Effects of Chronotherapy of Benazepril on the Diurnal Profile of RAAS and Clock Genes in the Kidney of 5/6 Nephrectomy Rats

Summary： This study investigated the effects of benazepril administered in the morning or evening on the diurnal variation of renin-angiotensin-aldosterone system （RAAS） and clock genes in the kidney. The male Wistar rat models of 5/6 subtotal nephrectomy （STNx） were established. Animals were ran- domly divided into 4 groups： sham STNx group （control）, STNx group, morning benazepril group （MB） and evening benazepril group （EB）. Benazepril was intragastfically administered at a dose of 10 mg/kg/day at 07：00 and 19：00 in the MB group and EB group respectively for 12 weeks. All the animals were synchronized to the light：dark cycle of 12：12 for 12 weeks. Systolic blood pressure （SBP）, 24-h urinary protein excretion and renal function were measured at 11 weeks. Blood samples and kidneys were collected every 4 h throughout a day to detect the expression pattern of renin activity （RA）, angio- tensin Ⅱ （Ang Ⅱ ） and aldosterone （Aid） by radioimmunoassay （RIA） and the mRNA expression profile of clock genes （bmall, dbp and per2） by real-time PCR at 12 weeks. Our results showed that no signifi- cant differences were noted in the SBP, 24-h urine protein excretion and renal function between the MB and EB groups. There were no significant differences in average Aid and RA content of a day between the MB group and EB group. The expression peak of bmall mRNA was phase-delayed by 4 to 8 h, and the diurnal variation of per2 and dbp mRNA diminished in the MB and EB groups compared with the control and STNx groups. It was concluded when the similar SBP reduction, RAAS inhibition and clock gene profile were achieved with optimal dose of benazepril, morning versus evening dosing of benazepril has the same renoprotection effects.

Clock genes:Their role in colorectal cancer

Clock genes create a complicated molecular time-keeping system consisting of multiple positive and negative feedback loops at transcriptional and translational levels.This circadian system coordinates and regulates multiple cellular procedures implicated in cancer development such as metabolism,cell cycle and DNA damage response.Recent data support that molecules such as CLOCK1,BMAL1 and PER and CRY proteins have various effects on c-Myc/p21 and Wnt/β-catenin pathways and influence multiple steps of DNA damage response playing a critical role in the preservation of genomic integrity in normal and cancer cells.Notably,all these events have already been related to the development and progression of colorectal cancer(CRC).Recent data highlight critical correlations between clock genes’expression and pathogenesis,progression,aggressiveness and prognosis of CRC.Increased expression of positive regulators of this circadian system such as BMAL1 has been related to decrease overall survival while decreased expression of negative regulators such as PER2 and PER3 is connected with poorer differentiation,increased aggressiveness and worse prognosis.The implications of these molecules in DNA repair systems explain their involvement in the development of CRC but at the same time provide us with novel targets for modern therapeutic approaches for patients with advanced CRC.

The Clock gene clone and its circadian rhythms in Pelteobagrus vachelli

The Clock gene,a key molecule in circadian systems,is widely distributed in the animal kingdom. We isolated a 936-bp partial c DNA sequence of the C lock gene( Pva- clock) from the darkbarbel catfish P elteobagrus vachelli that exhibited high identity with C lock genes of other species of fish and animals(65%–88%). The putative domains included a basic helix-loop-helix(b HLH) domain and two period-ARNT-single-minded(PAS) domains,which were also similar to those in other species of fish and animals. P va- Clock was primarily expressed in the brain,and was detected in all of the peripheral tissues sampled. Additionally,the pattern of P va- Clock expression over a 24-h period exhibited a circadian rhythm in the brain,liver and intestine,with the acrophase at zeitgeber time 21:35,23:00,and 23:23,respectively. Our results provide insight into the function of the molecular C lock of P. vachelli.

