系统性红斑狼疮患者循环微小RNAs差异表达的初步分析

目的 检测系统性红斑狼疮(SLE)患者血浆中差异表达的微小RNAs(miRNAs),为寻求一种新的无创性SLE生物标志物奠定基础.方法 采用Agilent human miRNA芯片检测并筛选出SLE患者与健康人血浆中表达丰度有显著变化miRNA,并通过实时荧光定量聚合酶链反应(RT-qPCR)对部分差异表达基因进行验证.2组间的比较用独立样本的t检验.结果 MiRNAs芯片检测发现,SLE患者与健康对照间存在明显差异的循环miRNAs共有51个,其中19个上调,32个下调;RT-qPCR对其中4个上调(miR- 126、miR-21、miR-223和miR-451)和3个下调(miR-125a-3p、miR-146a和miR-155)循环miRNAs的验证结果与芯片数据所示具有较好的一致性.结论 SLE患者和健康人体内循环miRNAs的表达谱存在着明显差异,这些差异的循环miRNAs可作为一种潜在的SLE候选生物标志物.     

胃癌外周血微小核糖核酸表达谱的初步分析

目的 研究胃癌患者外周血微小核糖核酸(miRNA)的表达,初步建立胃癌特征性的循环miRNA表达谱,为深入研究miRNA与胃癌发生、发展并寻找新的分子标志物提供依据.方法 选取6例胃癌患者和6例健康体检者,提取外周血总RNA,进行miRNA表达谱检测和生物信息学分析,采用实时定量PCR技术对芯片检测结果进行验证,对筛选出的显著差异表达miRNA的靶基因进行预测.结果 胃癌组与对照组对比共有54个差异表达miRNA,其中表达上调的miRNA为35个(miRNA-504、miRNA-183、miRNA-938、miRNA-1285、miRNA-576-3p、miRNA-663 等),表达下调的miRNA为19个(miRNA-433、miRNA-193b、miRNA-329、miRNA-409-3p、miRNA-154等).实时定量PCR对其中2个上调miRNA(miRNA-504和miRNA-183)和2个下调miRNA(miRNA-433和miRNA-193b)的验证结果与芯片检测结果间具有较好的一致性.结论 胃癌患者外周血中具有特异性的miRNA表达谱,这些差异表达的miRNA有可能成为新的胃癌诊断分子标志物.     

血浆miR-1228-5p、miR-34a-5p、miR-192-5p和miR-30a-3p水平与早发冠心病的相关性及其初筛价值

目的]探讨血浆miR-1228-5p、miR-34a-5p、miR-192-5p和miR-30a-3p水平与早发冠心病(PCAD)的相关性及其对PCAD的初筛价值。[方法]根据纳入标准及排除标准,纳入6例明确诊断的PCAD患者作为PCAD组,纳入6例健康受试者作为对照组,收集PCAD组和对照组血液,提取血清样本并保存,使用DNBseq平台检测两组血清中miRNA水平,筛选差异水平显著的miRNA。根据纳入标准及排除标准,收集78例PCAD患者、75例晚发冠心病患者和69例健康对照者的血液并对筛选的miRNA进行实时荧光定量PCR验证。分析PCAD患者冠状动脉造影报告,采用Gensini评分评估冠状动脉病变的严重程度。Spearman相关性检验分析有关miRNA水平与冠状动脉狭窄程度的相关性。ROC曲线分析血浆miR-1228-5p、miR-34a-5p、miR-192-5p及miR-30a-3p水平对PCAD的诊断价值,多因素Logistic回归分析PCAD发生的影响因素。[结果]DNBseq平台分析显示,差异表达miRNA 33个,其中上调miRNA 17个,下调miRNA 16个,差异水平最为显著的5个miRNA分别为miR-1228-5p、miR-34a-5p、miR-192-5p、miR-424-3p和miR-30a-3p;实时荧光定量PCR结果显示,与对照组相比,PCAD患者血浆miR-1228-5p升高1.7倍,miR-34a-5p升高1.4倍,miR-192-5p升高0.7倍,miR-30a-3p升高2.5倍(P＜0.05),两组间血浆miR-424-3p水平差异无统计学意义(P＞0.05);血浆miR-1228-5p和miR-34a-5p水平与PCAD患者冠状动脉狭窄程度均呈正相关(r＝0.307,P＝0.004;r＝0.238,P＝0.036);ROC曲线分析显示,miR-1228-5p、miR-34a-5p、miR-192-5p和miR-30a-3p诊断PCAD的ROC曲线下面积分别为0.903、0.832、0.731及0.798,其联合诊断PCAD的ROC曲线下面积为0.990,95%CI为0.976～1.000。[结论]PCAD患者血浆miR-1228-5p、miR-34a-5p、miR-192-5p和miR-30a-3p水平显著升高,其联合检测诊断PCAD具有较高的准确性,有望成为初筛PCAD的新型生物标志物。     

