神经多肽对肾上腺功能调控的研究进展

许多神经多肽通过旁分泌在下丘脑－垂体－肾上腺（HPA）轴不同水平调控肾上腺功能，在下丘脑水平精氨酸血管加压素（AVP），血管活性肠多肽（VIP）胆囊收缩素（CCK）刺激促肾上腺皮质激素释放激素（CRH）释放，而神经降压素（NT）诱导CRH基因表达，垂体水平垂体腺苷酸环化酶激活多肽（PACAP），尿促皮素（UC），神经介素（NM）及CCK刺激促肾上腺皮质激素（ACTH）分泌，相反黑色素浓集激素（MCH）和甘丙肽（GAL）抑制ACTH释放，在肾上腺水平PACAP，NM，CCK，GAL及肾上腺髓质素（ADM可直接作用于肾上腺皮质，在肾上腺髓质MCH，VIP，PACAP，GAL刺激儿茶酚胺释放，此外，一些多肽，如PACAP，VIP，NT和AVP尚可通过髓质内CRH／ACTH系统促进皮质激素分泌，总之，它们对肾上腺功能的调节既有协同作用，亦有拮抗作用,使肾上腺与中枢神经与体液等系统形成了广泛的联系。     

基于芯片数据分析丝裂原激活蛋白激酶信号通路基因在不同证候H22肝癌小鼠肾上腺的表达差异

目的:探索丝裂原激活蛋白激酶(MAPK)信号通路基因在不同证候H22肝癌荷瘤小鼠肾上腺的表达特征.方法:采用小鼠标准化四诊及辨证方法,以及GeneChip Mouse Exon 1.0 ST Array等技术,检测H22肝癌小鼠早期邪毒壅盛证和气虚证、中期阳气虚证、中晚期气阴阳虚证共4个证候肾上腺基因表达的差异,分析不同证候小鼠肾上腺组织MAPK信号通路的表达特征.结果:与正常小鼠比较,①肝癌小鼠各证候中,ERK通路、p38/MAPK通路、ERK5通路基因在肾上腺中表达升高;②JNK通路基因在各证候肝癌小鼠肾上腺中表达下降;③其他级联激酶基因在MAPK信号通路中表达也下降.结论:H22肝癌小鼠肿瘤发生后,其肾上腺组织MAPK信号转导中与TGF-β相关的p38/MAPK通路被激活,而JNK通路被抑制;另外两条ERK与ERK5通路同样也被激活.提示不同证候肝癌小鼠的肾上腺组织主动参与了MAPK信号转导过程.     

神经多肽对肾上腺功能调控的研究进展

许多神经多肽通过旁分泌在下丘脑 垂体 肾上腺 (HPA)轴不同水平调控肾上腺功能。在下丘脑水平精氨酸血管加压素 (AVP)、血管活性肠多肽 (VIP)、胆囊收缩素 (CCK)刺激促肾上腺皮质激素释放激素 (CRH)释放 ,而神经降压素 (NT)诱导CRH基因表达。垂体水平垂体腺苷酸环化酶激活多肽 (PACAP)、尿促皮素 (UC)、神经介素 (NM)及CCK刺激促肾上腺皮质激素 (ACTH)分泌 ,相反黑色素浓集激素 (MCH)和甘丙肽 (GAL)抑制ACTH释放。在肾上腺水平PACAP、NM、CCK、GAL及肾上腺髓质素 (ADM)可直接作用于肾上腺皮质。在肾上腺髓质MCH、VIP、PACAP、GAL刺激儿茶酚胺释放。此外 ,一些多肽 ,如PACAP、VIP、NT和AVP尚可通过髓质内CRH/ACTH系统促进皮质激素分泌。总之 ,它们对肾上腺功能的调节既有协同作用 ,亦有拮抗作用 ,使肾上腺与中枢神经与体液等系统形成了广泛的联系。     