Exploitation of host clock gene machinery by hepatitis viruses B and C

Many aspects of cellular physiology display circadian(approximately 24-h)rhythms.Dysfunction of the circadian clock molecular circuitry is associated with human health derangements,including neurodegeneration,increased risk of cancer,cardiovascular diseases and the metabolic syndrome.Viruses triggering hepatitis depend tightly on the host cell synthesis machinery for their own replication,survival and spreading.Recent evidences support a link between the circadian clock circuitry and viruses’biological cycle within host cells.Currently,in vitro models for chronobiological studies of cells infected with viruses need to be implemented.The establishment of such in vitro models would be helpful to better understand the link between the clock gene machinery and viral replication/viral persistence in order to develop specifically targeted therapeutic regimens.Here we review the recent literature dealing with the interplay between hepatitis B and C viruses and clock genes.

Effects of 72 Hours Sleep Deprivation on Liver Circadian Clock Gene Expression and Oxidative Stress in Rats

Objective: To investigate the effects of 72 hours continuous sleep deprivation (SD) on circadian clock gene expression and oxidative stress in the rat liver. Methods: Twenty healthy male Sprague-Dawley rats were divided into 2 groups (n = 10 each) using a random number table: normal control group (group C), sleep deprivation group (group SD). Group SD was treated with a modified multiple platform water environment method. After 72 hours sleep deprived, the levels of AST (Aspartate transaminase ) and ALT (Alanine aminotransferase) in serum were determined. The contents of malondialdehyde (MDA), the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the liver tissue of the rats were examined in both two groups. The expression levels of CLOCK, BMAL1 and CRY1 protein in liver tissue were examined by Western blotting. Results: Compared with group C, the content of MDA, and the levels of AST and ALT in serum were significantly increased (P Conclusion: 72 hours continuous sleep deprivation can downregulate the expression of circadian clock gene and promote oxidative stress in rats.

The clock gene,period,influences migratory flight and reproduction of the oriental armyworm,Mythimna separata(Walker)

The oriental armyworm,Mythimna separata,is a major long-distance migratory insect pest of grain crops in China and other Asian countries.Migratory flights and reproductive behavior usually occur at night,regulated by a circadian rhythm.However,knowledge about the linkages between adult flight,reproduction,and clock genes is still incomplete.To fill this important gap in our knowledge,a clock gene(designated Msper)was identified and phylogenetic analysis indicated that the encoded protein(MsPER)was highly similar to PER proteins from other insect species.Quantitative RT-PCR assays demonstrated that significantly different spatiotemporal and circadian rhythmic accumulations of mRNA encoding MsPER occurred during development under steady 14 h:10 h light:dark conditions.The highest mRNA accumulation occurred in adult antennae and the lowest in larvae.Msper was expressed rhythmically in adult antennae,relatively less in photophase and more entering scotophase.Injecting small interference RNA(siRNA)into adult heads effectively knocked down Msper mRNA levels within 72 h.Most siRNA-injected adults reduced their evening flight activity significantly and did not exhibit a normal evening peak of flight activity.They also failed to mate and lay eggs within 72 h.Adult mating behavior was restored to control levels by 72 h post injection.We infer that Msper is a prominent clock gene that acts in regulating adult migratory flight and mating behaviors of M.separata.Because of its influence on migration and mating,Msper may be a valuable gene to target for effective management of this migratory insect.

Co-regulation of circadian clock genes and microRNAs in bone metabolism

Mammalian bone is constantly metabolized from the embryonic stage,and the maintenance of bone health depends on the dynamic balance between bone resorption and bone formation,mediated by osteoclasts and osteoblasts.It is widely recognized that circadian clock genes can regulate bone metabolism.In recent years,the regulation of bone metabolism by non-coding RNAs has become a hotspot of research.MicroRNAs can participate in bone catabolism and anabolism by targeting key factors related to bone metabolism,including circadian clock genes.However,research in this field has been conducted only in recent years and the mechanisms involved are not yet well established.Recent studies have focused on how to target circadian clock genes to treat some diseases,such as autoimmune diseases,but few have focused on the co-regulation of circadian clock genes and microRNAs in bone metabolic diseases.Therefore,in this paper we review the progress of research on the co-regulation of bone metabolism by circadian clock genes and microRNAs,aiming to provide new ideas for the prevention and treatment of bone metabolic diseases such as osteoporosis.