人血浆及粪便中微小RNA-92a-1表达水平在结直肠肿瘤筛查中的意义

目的 探讨联合检测人血浆及粪便中微小RNA-92a-1(miRNA-92a-1)表达水平作为结直肠肿瘤筛查标志物的可行性及临床意义.方法 收集2011年8月至10月间60例结直肠癌患者、23例结直肠腺瘤患者及30名健康对照者的粪便及血浆标本,采用实时定量RT-PCR的方法检测miRNA-92a-1的表达水平.组间采用Mann-Whitney U检验进行差异性检验,然后根据受试者工作特征曲线(ROC曲线)确定截断点,对实验结果的敏感度及特异度进行分析.结果 结直肠癌和结直肠腺瘤患者血浆中miRNA-92a-1的表达水平均高于健康对照者(U=288.5和151.0,P均＜0.01).结直肠癌患者粪便中miRNA-92a-1的表达水平高于健康对照者(U=627.5,P=0.0199).根据ROC曲线分析,当截断点值＞1.22时,miRNA-92a-1在结直肠癌和结直肠腺瘤患者血浆中的敏感度分别为85.0％(51/60)和73.9％(17/23),在健康对照者血浆中的特异度为76.7％(23/30);当截断点值＞1.14时,miRNA-92a-1在结直肠癌和结直肠腺瘤患者粪便中的敏感度分别为31.7％(19/60)和26.1％(6/23),在健康对照者粪便中的特异度为90.0％(27/30).联合血浆及粪便中的检测结果,miRNA-92a-1在结直肠癌和结直肠腺瘤患者中的敏感度分别为88.3％(53/60)和82.6％(19/23),在健康对照者中的特异度为73.3％(22/30),敏感度高于单样本检测.结论 联合检测结直肠癌及结直肠腺瘤患者血浆及粪便中miRNA-92a-1的表达水平,具有较高的敏感度和特异度.miRNA-92a-1可作为结直肠癌诊断及筛查的一个潜在指标.     

T细胞来源的细胞外囊泡通过miR-193a诱导足细胞损伤

目的:探讨T细胞来源的循环细胞外囊泡(extracellular vesicles,EVs)中miR-193a在局灶节段性肾小球硬化(FSGS)患者足细胞损伤中的作用。方法:收集经肾活检确诊的FSGS患者和健康志愿者外周血,分离EVs。芯片和RT-PCR筛选出患者EVs中升高的microRNA(miRNA),再经体外足细胞试验,选出能导致足细胞骨架改变的miRNA。用目标miRNA转染T细胞系(Jurkat细胞),获得富集目标miRNA的EVs。给予Balb/c小鼠尾静脉注射Jurkat细胞EVs,测定尿蛋白/肌酐;用Jurkat细胞来源的EVs与足细胞共孵育,检测细胞骨架改变。验证miR-193a损伤足细胞的分子机制。结果:FSGS患者外周血EVs中miR-193a水平明显上调,并能导致足细胞骨架损伤。FSGS患者循环EVs可导致小鼠蛋白尿和足细胞骨架结构紊乱。富含miR-193a的Jurkat细胞EVs通过下调WT1,导致足细胞骨架结构损伤、诱导小鼠产生蛋白尿和足细胞足突融合。结论:本研究发现FSGS患者循环中T细胞来源的EVs通过miR-193a可诱导足细胞损伤。     

