褪黑激素受体1A基因外显子2与小尾寒羊高繁殖力的关联

以褪黑激素受体1A(melatonin receptor 1A,MTNR1A)基因为候选基因,通过2种限制性内切酶(MnlⅠ、RsaⅠ),采用PCR-RFLP技术检测MTNR1A基因第2外显子主要序列在高繁殖力绵羊品种小尾寒羊中的单核苷酸多态性,同时研究该基因对小尾寒羊高繁殖力的影响。结果表明,MTNR1A基因外显子2的605位碱基处表现出MnlⅠ酶切多态性,在小尾寒羊中检测到3种基因型:MM(236 bp/236 bp)、Mm(236 bp/303 bp)、mm(303 bp/303 bp),MM和Mm基因型频率明显高于mm基因型频率。MTNR1A基因外显子2的604位碱基处表现出RsaⅠ酶切多态性,在小尾寒羊中检测到3种基因型:RR(267 bp/267 bp)、Rr(267 bp/290 bp)、rr(290 bp/290 bp),RR和Rr基因型频率明显高于rr基因型频率。复合基因型MMRR、MMRr、MmRR、MmRr的频率明显高于MMrr、mmRR、mmRr的频率,没有检测到mmrr复合基因型。小尾寒羊产羔数在MnlⅠ和RsaⅠ酶切复合基因型之间没有显著差异,但至少携带一个拷贝等位基因M的小尾寒羊比不携带M的具有稍微较高的产羔数,基因型为mm的小尾寒羊产羔数比其他基因型的小尾寒羊平均低0.26只。

蓝狐与银狐褪黑激素受体1A基因的克隆及序列分析

根据其它物种褪黑激素受体（melatonin receptor,MTNR）1A基因核酸序列的同源性设计引物,PCR扩增蓝狐和银狐MTNR1A基因的546 bp DNA序列,GenBank登录号分别为EU170442和EU170443。蓝狐和银狐MTNR1A基因DNA序列同源性为99.1%,编码的氨基酸同源性为99.4%,与人、鼠及绵羊MTNR1A基因同源性为84%-86%,与鸡MTNR1A基因同源性为74%。

蓝狐褪黑激素受体1A基因的克隆及序列分析

根据已知的蓝狐MTNR1A基因部分序列和狗的MTNR1A基因序列,借助Primer5.0引物设计软件,结合PCR引物设计优化条件,设计、合成引物。用基于PCR的96孔板筛选方法从蓝狐脾脏cDNA文库中分离获得阳性克隆,其序列与已知的蓝狐MTNR1A基因部分序列有100%的同源性,与已报道的狗、马、猕猴等动物的MTNR1A的cDNA分别有98%、88%、85%以上的同源性,推测该cDNA为MTNR1A的cDNA。蓝狐MTNR1AcDNA的获得,为进一步研究Mel配体-受体间的相互作用和生物功能提供了条件。

褪黑素受体1A基因多态性与水牛繁殖性能的关联分析

为了探讨褪黑素受体1A(MTNR1A)基因多态性对水牛繁殖性能的影响,试验以86头摩拉水牛经产牛为研究对象,采用PCR方法扩增其MTNR1A基因外显子Ⅱ序列,测序后分析该序列的多态性位点;记录86头摩拉水牛经产牛在2019年1月份—2020年12月份期间的怀孕和产犊情况,分析MTNR1A基因外显子Ⅱ的多态性与繁殖性能间的关系。结果表明:成功克隆到MTNR1A基因外显子Ⅱ,测序发现其第72位碱基处存在C>T单核苷酸多态性(SNP)位点,该位点有3种基因型,即CC、CT和TT基因型;C和T等位基因频率分别为0.47和0.53,CC、CT、TT基因型频率分别为0.37,0.30,0.33。CC和CT基因型摩拉水牛在2019年1月份和8—12月份时的怀孕数量均高于2019年2—7月份,TT基因型水牛在2019年2—7月份时的怀孕数量高于2019年1月份和8—12月份。摩拉水牛产犊季节主要集中在2020年1月份及9—12月份,经计算产犊数量占总产犊数的56.98%;2020年2—8月份是摩拉水牛产犊的淡季,经计算产犊数量仅占全年产犊总数的43.02%。CC和CT基因型的摩拉水牛产犊时间主要集中在2020年1月份和8—12月份,TT基因型水牛的产犊时间则变化不大。说明MTNR1A基因外显子Ⅱ第72位碱基处存在C>T SNP位点,可以作为一种基因标记,加速水牛良种选育进程。