大负荷运动后骨骼肌核心时钟基因Bmal1/Clock、Cry1/Per1的节律性振荡变化趋势

目的:探讨大负荷运动对骨骼肌核心时钟基因节律性表达的影响。方法:将208只8周龄SD大鼠随机分为对照(control,C)组与运动(Exercise,E)组,E组予以一次大负荷运动训练。每6 h取各组比目鱼肌,采用透射电镜观察骨骼肌纤维超微结构,通过实时荧光定量PCR实验测定骨骼肌核心时钟基因Bmal1、Clock、Cry1、Per1表达量,并使用余弦分析软件circacompare(R package)获取拟合余弦曲线参数,分析其节律性振荡的变化趋势。结果:1)C组Bmal1、Clock、Cry1、Per1 mRNA在72 h内呈现3个完整近日节律周期;E组Bmal1 mRNA、Cry1和Per1mRNA在0~24 h、Clock mRNA在0~72 h近日节律消失。2)与C组相比,E组Bmal1 mRNA在0、6、12和18 h显著升高,Clock mRNA在0 h时显著升高;Cry1、Per1 mRNA在0、12 h显著降低。3)C组各时相肌纤维超微结构正常,E组0、24和48 h均出现不同程度肌小节结构破坏、线粒体肿胀及聚集等改变,72 h有所缓解。结论:一次大负荷运动可诱发骨骼肌核心时钟基因Bmal1/Clock、Cry1/Per1节律振荡紊乱,其变化趋势与肌纤维超微结构损伤时相基本一致。

生物钟相关基因CLOCK及褪黑素受体MT1和MT2基因与2型糖尿病患者肠道菌群及炎症因子相关性分析

目的:探讨生物钟基因CLOCK及褪黑素受体MT1和MT2基因与2型糖尿病(T2DM)患者肠道菌群及炎症因子的相关性。方法:选择T2DM患者80例为观察组,选择同期进行健康体检者80例为对照组。检测两组血清CLOCK、MT1和MT2基因水平,以及超敏C反应蛋白(hs-CRP)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)和IL-18水平。检测两组粪便中肠杆菌、双歧杆菌、乳杆菌、肠球菌、双歧杆菌/肠杆菌丰度。分析观察组血清CLOCK、MT1和MT2基因水平与T2DM患者肠道菌群及炎症因子的相关性。结果:观察组血清CLOCK、MT1、MT2基因水平明显较对照组低(均P<0.05)。观察组肠杆菌水平明显较对照组高,双歧杆菌、乳杆菌、双歧杆菌/肠杆菌水平明显较对照组低(均P<0.05)。观察组hs-CRP、IL-6、TNF-α、IL-18水平明显较对照组高(均P<0.05)。观察组血清CLOCK、MT1、MT2基因水平与肠杆菌、hs-CRP、IL-6、TNF-α、IL-18水平呈负相关,与双歧杆菌、乳杆菌、双歧杆菌/肠杆菌水平呈正相关(均P<0.05)。结论:血清生物钟相关基因CLOCK、MT1和MT2基因与T2DM患者肠道菌群及炎症因子均有一定相关性,可作为潜在的诊断指标及治疗靶点。

异齿裂腹鱼Clock基因cDNA的克隆与组织表达特征

异齿裂腹鱼广布于雅鲁藏布江,产卵期为每年的3—5月,表现出明显的季节性繁殖行为,Clock基因被认为是自然选择塑造日节律以及与动物繁殖等生活史特性有关潜在的目标基因,异齿裂腹鱼Clock蛋白C末端Poly-Q长度的变异可能与其产卵开始的时间关联。以采自雅鲁藏布江的异齿裂腹鱼为对象,采用反转录PCR和cDNA末端快速扩增技术克隆异齿裂腹鱼Clock基因cDNA全长序列,通过实时荧光定量PCR对其在异齿裂腹鱼雌、雄个体不同组织中的表达特征进行分析。试验结果显示:异齿裂腹鱼Clock基因cDNA全长为4291 bp(Genbank登录号MT774361),5′非编码区为539 bp,3′非编码区为1046 bp,编码序列为2706 bp,共编码901个氨基酸,所编码的氨基酸在末端具有典型的Poly-Q结构;Clock基因在异齿裂腹鱼雌、雄个体的肝脏、脾脏、心脏、脑、性腺和肌肉中均有表达,且以性腺中的相对表达量最高,肌肉次之,脾脏最低;Clock基因除在心脏组织中的相对表达量雄鱼高于雌鱼,在其他组织的相对表达量均表现为雌鱼高于雄鱼。异齿裂腹鱼雌、雄个体的Clock基因在性腺组织中均高表达,表明Clock基因在异齿裂腹鱼的繁殖过程中发挥着更大的作用,异齿裂腹鱼Clock蛋白大片段的插入也可能是为了适应高海拔某些条件而特化的。