小动脉闭塞性卒中患者血浆微小RNA表达谱

目的 探讨小动脉闭塞性卒中(small artery occlusion,SAO)患者与同期健康体检者血浆微小RNA(microRNAs,miRNAs)表达谱差异.方法 选取TOAST分型为小动脉闭塞性卒中患者8例,以8例同期健康体检者作为对照组,应用高通量测序技术检测血浆miRNAs表达谱,筛选出差异表达的miRNAs,采用实时荧光定量聚合酶链反应验证结果,并进行靶基因预测和生物信息学分析.结果 miRNAs差异化分析显示,SAO组miRNA-127、miRNA-99b-5p和miRNA-320等19个miRNAs 较对照组表达显著性上调(P均＜0.01),而miRNA-451a等5个miRNAs较对照组表达显著性下调(P均＜0.01).实时荧光定量聚合酶链反应对其中miRNA-127、miRNA-99b-5p、miRNA-320和miRNA-451a的验证结果与高通量测序结果一致.生物信息学分析显示,差异化表达的miRNAs调控的靶基因主要与细胞增殖、黏附、系统发育、高分子代谢等生物学功能相关.结论 SAO患者与健康体检者血浆miRNAs表达谱存在显著性差异,提示miRNAs可能通过靶基因在SAO的发病过程中发挥调节作用.     

怀有先天性心脏病胎儿孕妇血浆microRNA的表达差异

目的检测血浆miRNA在怀有先天性心脏病(先心病)胎儿的孕妇和怀有正常心脏胎儿的孕妇之间的表达差异,筛选出表达显著差异的miRNAs,为产前筛查先心病提供生物标记物和为先心病的发生机制研究提供分子生物学依据。方法采集60例怀有先心病胎儿的孕妇及60例对照组的血样标本,用Solexa测序方法筛选出具有显著差异性的血浆miRNA。结果 Solexa测序结果显示,在病例组及对照组中分别检测出545个及580个miRNA的表达,以拷贝数≥30,病例组与对照组相比miRNA表达差异倍数≥9为标准,筛选出7个表达上调的miRNA:hsa-miR-137、hsa-miR-206、hsa-miR-224-3p、hsa-miR-34a-3p、hsa-miR-485-3p、hsa-miR-5094、hsa-miR-653-3p;4个表达下调的miRNA:hsa-miR-100-3p、hsa-miR-188-5p、hsa-miR-190a-3p、hsa-miR-216a-5p。结论怀有先心病胎儿的孕妇血浆中存在miRNA表达差异。     

抗结核药物肝损伤与无肝损伤患者化疗前血循环miRNA分子的差异性表达

目的:研究抗结核药物肝毒性(anti-tuberculosis drug-induced hepatotoxicity,ATDH)与非ATDH患者化疗前血浆差异性表达的miRNA分子,为ATDH易感者筛检提供新的生物学指标.方法:采用miRNA芯片检测ATDH及对照患者化疗前血浆标本.对存在差异表达趋势的25种miRNA分子,采用高通量实时荧光定量PCR进行分析验证.应用互联网上miRNA靶基因预测软件对表达上调的miRNA进行靶基因预测,并采用PANTHER tool查找靶蛋白GO功能分类.结果:与对照组相比,ATDH患者化疗前血浆中共检出7个差异性表达的miRNA分子,其中表达上调的miRNA有4个,包括miR-4284、miR-3620、miR-652-5p和miR-4800-5p;表达下调的miRNA有3个,包括miR-338-3p、miR-424-5p和miR-194-5p.结论:ATDH患者化疗前血浆内存在差异性表达的miRNA分子,其中表达上调的miRNA分子(特别是miR-4284)可能作为ATDH易感者筛检的生物学指标.     

