夜节律异常组发生MI者明显高于血压昼夜节律正常组(P<0.05)。结论 高血压病患者中伴有血压昼夜节律异常者更易导致心肌缺血的发生。对高血压病患者的治疗,除积极控制血压外,还应注意检测和逆转异常的昼夜血压节律,对减少心肌缺血的发生和靶器官损害具有重要意义。     

高血压室左室肥厚与血压昼夜节律及血液流变学的关系

本文观察了８４例高血压病患者左室患者重量指数与血液流变指标及动态血压昼夜节律的相互关系，结果发现：高血压病左室肥厚组高切粘度，低切粘度和血浆粘度明显升高，血压昼夜节律消失率升高，提示高血压病左室肥厚与血液流变学指标异常改变和血压昼夜节律消失密切相关。     

Pinealectomy does not affect diurnal PER2 expression in the rat limbic forebrain

A role for the pineal hormone, melatonin, in the regulation of the rhythmic expression of circadian clock genes is suggested by the finding that surgical removal of the pineal gland abolishes the rhythm of expression of clock genes such as Per1 in several neural and endocrine tissues in rodents, including the caudate-putamen (CP) and nucleus accumbens, the hypophyseal pars tuberalis and adrenal cortex. Pinealectomy has no effect on clock gene rhythms in the suprachiasmatic nucleus (SCN), the master circadian clock, as well as in the eyes and heart, indicating that the effect of melatonin on clock gene rhythms is tissue specific. To further study the role of melatonin in the regulation of the rhythm of clock genes, we assessed in rats the effect of pinealectomy on the rhythm of expression of the clock protein, PER2, in a number of key limbic forebrain structures, the oval nucleus of the bed nucleus of the stria terminalis (BNST-OV), the central nucleus of the amygdala (CEA) and the hippocampus (HIPP). Despite previous evidence showing that these regions are sensitive to melatonin, pinealectomy had no effect on the daily rhythm of expression of PER2 within these structures, further supporting the view that the role of endogenous melatonin in the regulation of clock gene expression is tissue specific.     

The central clock controls the daily rhythm of <Emphasis Type="Italic">Aqp5</Emphasis> expression in salivary glands

Salivary secretion displays day–night variations that are controlled by the circadian clock. The central clock in the suprachiasmatic nucleus (SCN) regulates daily physiological rhythms by prompting peripheral oscillators to adjust to changing environments. Aquaporin 5 (Aqp5) is known to play a key role in salivary secretion, but the association between Aqp5 and the circadian rhythm is poorly understood. The aim of our study was to evaluate whether Aqp5 expression in submandibular glands (SMGs) is driven by the central clock in the SCN or by autonomous oscillations. We observed circadian oscillations in the activity of period circadian protein homolog 2 and luciferase fusion protein (PER2::LUC) in cultured SMGs with periodicity depending on core clock genes. A daily rhythm was detected in the expression profiles of Aqp5 in SMGs in vivo. In cultured SMGs ex vivo, clock genes showed distinct circadian rhythms, whereas Aqp5 expression did not. These data indicate that daily Aqp5 expression in the mouse SMG is driven by the central clock in the SCN.     

Restricted access to food, but not sucrose, saccharine, or salt, synchronizes the expression of Period2 protein in the limbic forebrain

Restricted feeding schedules (RF) in which daily access to food is limited to a few hours each day can entrain the rhythms of expression of circadian clock genes in the brain and periphery in rodents. The critical factors mediating the effect of RF on rhythms of clock gene expression are unknown. Previously, we demonstrated that daytime RF shifts the phase of expression of the clock protein, Period2 (PER2) in the oval nucleus of the bed nucleus of the stria terminalis in rats kept on a 12-h light/dark cycle, and restored the rhythm of PER2 expression in rats housed in constant light. We now report that RF also modifies the rhythms of PER2 expression in the central and basolateral nuclei of the amygdala and in the dentate gyrus, such that all three areas become synchronized, peaking 12 h after the time of food presentation. Daily limited access to sucrose or saccharine in freely fed rats or scheduled access to saline in sodium-deprived rats had no effect on these PER2 rhythms. Thus, it would appear that the rhythms of PER2 in limbic forebrain structures are sensitive to signals that arise from the alleviation of a negative metabolic state associated with scheduled feeding and that access to rewarding substances in the absence of food deprivation or metabolic challenges, per se, is not sufficient to alter the rhythms of PER2 expression in these regions.     

Nighttime dim light exposure alters the responses of the circadian system

The daily light dark cycle is the most salient entraining factor for the circadian system. However, in modern society, darkness at night is vanishing as light pollution steadily increases. The impact of brighter nights on wild life ecology and human physiology is just now being recognized. In the present study, we tested the possible detrimental effects of dim light exposure on the regulation of circadian rhythms, using CD1 mice housed in light/dim light (LdimL, 300 lux:20 lux) or light/dark (LD, 300 lux:1 lux) conditions. We first examined the expression of clock genes in the suprachiasmatic nucleus (SCN), the locus of the principal brain clock, in the animals of the LD and LdimL groups. Under the entrained condition, there was no difference in PER1 peak expression between the two groups, but at the trough of the PER 1 rhythm, there was an increase in PER1 in the LdimL group, indicating a decrease in the amplitude of the PER1 rhythm. After a brief light exposure (30 min, 300 lux) at night, the light-induced expression of mPer1 and mPer2 genes was attenuated in the SCN of LdimL group. Next, we examined the behavioral rhythms by monitoring wheel-running activity to determine whether the altered responses in the SCN of LdimL group have behavioral consequence. Compared to the LD controls, the LdimL group showed increased daytime activity. After being released into constant darkness, the LdimL group displayed shorter free-running periods. Furthermore, following the light exposure, the phase shifting responses were smaller in the LdimL group. The results indicate that nighttime dim light exposure can cause functional changes of the circadian system, and suggest that altered circadian function could be one of the mechanisms underlying the adverse effects of light pollution on wild life ecology and human physiology.     