电针对昼夜节律紊乱C57BL/6J小鼠肝脏基因Clock、Bmal1表达的影响

目的 观察电针肝俞对昼夜节律紊乱小鼠外周时钟基因的影响,分析各实验条件下,不同时间点Clock、Bmal1基因的分布规律,为外周生物钟基因时间分布提供参考,并探讨电针治疗睡眠节律紊乱的潜在机制。方法 将72只C57BL/6J小鼠按随机数字表法分成空白组、模型组、肝俞组和非经非穴组,每组18只。空白组常规饲养;其余3组采取光暗交替法L(光)∶D(暗)=2 h∶2 h造模法持续5 d,模型组只捆绑不针刺;肝俞组针刺肝俞穴;非经非穴组选取非经非穴部位进行针刺。电针干预5 d后,于第6天02∶00开始,每组每4 h取材1次,共取材6次。采用PCR法以及Western blot法检测各组小鼠肝脏内的Clock和Bmal1基因mRNA的表达水平和蛋白表达含量,同时用余弦函数计算出各组基因的节律性。结果 与模型组比较,肝俞组中Clock与Bmal1基因表达恢复时间节律(P<0.05)。与模型组比较,非经非穴组内Clock与Bmal1基因表达无时序性(P>0.05);肝俞组Clock、Bmal1的蛋白表达量较模型组明显降低且恢复节律性(P<0.05)。结论 小鼠肝脏中Clock和Bmal1时钟基因呈时序性变化,电针肝俞穴可改善小鼠睡眠,其机制可能与调节时钟基因Clock和Bmal1,降低两种基因及蛋白表达,恢复正常睡眠节律有关。

Circadian Clock Genes Universally Control Key Agricultural Traits

Circadian clocks are endogenous timers that enable plants to synchronize biological processes with daily and seasonal environmental conditions in order to allocate resources during the most beneficial times of day and year. The circadian clock regulates a number of central plant activities, including growth, develop- ment, and reproduction, primarily through controlling a substantial proportion of transcriptional activity and protein function. This review examines the roles that alleles of circadian clock genes have played in domestication and improvement of crop plants. The focus here is on three groups of circadian clock genes essential to clock function in Arabidopsis thaliana： PSEUDO-RESPONSE REGULATORs, GIGANTEA, and the evening complex genes EARL Y FLOWERING 3, EARLY FLOWERING 4, and LUX ARRHYTHMO. Homol- ogous genes from each group underlie quantitative trait loci that have beneficial influences on key agricul- tural traits, especially flowering time but also yield, biomass, and biennial growth habit. Emerging insights into circadian clock regulation of other fundamental plant processes, including responses to abiotic and biotic stresses, are discussed to highlight promising avenues for further crop improvement.

Knockdown the Circadian Clock Gene period and timeless arrest the Photoperiodic Induction of Summer Diapause in Colaphellus bowringi Baly