血浆Ki67联合miRNA-26b检测诊断肝细胞癌价值分析

目的 探讨肝细胞癌(HCC)患者血浆Ki67和miRNA-26b水平变化,分析其诊断效能。方法 我院收治的HCC患者90例和乙型肝炎肝硬化患者60例,采用RT-qPCR检测血浆Ki67和miRNA-26b水平,采用ROC曲线分析血浆Ki67和miRNA-26b水平诊断HCC的效能。结果 HCC患者血浆Ki67水平为【1.6(1.1～2.2)】,显著高于肝硬化组【0.4(0.1～1.2),P＜0.05】;HCC患者血浆miRNA-26b水平为【0.7(0.3～1.4)】,显著低于肝硬化组【2.0(1.4～3.0),P＜0.05】;不同肿瘤Edmonson分级、早期是否复发和肿瘤转移患者血浆Ki67和miRNA-26b水平分布差异具有统计学意义(P＜0.05);血浆Ki67诊断HCC的ROC曲线下面积为0.8(95%置信区间为0.7～0.9),血浆miRNA-26b诊断HCC的ROC曲线下面积为0.8(95%置信区间为0.8～0.9)。ROC曲线结果显示,血浆Ki67和miRNA-26b水平诊断HCC的灵敏度分别为55.6%和70.0%,特异度分别为95.0%和83.3%,血浆Ki67和miRNA-26b串联试验诊断的特异度为96.7%,并联试验的灵敏性为87.8%,两指标联合诊断HCC的准确性为85.3%,显著高于单个指标的71.3%和75.3%。结论 联合检测血浆Ki67和miRNA-26b水平可帮助诊断HCC,值得临床进一步探讨。     

系统性红斑狼疮患者外周血单个核细胞miRNA-199a-3p表达及调控机制

目的 探究系统性红斑狼疮(systemic lupus erythematosus,SLE)患者外周血单个核细胞miRNA-199a-3p的表达及对PI3K/Akt/mTOR信号通路调控机制。方法 选取本院就诊的SLE患者50例,包括缓解期患者20例(缓解期组)、活动期患者30例(活动期组),同时选择25例健康志愿者作为对照组。采用RT-PCR检测入选者外周血单个核细胞miRNA-199a-3p表达情况;应用Pearson相关性分析方法了解miRNA-199a-3p表达水平与SLE疾病活动程度,并进一步分析miRNA-199a-3p表达水平与脏器受累的相关性;利用流式细胞仪检测miRNA-199a-3p对细胞凋亡的影响。通过生物信息软件和荧光素酶报告实验验证miRNA-199a-3p的靶基因。上调miRNA-199a-3p后采用Western Blot检测PI3K/Akt/mTOR信号通路相关蛋白表达情况。结果 缓解期SLE患者外周血单个核细胞miRNA-199a-3p相对表达水平(1. 25±0. 19)显著低于健康志愿者(2. 38±0. 24),但显著高于活动期患者(0. 34±0. 07)(P 0. 05)。Pearson相关性分析表明miRNA-199a-3p相对表达水平与系统性红斑狼疮疾病活动度指数(systemic lupus erythematosus disease activity index,SLEDAI)评分呈负相关性(R=-0. 704,P 0. 05),浆膜炎、血尿、血管炎、脱发和光过敏患者miRNA-199a-3p水平与无以上临床表现患者相近(P 0. 05);皮疹、白细胞降低和肾炎患者miRNA-199a-3p水平显著高于无以上临床表现患者(P 0. 05)。凋亡检测发现上调miRNA-199a-3p能够显著提高单个核细胞凋亡率(P 0. 05)。生物信息学及荧光素酶报告实验表明miRNA-199a-3p的靶基因为mTOR mRNA 3'UTR。WB检测表明外周血单个核细胞转染miRNA-199a-3p mimics后,能够显著下调mTOR、PI3K、p AKt和Bcl-2表达水平(P 0. 05),同时能够显著上调Bax与caspase-3表达水平(P 0. 05)。结论 SLE患者外周血单个核细胞miRNA-199a-3p低表达,且其能够影响PI3K/Akt/mTOR信号通路分子的表达。     