褪黑素受体Mel_(1a)和 ROR_β的基因克隆和 cRNA探针的制备(英文)

为了研究褪黑素受体在人脑中的表达状况以及褪黑素在老年性痴呆中的作用 ,本研究克隆了 Mel1 a和 RORβ等两种褪黑素受体的 c DNA并将其转入 p Bluescript ( KS+)质粒载体中。重组质粒通过限制性内切酶鉴定分析并测序。结果表明 ,克隆的序列与 Gen Bank中的序列一致。利用线性化的质粒模板反转出 c RNA探针 。

褪黑素受体Mel 1a在皖西白鹅各组织中的表达与分布

【目的】探究褪黑素受体Mel 1a在皖西白鹅各组织中的分布特点和表达水平,为褪黑素在皖西白鹅的功能调控奠定理论基础。【方法】以皖西白鹅作为研究对象,提取各组织器官的总RNA,采用Real-time PCR和Western blot检测褪黑素受体Mel 1a mRNA和蛋白在鹅组织中的分布和差异性表达,免疫组化检测褪黑素受体Mel 1a蛋白在鹅的组织中的表达。【结果】在心脏、肝脏、脾脏、肺脏、肾脏、胰脏、大脑、小脑、肌肉、卵巢各组织中均存在Mel 1a mRNA表达;Mel 1a蛋白广泛分布于大脑、心脏、肾脏、肝脏、肺脏、脾脏、胰脏和肌肉组织细胞中,利用Image-pro plus 6软件分析免疫组化阳性信号的IOD值显示,脾脏阳性信号最强,其次是胰腺。脾脏显著强于大脑,肺脏中的信号显著强于心脏和肌肉组织。采用Real time PCR和Western blot技术检测各组织中Mel 1a mRNA和蛋白表达水平,结果表明,卵巢中表达水平最高,其次是胰腺、脾脏、肾脏、肝脏、心脏和脑组织中,肺脏和肌肉中的表达水平最低,另外在各级卵泡中Mel 1a mRNA的表达水平随卵泡发育逐渐增加,直到卵泡发育到直径超过3 cm后,其表达量下降。【结论】皖西白鹅各组织中均存在褪黑素受体,其中在卵巢中表达最高,褪黑素可通过受体介导调控皖西白鹅多种生物功能,尤其是对皖西白鹅卵泡发育的调控。

The Regulatory Role and Mechanism of Circadian Rhythm in Hemoglobin Co-cultured Neurovascular Unit

Objective Intracranial hemorrhage(ICH),the second most common subtype of stroke,exacerbates the disruption of the blood-brain barrier(BBB),leading to vasogenic edema,plasma protein extravasation,and infiltration of neurotoxic substances.The clearance capacity of the brain plays a crucial role in maintaining BBB homeostasis and facilitating patient recovery after hemorrhage.This study aimed to investigate the effect of circadian rhythms on BBB function,neuronal damage,and clearance capabilities.Methods The transwell model and hemoglobin were co-cultured to simulate the BBB environment after ICH.After intervention with different light groups,neuronal apoptosis was determined,glial phagocytosis was analyzed,the expression of endogenous clearing-related proteins aquaporin 4(AQP4)and low-density lipoprotein receptor-related protein 1(LRP1)was detected by western blotting and immunofluorescence dual standard method,and the expression of the tight junction protein occludin and melatonin receptor 1A(MTNR1A)was quantitatively analyzed.Results Circadian rhythms play a key role in maintaining the integrity of the BBB,reducing oxidative stress-induced neuronal damage,and improving microglial phagocytosis.Meanwhile,the expression of occludin and MTNR1A in neurovascular unit(NVU)co-cultured with hemoglobin improved the expression of AQP4 and LRP1,the key proteins in the NVU's endogenous brain clearance system.Conclusion Circadian rhythm(alternating black and white light)protects the NVU BBB function after ICH,promotes the expression of proteins related to the clearance of the hematoma,provides new evidence for the clinical treatment of patients recovering from ICH,and improves the circadian rhythm to promote brain metabolism and hematoma clearance.