In insects,facultative diapause is a state of developmental arrest mainly induced by photoperiod or temperature that allows insects to survive adverse environmental conditions.Understanding how insect initiates facultative diapause and prepares diapause can provide us new insights to study developmental and evolutionary biology.It has been shown that the circadian clock genes can participate in photoperiodic measurement and regulate reproductive diapause initiation through JH signaling in short-day-induced winter diapause.However,how circadian clock genes translate photoperiodic information into downstream JH signaling for diapause destiny and then affect diapause preparation remains largely unknown.In the present study,we investigate this in the cabbage beetle Colaphellus bowringi which undergoes reproductive diapause under long-day condition.We respectively knocked down two circadian clock negative regulators,period(per)and timeless(tim),in the 3-day-old larvae(most sensitive to photoperiod),and dsgfp treatment was served as a control.Under the diapause-inducing photoperiod(16L:8D),knocking down per and tim significantly decreased the rate of burrowing behavior.And mtany female beetles of the per and tim RNAi showed developed ovary,decreased lipid accumulation and downregulated expression of stress resistance genes.The JHinduced genes,Kr-h1,JHE1,Vg1,and Vg2, significantly increased in the females with suppression of per and tim.It implied that suppression of per and tim during diapause initiation phase(DIP)could activate the JH signaling in the female adults.Before the beetles enter into diapause preparation phase(DPP),we used RNA sequencing to analyize gene expression profiles after per and tim RNAi.It showed that many differentially expressed genes were enriched in environmental information processing,such as mTOR and TGF-beta signaling pathway.To ask whether per and tim also regulate diapause preparation,we knocked down these two genes in the female adults during DPP.It showed that the diapause destiny was not af-fected,but the lipid storage in diapause-destined females was significantly reduced after per and tim RNAi.Interestingly,per-and tim-regulated lipid storage during DPP was independent on JH signaling.We further found that per and tim promoted lipid storage by regulating the expression of genes that control lipogenesis and lipolysis.In summary,these results suggest that per and tim participate in photoperiodic measurement and initiate reproductive diapause through JH signaling during DIP in long-day-induced summer diapause.The mTOR and TGF-beta signaling pathways may be involved in the regulation of JH signaling by circadian clock genes.Meanwhile,per and tim transduce photoperiodic signal and promote lipid storage during DPP in a JH-independent manner.These results provide us new clues to study the molecular mechanism of photoperiod-regulated diapause induction in insects.

生物节律与子痫前期的相关性

子痫前期(preeclampsia,PE)是一种常见的妊娠并发症,危害母婴健康,更是一个严重的国际公共卫生问题。尽管已证实PE的高危因素包括肥胖、糖尿病和妊娠前高血压等,但其发病机制仍未完全阐明。哺乳动物中绝大多数的生理过程由生物节律调控,生物节律的破坏被证实与众多疾病相关,如心血管疾病、糖尿病和乳腺癌等。尽管PE具有一系列时间生物节律的特征性表现,如PE患者的血压24 h节律变异性、“反勺状”血压与更严重的靶器官损伤有关、夜间服用阿司匹林的预防保护作用更优异等,但生物节律的变异是否为PE的危险因素,抑或PE本身是否与异常的生物节律相关,目前尚未明确。综述生物节律与PE的相关性,包括胎盘的钟基因表达及其与PE的关系、血压的生物节律、PE预防的时间治疗学、生物节律的破坏与PE风险性,以启示今后深入研究生物节律/钟基因与PE之间潜在关联的必要性。

生物钟基因Bmal1、Per2在血液肿瘤细胞株Raji、Hut-78、OCI-LY8及HL-60中的昼夜表达规律

目的通过检测生物钟基因Bmal1、Per2在血液肿瘤细胞株Raji、Hut-78、OCI-LY8及HL-60细胞中不同时间点的mRNA表达情况,探讨其与血液肿瘤发生发展的可能关系。方法血液肿瘤细胞株Raji、Hut-78、LY-8、HL-60经血清休克法诱导节律后,RT-PCR法检测生物钟基因Bmal1及Per2在不同时间点的mRNA转录水平,以时间为变量分别对其转录量进行余弦函数拟合,通过余弦函数预测基因转录高峰及低谷时段。结果Bmal1和Per2基因表达在OCI-LY8、Hut-78、HL-60细胞中呈现昼夜节律(P<0.05);Raji细胞内Bmal1基因无节律性表达(P>0.05),而Per2基因呈昼夜节律表达(P<0.05)。结论生物钟基因Per2和(或)Bmal1在多种血液肿瘤细胞株内呈昼夜节律性表达,其可能参与血液肿瘤的发生发展。