Three layer functional model and energy exchange concept of aging process

Relying on a certain degree of abstraction, we can propose that no particular distinction exists between animate or living matter and inanimate matter. While focusing attention on some specifics, the dividing line between the two can be drawn. The most apparent distinction is in the level of structural and functional organization with the dissimilar streams of ‘energy flow’ between the observed entity and the surrounding environment. In essence, living matter is created from inanimate matter which is organized to contain internal intense energy processes and maintain lower intensity energy exchange processes with the environment. Taking internal and external energy processes into account, we contend in this paper that living matter can be referred to as matter of dissipative structure, with this structure assumed to be a common quality of all living creatures and living matter in general. Interruption of internal energy conversion processes and terminating the controlled energy exchange with the environment leads to degeneration of dissipative structure and reduction of the same to inanimate matter, (gas, liquid and/or solid inanimate substances), and ultimately what can be called ‘death.’ This concept of what we call dissipative nature can be extended from living organisms to social groups of animals, to mankind. An analogy based on the organization of matter provides a basis for a functional model of living entities. The models relies on the parallels among the three central structures of any cell (nucleus, cytoplasm and outer membrane) and the human body (central organs, body fluids along with the connective tissues, and external skin integument). This three-part structural organization may be observed almost universally in nature. It can be observed from the atomic structure to the planetary and intergalactic organizations. This similarity is corroborated by the membrane theory applied to living organisms. According to the energy nature of living matter and the proposed functional model, the decreased integrity of a human body's external envelope membrane is a first cause of the structural degradation and aging of the entire organism. The aging process than progresses externally to internally, as in single cell organisms, suggesting that much of the efforts towards the restoration and maintenance of the mechanisms responsible for structural development should be focused accordingly, on the membrane, i.e., the skin. Numerous reports indicate that all parts of the human body, like: bones, blood with blood vessels, muscles, skin, and so on, have some ability for restoration. Therefore, actual revival of not only aging tissue of the human body's membrane, but the entire human body enclosed within, with all internal organs, might be expected. We assess several aging theories within the context of our model and provide suggestions on how to activate the body's own anti-aging mechanisms and increase longevity. This paper presents some analogies and some distinctions that exist between the living dissipative structure matter and inanimate matter, discusses the aging process and proposes certain aging reversal solutions.     

Unusual responses of nocturnal pineal melatonin synthesis and secretion to swimming: Attempts to define mechanisms

Abstract: The effect of swimming at night on rat pineal melatonin synthesis was compared with that of light exposure at night. Rats were forced to swim at 0030 hr (lights out at 2000 hr) and sacrificed by decapitation 15 and 30 min later, immediately after swimming. Other groups of animals were exposed to white light (650μW/cm²) for 15 and 30 min at same time. Swimming caused a rapid and highly significant drop in the melatonin content in the pineal gland; however, the activity of N-acetyltransferase (NAT), the supposed rate limiting enzyme in the melatonin production, was not changed. Despite the drop in pineal melatonin levels, serum concentrations of the indole remained elevated in the rats that swam. In contrast, melatonin levels in the pineal and serum of light exposed rats fell precipitously, accompanied by a significant suppression of NAT activity. Since we anticipated that the strenuous exercise associated with swimming may induce release of artrial natriuretic peptide (ANP) from the heart, which in turn could cause the release of pineal melatonin, in a second study we injected physiological saline intravenously to stretch the cardiac muscle and release ANP. Three milliliters of normal saline was injected during the day into the jugular vein of anesthetized rats that were pretreated with isoproterenol to stimulate pineal melatonin production. Animals were killed 15 min after the saline injection, and pineal NAT activity and pineal melatonin levels were measured. The saline injections caused no alteration in the elevated levels of either NAT or melatonin. These data suggest that the disparity in pineal NAT activity (which was high) and pineal melatonin (which was low), in animals swum at night, may not be caused by ANP which is released during strenuous exercise such as swimming.     

Melatonin and photoperiodic time measurement: Seasonal breeding in the sheep

Abstract: Well-established circadian physiology supports the view that photoperiodic time measurement utilizes the coincidence between the presence of light and a photosensitive phase of a 'biological clock' to alter reproductive status—the so-called external coincidence model of seasonal breeding. In this review, we examine the mechanism whereby photoperiod interacts with presumed suprachiasmatic nuclei activity to allow endogenous melatonin to normally synchronize reproductive activity to the optimal time of year. The Romney Marsh sheep is particularly explored as an experimental model. It is suggested that the on/off activity of seasonal reproduction may be a robust mechanism able to be predictably manipulated by the judicious use of the light/dark cycle and exogenous melatonin, but firmly based on circadian principles.     