E1A激活基因阻遏子基因表达与血管外膜成纤维细胞表型转化的关系

目的探讨E1A激活基因阻遏子（CREG）基因表达与血管外膜成纤维细胞（AFs）表型转化间的关系。方法制作小鼠颈动脉损伤模型,免疫荧光染色观察血管外膜中CREG与平滑肌肌动蛋白（α-SMA）表达变化;培养小鼠原代AFs,外源性应用血管紧张素Ⅱ（AngⅡ）诱导其表型转化,RT-PCR和Western-blot检测不同作用时间及不同浓度AngⅡ刺激AFs后CREG和α-SMA mRNA和蛋白质表达。结果血管损伤后1 d、3 d血管外膜增生显著,外膜中α-SMA表达显著上调,而CREG表达显著下调;血管损伤后7 d、14 d外膜增生有所缓解,α-SMA表达水平逐渐下降,而CREG表达也有所恢复。应用AngⅡ诱导AFs发生表型转化,AFs中α-SMA mRNA和蛋白水平显著上调,而CREG mRNA和蛋白水平显著下调,并且呈时间和剂量依赖性。结论在体和离体模型均表明AFs表型转化过程中伴随CREG基因表达下调,提示CREG基因可能参与AFs的表型转化。     

不同证候H22荷瘤小鼠垂体高表达基因分析

目的:筛选不同证候荷瘤小鼠垂体高表达基因.方法:采用荷瘤小鼠及其标准化四诊及辨证方法,以及GeneChip Mouse Exon 1.0 ST Array等技术,检测正常小鼠及不同证候H22荷瘤小鼠垂体的基因表达,筛选各证候小鼠垂体芯片读数均≥6000的基因.结果:各证候荷瘤小鼠芯片读数均≥6000的基因共28个,其中实证下调、虚证均上调的基因5个:Dapk3、Spint2、Gnas、LOC677374、Pdia6;早期下调,中期、中晚期上调的基因12个:Pomc1、Scg5、Cga、Prl、Gh、Scg2、LOC672819、Rps5、Pdia3、Gnb2l1、Mdh1、Itm2b;各证候一致上调的基因9个:Ctsb、Bsg、Hsp90b1、Hspa5、Sec11l1、Serinc1、Psap、LOC671023、LOC546840;不符合以上3种规律的两个:Slc25a4、Laptm4a.结论:肿瘤发生后,不同证候H22荷瘤小鼠垂体存在大量的高表达基因,与垂体的功能密切相关,其共同的特点为早期特别是邪毒证小鼠垂体的功能稍有抑制,至中期和中晚期垂体的功能非常活跃.     

肥胖大鼠前增食欲素原及增食欲素受体基因表达与下丘脑-垂体-肾上腺轴有关

饮食性肥胖大鼠下丘脑前增食欲素原基因表达降低；肾上腺增食欲素受体2mRNA表达上调并且与下丘脑促肾上腺皮质激素释放激素基因表达水平、血皮质酮水平间存在相关性。     

不同程度邪毒证H22荷瘤小鼠肾上腺皮质酮合成酶及其转录因子表达的差异

目的：探讨小鼠荷瘤状态下不同程度邪毒证肾上腺皮质酮合成与分泌的差异。方法：在220只昆明种小鼠右腋下接种H22肝癌腹水细胞,复制肝癌荷瘤小鼠模型,于接种肿瘤后第9天进行小鼠四诊检测,依据邪毒程度将各荷瘤小鼠排序,并排除气血阴阳虚证小鼠,筛选邪毒壅盛证组18只、邪毒微弱证组18只,并以18只正常小鼠为对照组。第11天处死各组小鼠,剥离肿瘤称瘤重,用ELISA方法检测血浆激素含量;应用实时荧光定量PCR检测各组小鼠肾上腺组织Cyp11b1、Cyp11a1、Star、Fdx1、Fdxr、Mc2r、Nr4a1、Nr5a1、Nr0b1、Gata6基因mRNA表达,并以Gapdh和β-actin两个基因作内参照;应用Western blot方法检测St AR和CYP11A1蛋白表达。结果：邪毒壅盛证小鼠肿瘤质量明显大于邪毒微弱证组（P〈0.05）;不同程度邪毒证肝癌小鼠血浆促肾上腺皮质激素（ACTH）和皮质酮（CORT）均高于正常组（P〈0.05）;皮质酮合成最后一步关键酶Cyp11b1、促肾上腺皮质激素受体（Mc2r）及铁氧还蛋白还原酶（Fdxr）基因表达在邪毒壅盛证组显著升高（P〈0.05）;转录抑制因子Nr0b1在邪毒壅盛证组明显低于邪毒微弱证组（P〈0.05）;转录因子Nr4a1、Nr5a1和Gata6基因表达在不同程度邪毒证肝癌小鼠均高于正常组（P〈0.05）;St AR蛋白表达在不同程度邪毒证肝癌小鼠均显著升高（P〈0.05）、且邪毒壅盛证组显著高于邪毒微弱证组（P〈0.05）。结论：小鼠肿瘤形成后,邪毒程度越重者,为适应机体慢性应激反应,其肾上腺皮质功能呈现失代偿性增加,导致垂体与肾上腺皮质激素储备功能减少,可能与邪毒壅盛证荷瘤小鼠带瘤生存期缩短有关,提示肿瘤早期治疗也需注意扶助正气。     