时钟基因调控低氧训练肥胖大鼠白色脂肪组织棕色化

背景:低氧训练可以促进机体脂肪的降解,外界环境变化可影响动物昼夜节律,但昼夜节律变化对脂肪组织棕色化及脂肪降解的调控机制尚不明确。目的:阐明时钟基因对低氧训练大鼠附睾脂肪组织棕色化的调控机制。方法:喂养高脂饲料构建肥胖大鼠模型,造模成功后抽取40只肥胖大鼠随机分为4组,即常氧安静组、低氧安静组、常氧训练组、低氧训练组,每组10只,进行4周的干预。安静组大鼠不实施干预;低氧组大鼠全天生活在氧浓度为13.6%的低氧仓中;训练组第1周为适应性训练,后3周训练速度和时长保持不变。测量肥胖大鼠体质量、体长和肾周脂肪质量;并使用血脂生化检测试剂盒检测肥胖大鼠血清中三酰甘油、总胆固醇、低密度脂蛋白胆固醇以及高密度脂蛋白胆固醇水平;油红O染色观察肝脏脂肪含量变化;苏木精-伊红染色评价各组大鼠附睾脂肪组织棕色化变化;转录组测序技术结合生物信息学分析脂肪组织中转录组水平变化;采用qRT-PCR检测附睾脂肪组织中PGC-1α、Beclin1、KLF2、Perilipin1 mRNA的表达变化情况。结果与结论:①低氧训练干预使营养性肥胖大鼠体质量、脂体比、Lees’s指数、血清总胆固醇、三酰甘油以及低密度脂蛋白胆固醇浓度显著降低(P<0.01),高密度脂蛋白胆固醇浓度显著升高(P<0.01);②油红O染色和苏木精-伊红染色显示,相比于常氧安静组、低氧安静组和常氧训练组,低氧训练更能有效促进大鼠肝脏组织中脂肪动员和附睾旁脂肪组织棕色化;③转录组测序结果显示,低氧训练时钟基因Dbp、Nr1d1、Sik1以及脂肪组织棕色化基因Ppargc1a(PGC-1α)等表达显著上调(P<0.05),而Arntl(Bmal1)显著下调(P<0.05),同时伴随物质代谢相关基因的表达增强;④qRT-PCR结果证实低氧训练时脂肪组织棕色化基因PGC-1α和脂代谢基因Perilipin1表达量均显著升高(P<0.01);⑤结果证实时钟基因在低氧训练促进脂肪组织棕色化过程中起到重要作用。

大鼠视交叉上核与松果体中Clock基因转录的昼夜节律性及不同光反应性(英文)

本研究旨在观察和比较视交叉上核(suprachiasmatic nucleus,SCN)与松果体(pineal gland,PG)中Clock基因内源性昼夜转录变化规律以及光照对其的影响。Sprague-Dawley大鼠在持续黑暗(constant darkness,DD)和12 h光照：12 h黑暗交替(12 hour- light：12 hour-dark cycle,LD)光制下分别被饲养8周(n=36)和4周(n=36)后,在一昼夜内每隔4 h采集一组SCN和PG组织(n=6),提取总RNA,用竞争性定量RT-PCR测定不同昼夜时点(circadian times,CT or zeitgeber times,ZT)各样品中Clock基因的mRNA相对表达量,通过余弦法和Clock Lab软件获取节律参数,并经振幅检验是否存在昼夜节律性转录变化。结果如下：(1)SCN中Clock基因mRNA的转录在DD光制下呈现昼低夜高节律性振荡变化(P＜0．05),PG中Clock基因的转录也显示相似的内源性节律外观,即峰值出现于主观夜晚(SCN为CT15,PG为CT18),谷值位于主观白天(SCN为CT3,PG为CT6)(P＞0．05)。(2) LD光制下SCN中Clock基因的转录也具有昼夜节律性振荡(P＜0．05),但与其DD光制下节律外观相比,呈现反时相符律变化(P＜0．05),且其表达的振幅及峰值的mRNA水平均增加(P＜0．05),而PG中Clock基因在LD光制下转录的相应节律参数变化却恰恰相反(P＜0．05)。(3)在LD光制下,光照使PG中Clock基因转录的节律外观反时相于SCN(P＜0．05),即在SCN和PG的峰值分别出现于光照期ZT10和黑暗期ZT17,谷值分别位于黑暗期ZT22和光照期ZT5。结果表明,Clock基因的昼夜转录在SCN和PG中存在同步的内源性节律本质,而光导引在这两个中枢核团调节Clock基因昼夜节律性转录方面有着不同的作用。