The immunoneuroendocrine role of melatonin

Abstract: A tight, physiological link between the pineal gland and the immune system is emerging from a series of experimental studies. This link might reflect the evolutionary connection between self-recognition and reproduction. Pinealectomy or other experimental methods which inhibit melatonin synthesis and secretion induce a state of immunodepression which is counteracted by melatonin. In general, melatonin seems to have an immunoenhancing effect that is particularly apparent in immunodepressive states. The negative effect of acute stress or immunosuppressive pharmacological treatments on various immune parameters are counteracted by melatonin. It seems important to note that one of the main targets of melatonin is the thymus, i.e., the central organ of the immune system. The clinical use of melatonin as an immunotherapeutic agent seems promising in primary and secondary immunodeficiencies as well as in cancer immunotherapy. The immunoenhancing action of melatonin seems to be mediated by T-helper cell-derived opioid peptides as well as by lymphokines and, perhaps, by pituitary hormones. Melatonin-induced-immuno-opioids (MHO) and lymphokines imply the presence of specific binding sites or melatonin receptors on cells of the immune system. On the other hand, lymphokines such as -γ-interferon and interleukin-2 as well as thymic hormones can modulate the synthesis of melatonin in the pineal gland. The pineal gland might thus be viewed as the crux of a sophisticated immunoneuroendocrine network which functions as an unconscious, diffuse sensory organ.     

Serological survey of HIV and syphilis in pregnant women in Madagascar

Objectives Peripartal transmission of human immunodeficiency virus (HIV) and Treponema pallidum, the causative agent of syphilis, leads to severe consequences for newborns. Preventive measures require awareness of the maternal infection. Although HIV and syphilis testing in Madagascar could be theoretically carried out within the framework of the national pregnancy follow‐up scheme, the required test kits are rarely available at peripheral health centres. In this study, we screened blood samples of pregnant Madagascan women for HIV and syphilis seroprevalence to estimate the demand for systemic screening in pregnancy. Methods Retrospective anonymous serological analysis for HIV and syphilis was performed in plasma samples from 1232 pregnant women that were taken between May and July 2010 in Ambositra, Ifanadiana, Manakara, Mananjary, Moramanga and Tsiroanomandidy (Madagascar) during pregnancy follow‐up. Screening was based on Treponema pallidum haemagglutination tests for syphilis and rapid tests for HIV, with confirmation of positive screening results on line assays. Results Out of 1232 pregnant women, none were seropositive for HIV and 37 (3%) were seropositive for Treponema pallidum. Conclusions Our findings are in line with previous studies that describe considerable syphilis prevalence in the rural Madagascan population. The results suggest a need for screening to prevent peripartal Treponema pallidum transmission, while HIV is still rare. If they are known, Treponema pallidum infections can be easily, safely and inexpensively treated even in pregnancy to reduce the risk of transmission.     

Response of rat pineal melatonin to calcium, magnesium, and lithium is circadian stage dependent

Abstract: Herein we documented the response of pineal melatonin production to electrolytes known to be effective on pineal function in view of a possible circadian stage dependence. We studied the release of melatonin by perifused rat pineal glands at 2 different circadian stages corresponding to the middle of the light and dark periods, i.e., respectively, 7 and 19 HALO (Hours After Light Onset, L:D = 12:12). The initial efflux rates were, as expected, much higher in the perifusates of glands removed from rats sacrificed during the dark phase than of those removed during the light phase. After 3 hr of perifusion, melatonin release reached similar levels which were found constant up to the 8th hr of perifusion, whatever the circadian stage. Perifusion of the glands with physiological concentrations for the rat of calcium (5.2 mmol/1) and magnesium (1.34 mmol/1) resulted in a stimulatory effect on the pineal glands removed from rats sacrificed in the middle of the dark period (19 HALO), whereas no effects were observed on the pineal glands removed from rats sacrificed during the light (7 HALO). Lithium (0.28 and 0.55 mmol/1) was ineffective on melatonin release in pineal glands removed 7 and 19 HALO. Our results show differences in the initial efflux rates of melatonin and in the response of perifused pineal glands to calcium and magnesium according to the circadian stage.     

Conservative management of perforated duodenal diverticulum： A case report and review of the literature

Duodenal diverticula are a relatively common condition. They are asymptomatic, unless they become complicated, with perforation being the rarest but most severe complication. Surgical treatment is the most frequently performed approach. We report the case of a patient with a perforated duodenal diverticulum, which was diagnosed early and treated conservatively with antibiotics and percutaneous drainage of secondary retroperitoneal abscesses. We suggest this method could be an acceptable option for the management of similar cases, provided that the patient is in good general condition and without septic signs.