内皮素1对人血管内皮细胞基因表达谱的影响

目的探讨内皮素1对人血管内皮细胞基因表达谱的影响，进一步阐明内皮素1导致内皮细胞功能异常的分子机制。方法培养人血管内皮细胞，提取总RNA，采用人内皮细胞生物学功能基因芯片（含96个基因），检测内皮素1作用12h后人血管内皮细胞主要功能基因表达谱的变化。结果与未用内皮素处理的对照组相比，人血管内皮细胞在内皮素1作用12h后，共有53个基因出现差异表达，其中抗凝血和抗氧化基因等22个基因表达下调，促凝和炎性因子基因等31个基因表达上调。结论内皮素通过诱导人血管内皮细胞凝血和纤溶系统基因表达失衡、下调抗氧化酶基因表达和上调促炎因子基因表达，损伤人血管内皮细胞，造成其生物学功能异常。     

降钙素基因相关肽抑制血管平滑肌细胞能量代谢的AMP激活蛋白激酶信号通路

目的观察降钙素基因相关肽对血管紧张素Ⅱ诱导血管平滑肌细胞增殖过程中能量代谢的影响及机制,探索细胞外信号调节激酶和AMP激活蛋白激酶相关信号蛋白在该作用中的地位。方法组织贴块法体外培养SD大鼠胸主动脉血管平滑肌细胞,取第3～10代用于实验。分别用降钙素基因相关肽或/和血管紧张素Ⅱ处理细胞。MTT法及细胞计数法观察降钙素基因相关肽对血管紧张素Ⅱ诱导大鼠血管平滑肌细胞增殖的影响;詹纳斯绿B染色观察细胞线粒体形态及体积;ATP检测试剂盒检测细胞内ATP水平;Western Blotting检测细胞磷酸化AMP激活蛋白激酶的表达。结果降钙素基因相关肽预处理能降低血管紧张素Ⅱ诱导的大鼠血管平滑肌细胞的增殖(P<0.05),同时拮抗血管紧张素Ⅱ引起细胞内线粒体肿胀、ATP水平升高,在此过程中伴随细胞内磷酸化AMP激活蛋白激酶表达下调(P<0.05);降钙素基因相关肽受体拮抗剂CGRP8-37能拮抗降钙素基因相关肽对血管紧张素Ⅱ促增殖和促代谢的抑制作用(P<0.05);细胞外信号调节激酶抑制剂PD98059能部分拮抗降钙素基因相关肽对磷酸化AMP激活蛋白激酶的抑制作用(P<0.05)。结论降钙素基因相关肽能显著抑制血管紧张素Ⅱ诱导大鼠血管平滑肌细胞增殖过程中的能量代谢,其细胞内信号通路可能涉及到降钙素基因相关肽/降钙素基因相关肽1型受体-丝裂原细胞外激酶/细胞外信号调节激酶-磷酸化AMP激活蛋白激酶信息传递链。     

老年大鼠下丘脑-垂体-肾上腺轴炎性衰老相关基因表达特征

目的探讨炎性衰老的炎性细胞因子网络机制。方法清洁级健康雄性SD大鼠分为4月龄（4m）和24月龄（24m）二组,每组各10只。分别取各组大鼠下丘脑、垂体和肾上腺组织,应用炎症细胞因子与受体基因芯片进行基因表达检测,绘制基冈表达谱,筛选有表达差异的基因。结果与4m组相比,24m组下丘脑、垂体和肾上腺组织中表达上调的促炎性反应细胞因子与受体基因分别有6个、7个和9个,表达下调的抗炎细胞因子与受体基因分别有3个、4个和3个（P〈0．05）。三种组织中表达都上调的促炎性反应细胞因子与受体基因有白介素、肿瘤坏死因子和趋化因子。结论老年大鼠下丘脑－垂体－肾上腺轴组织存在促炎性细胞因子基因表达上调,导致促－抗炎性细胞因子网络和促－抗炎性反应体系平衡失调,这可能是炎性衰老中出现高促炎性反应状态的原因和炎性衰老发生的新机制。     

Pituitary adenylate cyclase-activating polypeptide is a sympathoadrenal neurotransmitter involved in catecholamine regulation and glucohomeostasis.