山羊clock和cry1基因的克隆及其在大脑和垂体中表达差异的研究

本研究旨在探讨生物钟基因clock和cry1在山羊出生后不同时期的大脑和垂体中的表达差异。在出生后30、60、90和120d共屠宰24只南江黄羊羔羊(公母各12只),取其大脑皮质层和垂体组织样品用于抽提RNA,进行分子克隆和实时荧光定量PCR分析。结果,分离克隆得到了山羊clock基因外显子1到外显子8以及cry1基因外显子6到外显子15的CDS区序列,其长度分别为1 479bp和993bp,其中clock基因部分CDS区序列与绵羊、牛、人和小鼠的对应序列相似性分别为99%、90%、94%和92%,cry1基因部分CDS区序列与绵羊、牛、人和小鼠的对应序列相似性分别为99%、98%、94%和87%。组织表达分析结果表明,性别对clock和cry1基因的表达影响不显著(P>0.05),2个基因在垂体中的mRNA表达量极显著高于大脑中的表达量(P<0.01);在不同时期的垂体组织中,clock和cry1基因表达量均呈现先上升后下降的趋势;在不同时期的大脑组织中,clock基因的表达量趋于波动平衡,呈现上升趋势,cry1基因的相对表达量呈细微的上升趋势。结果提示:clock和cry1基因的CDS区在物种间保守性较高。clock和cry1基因在山羊大脑和垂体组织中起到重要的作用,且其mRNA发育性表达模式具有组织特异性。

摩腹联合电针透穴法对失眠大鼠生物钟相关基因及神经递质表达的影响

目的探讨摩腹联合电针透穴法对失眠大鼠生物钟基因Clock、BMAL1、PER1表达及神经递质乙酰胆碱(ACh)、谷氨酸(Glu)含量的影响及可能的作用机制。方法50只SD雄性大鼠随机分为正常组、模型组、摩腹组、电针透穴组和联合组,每组10只。除正常组外,采用腹腔注射对氯苯丙氨酸建立失眠大鼠模型。摩腹组进行腹部按摩,电针透穴组采用平刺法,百会透神庭、三阴交透阴陵泉(双侧)、神门透内关(双侧),电针仪疏密波,频率1Hz/20Hz,联合组摩腹后进行电针透穴治疗,各组均1次/d,连续7d,正常组和模型组不予干预。HE染色观察下丘脑组织神经元细胞形态变化,RT-qPCR检测下丘脑组织Clock、BMAL1、PER1 mRNA表达,免疫组化染色检测下丘脑组织Clock、BMAL1、PER1表达,Western blot检测下丘脑组织Clock、BMAL1、PER1蛋白表达,ELISA检测血清ACh、Glu含量。结果与正常组比较,模型组大鼠入睡潜伏期延长,睡眠持续时间缩短,下丘脑组织细胞结构破坏严重、呈空泡样变化,神经元细胞数量减少,下丘脑组织Clock、BMAL1mRNA和蛋白表达升高,PER1mRNA和蛋白表达降低,血清ACh、Glu含量增加,差异均有统计学意义(P<0.05);与模型组比较,摩腹组、电针透穴组和联合组均能缩短入睡潜伏期,延长睡眠持续时间,改善下丘脑神经元细胞形态,降低下丘脑组织Clock、BMAL1mRNA和蛋白表达,上调PER1mRNA和蛋白表达,减少血清ACh、Glu含量,差异均有统计学意义(P<0.05),以联合组作用最显著(P<0.05)。结论摩腹联合电针透穴法能改善大鼠失眠状况,其机制可能与调控生物钟基因Clock、BMAL1、PER1mRNA和蛋白表达及神经递质含量有关。