Chronic sleep deprivation altered the expression of circadian clock genes and aggravated Alzheimer's disease neuropathology

Circadian disruption is prevalent in Alzheimer''s disease (AD) and may contribute to cognitive impairment, psychological symptoms, and neurodegeneration. This study aimed to evaluate the effects of environmental and genetic factors on the molecular clock and to establish a link between circadian rhythm disturbance and AD. We investigated the pathological effects of chronic sleep deprivation (CSD) in the APP^swe/PS1^ΔE9 transgenic mice and their wild‐type (WT) littermates for 2 months and evaluated the expression levels of clock genes in the circadian rhythm‐related nuclei. Our results showed that CSD impaired learning and memory, and further exaggerated disease progression in the AD mice. Furthermore, CSD caused abnormal expression of Bmal1, Clock, and Cry1 in the circadian rhythm‐related nuclei of experimental mice, and these changes are more significant in AD mice. Abnormal expression of clock genes in AD mice suggested that the expression of clock genes is affected by APP/PS1 mutations. In addition, abnormal tau phosphorylation was found in the retrosplenial cortex, which was co‐located with the alteration of BMAL1 protein level. Moreover, the level of tyrosine hydroxylase in the locus coeruleus of AD and WT mice was significantly increased after CSD. There may be a potential link between the molecular clock, Aβ pathology, tauopathy, and the noradrenergic system. The results of this study provided new insights into the potential link between the disruption of circadian rhythm and the development of AD.     

Inter-individual differences in habitual sleep timing and entrained phase of endogenous circadian rhythms of BMAL1, PER2 and PER3 mRNA in human leukocytes

STUDY OBJECTIVES: Individual sleep timing differs and is governed partly by circadian oscillators, which may be assessed by hormonal markers, or by clock gene expression. Clock gene expression oscillates in peripheral tissues, including leukocytes. The study objective was to determine whether the endogenous phase of these rhythms, assessed in the absence of the sleep-wake and light-dark cycle, correlates with habitual sleep-wake timing. DESIGN: Observational, cross-sectional. SETTING: Home environment and Clinical Research Center. PARTICIPANTS: 24 healthy subjects aged 25.0 +/- 3.5 (SD) years. MEASUREMENTS: Actigraphy and sleep diaries were used to characterize sleep timing. Circadian rhythm phase and amplitude of plasma melatonin, cortisol, and BMAL1, PER2, and PER3 expression were assessed during a constant routine. RESULTS: Circadian oscillations were more robust for PER3 than for BMAL1 or PER2. Average peak timings were 6:05 for PER3, 8:06 for PER2, 15:06 for BMAL1, 4:20 for melatonin, and 10:49 for cortisol. Individual sleep-wake timing correlated with the phases of melatonin and cortisol. Individual PER3 rhythms correlated significantly with sleep-wake timing and the timing of melatonin and cortisol, but those of PER2 and BMAL1 did not reach significance. The correlation between sleep timing and PER3 expression was stronger in individuals homozygous for the variant of the PER3 polymorphism that is associated with morningness. CONCLUSIONS: Individual phase differences in PER3 expression during a constant routine correlate with sleep timing during entrainment. PER3 expression in leukocytes represents a useful molecular marker of the circadian processes governing sleep-wake timing.     

Suggestive evidence for association of the circadian genes PERIOD3 and ARNTL with bipolar disorder.

Bipolar affective disorder (BPAD) is suspected to arise in part from malfunctions of the circadian system, a system that enables adaptation to a daily and seasonally cycling environment. Genetic variations altering functions of genes involved with the input to the circadian clock, in the molecular feedback loops constituting the circadian oscillatory mechanism itself, or in the regulatory output systems could influence BPAD as a result. Several human circadian system genes have been identified and localized recently, and a comparison with linkage hotspots for BPAD has revealed some correspondences. We have assessed evidence for linkage and association involving polymorphisms in 10 circadian clock genes (ARNTL, CLOCK, CRY2, CSNK1epsilon, DBP, GSK3beta, NPAS2, PER1, PER2, and PER3) to BPAD. Linkage analysis in 52 affected families showed suggestive evidence for linkage to CSNK1epsilon. This finding was not substantiated in the association study. Fifty-two SNPs in 10 clock genes were genotyped in 185 parent proband triads. Single SNP TDT analyses showed no evidence for association to BPAD. However, more powerful haplotype analyses suggest two candidates deserving further studies. Haplotypes in ARNTL and PER3 were found to be significantly associated with BPAD via single-gene permutation tests (PG = 0.025 and 0.008, respectively). The most suggestive haplotypes in PER3 showed a Bonferroni-corrected P-value of PGC = 0.07. These two genes have previously been implicated in circadian rhythm sleep disorders and affective disorders. With correction for the number of genes considered and tests conducted, these data do not provide statistically significant evidence for association. However, the trends for ARNTL and PER3 are suggestive of their involvement in bipolar disorder and warrant further study in a larger sample.