The adrenal gland is important for homeostatic responses to metabolic stress: hypoglycemia stimulates the splanchnic nerve, epinephrine is released from adrenomedullary chromaffin cells, and compensatory glucogenesis ensues. Acetylcholine is the primary neurotransmitter mediating catecholamine secretion from the adrenal medulla. Accumulating evidence suggests that a secretin-related neuropeptide also may function as a transmitter at the adrenomedullary synapse. Costaining with highly specific antibodies against the secretin-related neuropeptide pituitary adenylate cyclase-activating peptide (PACAP) and the vesicular acetylcholine transporter (VAChT) revealed that PACAP is found in nerve terminals at all mouse adrenomedullary cholinergic synapses. Mice with a targeted deletion of the PACAP gene had otherwise normal cholinergic innervation and morphology of the adrenal medulla, normal adrenal catecholamine and blood glucose levels, and an intact initial catecholamine secretory response to insulin-induced hypoglycemia. However, insulin-induced hypoglycemia was more profound and longer-lasting in PACAP knock-outs, and was associated with a dose-related lethality absent in wild-type mice. Failure of PACAP-deficient mice to adequately counterregulate plasma glucose levels could be accounted for by impaired long-term secretion of epinephrine, secondary to a lack of induction of tyrosine hydroxylase, normally occurring after insulin hypoglycemia in wild-type mice, and a consequent depletion of adrenomedullary epinephrine stores. Thus, PACAP is needed to couple epinephrine biosynthesis to secretion during metabolic stress. PACAP appears to function as an "emergency response" cotransmitter in the sympathoadrenal axis, where the primary secretory response is controlled by a classical neurotransmitter but sustained under paraphysiological conditions by a neuropeptide.     

Mice, melatonin and the circadian system

Melatonin effects are discussed by reviewing results from mice with intact or disrupted melatonin signaling. Melatonin, the neuroendocrine hand of the clock produced in the pineal gland during night, acts upon two receptor subtypes. Melatonin receptors are found in the suprachiasmatic nuclei (SCN), hypophysial pars tuberalis (PT) and adrenal gland. In SCN, melatonin interacts with PACAP, a neuropeptide of the retinohypothalamic tract. Moreover, melatonin acts on the SCN to modulate the activity of the sympathetic nervous system. Melatonin is not required to maintain rhythmic clock gene expression in SCN. By contrast, the rhythmic clock gene expression in PT depends on a melatonin signal interacting with adenosine. Melatonin may also affect clock gene protein levels in the adrenal cortex and influence adrenal functions. In conclusion, melatonin may serve the synchronization of peripheral oscillators by interacting with other neuroactive substances. A stress-reducing potency of melatonin needs to be explored in further studies.     

稳定期冠心病患者中医证候演变规律的研究

目的探讨稳定期冠心病患者发生急性心血管事件的中医证候演变规律及其与随访发生急性心血管事件的关系。方法纳入稳定期冠心病患者303例,详细记录入选时、半年、1年的中医证候情况及2年随访时事件发生情况。应用频数分布法,观察两组患者整体的证候分布。应用多因子降维(MDR)数据挖掘方法探讨中医证候的演变规律。结果频数法显示3个时间点(两个时间段)的演变趋势:两组的气滞、寒痰(第2时段)均呈现降低趋势,痰热证在事件组呈现逐渐增加。阴虚、气虚的变化趋势基本相同,而阳虚的变化两组则呈现相反的方向。应用MDR来研究证候之间的转化,结果提示,第2个时点的阳虚转化为第3个时点的痰热、第2个时点的气虚转化为第3个时点的痰热与发生心血管事件呈正相关。结论证候演变中以气虚—痰热、阳虚—痰热的转化易引发心血管事件。     