多胎与单胎山羊发情期卵巢褪黑素受体基因的差异表达研究

为探明褪黑素受体(MTR1A)对山羊产羔数等繁殖性状的调控作用,并比较多胎高产的济宁青山羊与单胎的沂蒙黑山羊发情期卵巢组织中MTR1A的差异表达量。采用荧光定量差异显示PCR(DDRT-qPCR)技术,以持家基因GAPDH为分子内标,对比研究了褪黑素受体1A(MTR1A)基因在多胎的济宁青山羊和单胎的沂蒙黑山羊发情期卵巢组织中的差异表达量。研究发现,MTR1A在两种山羊发情期的卵巢组织中均有表达,但是,在青山羊发情期卵巢组织中的表达量显著低于在沂蒙黑山羊卵巢组织中的表达量(P<0.05)。说明,发情期褪黑素受体基因在卵巢组织中的高表达是限制山羊卵巢排卵和产羔数的重要因素之一。研究结果提示:卵巢组织是褪黑素作用的重要靶器官,褪黑素对于山羊卵巢组织的发育、成熟和排卵具有重要影响。研究结果对于阐明褪黑素受体调控山羊生殖机能的作用机理提供了基础研究数据。

多胎与单胎山羊发情期卵巢褪黑素受体基因的克隆测序分析

为揭示褪黑素影响山羊繁殖性状的遗传机制,本研究采用差异显示PCR(DDRT-PCR)和克隆测序技术,研究了褪黑素受体1a(MelR1a)基因在多胎的济宁青山羊和单胎的沂蒙黑山羊发情期卵巢组织中的表达情况,并对MelR1a基因进行了克隆测序分析。检测结果表明,MelR1a在两种山羊卵巢中均有表达,说明,褪黑素可以通过分布于卵巢中的褪黑素受体而对山羊的生殖发挥重要的调控作用。克隆测序分析结果表明,两种山羊发情期卵巢组织中褪黑素受体1a基因的碱基组成没有变异,说明山羊褪黑素受体基因序列具有高度的保守性。

Orofacial inflammatory pain affects the expression of MT1 and NADPH-d in rat caudal spinal trigeminal nucleus and trigeminal ganglion

Very little is known about the role of melatonin in the trigeminal system, including the function of melatonin receptor 1. In the present study, adult rats were injected with formaldehyde into the right vibrissae pad to establish a model of orofacial inflammatory pain. The distribution of melatonin re- ceptor 1 and nicotinamide adenine dinucleotide phosphate diaphorase in the caudal spinal trigeminal nucleus and trigeminal ganglion was determined with immunohistochemistry and histo- chemistry. The results show that there are significant differences in melatonin receptor 1 expression and nicotinamide adenine dinucleotide phosphate diaphorase expression in the trigeminal ganglia and caudal spinal nucleus during the early stage of orofacial inflammatory pain. Our findings sug- gest that when melatonin receptor 1 expression in the caudal spinal nucleus is significantly reduced, melatonin＇s regulatory effect on pain is attenuated.

Role of melatonin receptor 1B gene polymorphism and its effect on the regulation of glucose transport in gestational diabetes mellitus

Melatonin receptor 1B(MT2,encoded by the MTNR1B gene),a high-affinity receptor for melatonin,is associated with glucose homeostasis including glucose uptake and transport.The rs10830963 variant in the MTNR1B gene is linked to glucose metabolism disorders including gestational diabetes mellitus(GDM);however,the relationship between MT2-mediated melatonin signaling and a high birth weight of GDM infants from maternal glucose abnormality remains poorly understood.This article aims to investigate the relationship between rs10830963 variants and GDM development,as well as the effects of MT2 receptor on glucose uptake and transport in trophoblasts.TaqMan-MGB(minor groove binder)probe quantitative realtime polymerase chain reaction(qPCR)assays were used for rs10930963 genotyping.MT2 expression in the placenta of GDM and normal pregnant women was detected by immunofluorescence,western blot,and qPCR.The relationship between MT2 and glucose transporters(GLUTs)or peroxisome proliferator-activated receptorγ(PPARγ)was established by western blot,and glucose consumption of trophoblasts was measured by a glucose assay kit.The results showed that the genotype and allele frequencies of rs10830963 were significantly different between GDM and normal pregnant women(P<0.05).The fasting,1-h and 2-h plasma glucose levels of G-allele carriers were significantly higher than those of C-allele carriers(P<0.05).Besides,the protein and messenger RNA(mRNA)expression of MT2 in the placenta of GDM was significantly higher than that of normal pregnant women(P<0.05).Melatonin could stimulate glucose uptake and GLUT4 and PPARγprotein expression in trophoblasts,which could be attenuated by MT2 receptor knockdown.In conclusion,the rs10830963 variant was associated with an increased risk of GDM.The MT2 receptor is essential for melatonin to raise glucose uptake and transport,which may be mediated by PPARγ.