慢性心力衰竭不同原发病与中医证型特点分析

目的分析不同原发病所致慢性心力衰竭(CHF)的中医证型特点和规律,为CHF病机探索和认识提供参考。方法对117例CHF患者进行回顾性分析,根据不同原发病和心功能分级情况进行证型归纳和统计分析,分析中医证型与原发病、心功能分级的相关性。结果原发病中冠心病占31.6%,高血压占30.8%,肺心病占13.7%,风心病占11.1%,心肌病占6.8%,老年性心脏瓣膜病占4.3%;冠心病组中气虚血瘀证、心脾肾阳虚水泛兼血瘀证最多,高血压病组气虚血瘀证比例最大,风心病心脾肾阳虚水泛兼血瘀证最多,肺心病组心肺气虚证为多,心肌病以心脾阳虚兼血瘀水停证为多,老年瓣膜病以气虚血瘀证为多;心功能Ⅱ级中以心肺气虚证为主,心功能Ⅲ级中以气虚血瘀和心脾阳虚兼血瘀水停证居多,心功能IV级中以心脾肾阳虚水泛兼血瘀证居多,心功能等级以心肺气虚、心气阴虚兼血瘀,气虚血瘀,心脾阳虚兼血瘀水停,心脾肾阳虚水泛兼血瘀逐级递增(r=0.508,P<0.001);血浆脑钠肽(BNP水平)按心肺气虚、心气阴虚兼血瘀,气虚血瘀,心脾阳虚兼血瘀水停,心脾肾阳虚水泛兼血瘀逐级递增(r=0.773,P<0.001);结论引起CHF最常见的原发病是冠心病,其中气虚血瘀证和心脾肾阳虚水泛兼血瘀证常见,说明气虚、阳虚、血瘀是CHF的最突出的病理改变。     

Targeted pituitary overexpression of pituitary adenylate-cyclase activating polypeptide alters postnatal sexual maturation in male mice

The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) is present in high concentrations within the hypothalamus, suggesting that it may be a hypophysiotropic factor, whereas pituitary expression suggests a paracrine function. PACAP stimulates gonadotropin secretion and enhances GnRH responsiveness. PACAP increases gonadotropin α-subunit (αGSU), lengthens LHβ, but reduces FSHβ mRNA levels in adult pituitary cell cultures in part by increasing follistatin. PACAP stimulates LH secretion in rats; however, acceptance of PACAP as a regulator of reproduction has been limited by a paucity of in vivo studies. We created a transgenic mouse model of pituitary PACAP overexpression using the αGSU subunit promoter. Real-time PCR was used to evaluate PACAP, follistatin, GnRH receptor, and the gonadotropin subunit mRNA in male transgenic and wild-type mice of various ages. Transgenic mice had greater than 1000-fold higher levels of pituitary PACAP mRNA; and immunocytochemistry, Western blot, and ELISA analyses confirmed high peptide levels. FSH, LH, and testosterone levels were significantly suppressed, and the timing of puberty was substantially delayed in PACAP transgenic mice in which gonadotropin subunit and GnRH receptor mRNA levels were reduced and pituitary follistatin expression was increased. Microarray analyses revealed 1229 of 45102 probes were significantly (P < 0.01) different in pituitaries from PACAP transgenic mice, of which 83 genes were at least 2-fold different. Genes involved in small molecule biochemistry, cancer, and reproductive system diseases were the top associated networks. The GnRH signaling pathway was the top canonical pathway affected by pituitary PACAP excess. These experiments provide the first evidence that PACAP affects gonadotropin expression and sexual maturation in vivo.     

Immunocytochemical demonstration of day/night changes of clock gene protein levels in the murine adrenal gland: differences between melatonin-proficient (C3H) and melatonin-deficient (C57BL) mice

The circadian system comprises several peripheral oscillators and a central rhythm generator that, in mammals, is located in the suprachiasmatic nucleus of the hypothalamus. Expression of clock genes is a characteristic feature of the central rhythm generator and the peripheral oscillators. With regard to the rhythmic production of glucocorticoids, the adrenal gland can be considered as peripheral oscillator, but little is known about clock gene expression in this tissue. Therefore, the present study investigates the levels of three clock gene proteins PER1, BMAL1 and CRY2 in the murine adrenal cortex and medulla at seven different time points of a 12-hr light/12-hr dark cycle. To determine a potential role of melatonin we compared the patterns of clock gene proteins in the adrenal gland of melatonin-proficient mice (C3H) with those of melatonin-deficient mice (C57BL). In C3H mice, both, the adrenal cortex and medulla displayed day/night variation in PER1-, CRY2- and BMAL1-protein levels. PER1 and CRY2 peaked in the middle of the light phase, whereas BMAL1 peaked in the dark phase. This pattern was also observed in the adrenal medulla of C57BL, but in the adrenal cortex of C57BL clock gene protein levels did not change with time and were consistently lower than in C3H mice. These results support the hypothesis that the adrenal gland is a peripheral oscillator and raise the possibility that melatonin may be involved in the control of clock gene protein levels in the adrenal cortex of mice.