Melatonin Ameliorates Abnormal Sleep-Wake Behavior via Facilitating Lipid Metabolism in a Zebrafish Model of Parkinson’s Disease

Sleep-wake disorder is one of the most common nonmotor symptoms of Parkinson's disease(PD).Melatonin has the potential to improve sleep-wake disorder,but its mechanism of action is still unclear.Our data showed that melatonin only improved the motor and sleep-wake behavior of a zebrafish PD model when melatonin receptor 1 was present.Thus,we explored the underlying mechanisms by applying a rotenone model.After the PD zebrafish model was induced by 10 nmol/L rotenone,the motor and sleep-wake behavior were assessed.In situ hybridization and real-time quantitative PCR were used to detect the expression of melatonin receptors and lipid-metabolism-related genes.In the PD model,we found abnormal lipid metabolism,which was reversed by melatonin.This may be one of the main pathways for improving PD sleep-wake disorder.

褪黑素受体1A基因多态性与青少年特发性脊柱侧凸相关性研究

目的研究褪黑素受体1A基因(MTNR1A)多态性与青少年特发性脊柱侧凸(AIS)发生发展的关系。方法共纳入226例 AIS 患者及277例正常对照者。记录 AIS 患者组最大 Cobb 角。采用 PCR-RFLP 的方法对 MTNR1A 基因启动子区域多态性位点 rs2119882进行基因分型。结果AIS 患者组 MTNR1A 基因多态性位点 rs2119882的基因型及等位基因分布与正常对照组比较没有明显差异。在 AIS 组内,多态性位点 rs2119882不同基因型所对应的最大 Cobb 角没有明显差异。结论MTNR1A 基因 rs2119882位点多态性与 AIS 的发生发展都没有关系。

Role of the Volatile Components in the Anti-insomnia Effect of Jiao-Tai-Wan in PCPA-induced Insomnia Rats

Background:Jiao-Tai-Wan(JTW)is widely used for insomnia in traditional Chinese medicine.It has been found that berberine in JTW has a potential anti-insomnia effect,but the anti-insomnia effect of volatile oil,which is another major component in JTW,remains unclear.Objective:To comprehensively analyze the anti-insomnia effect of volatile oil through chemical analysis,phar-macological research and network pharmacology methods.Methods:Gas chromatography-mass spectrometry(GC-MS)analysis and network pharmacology platform were used to elucidate the material basis,effect and mechanism of JTW in the treatment of insomnia.A rat model of insomnia induced by p-Chlorophenylalanine(PCPA)was used to verify its anti-insomnia effect.The levels of Melatonin(MT)and Melatonin receptor type 1b(MTNR1B)in the brain and serum of rats were detected by ELISA,Western blot and Real-time PCR methods.Results:Eighteen volatile oil ingredients of JTW were identified by GC-MS,containing six new components which were found in JTW for the first time.Cinnamaldehyde,Behenic alcohol,Tetradecanal,and Gleenol can promote sleep by targeting MTNR1B according to the predicted results of network-pharmacology.Compared with the model group,JTW can increase the level of MTNR1B in rats’prefrontal cortex and brain stem,while reducing the level of MT in rats’brain stem.Conclusion:The volatile oil components,such as Cinnamaldehyde,Tetradecanal,and Gleenol,might also be playing a critical role in the anti-insomnia effect of JTW by target MTNR1B.