基于非靶向代谢组学技术的胆石症血浆代谢标志物的初步探讨

背景:胆石症是胆道系统最常见的疾病之一,有关胆石症代谢组学的研究目前尚无报道。目的:应用非靶向代谢组学技术筛选胆石症患者的血浆代谢标志物。方法:基于上海市男、女性健康队列开展胆石症血浆代谢谱预初实验。应用超高效液相色谱-四级杆飞行时间质谱(UPLC-QTOFMS)和气相色谱-飞行时间质谱(GC-TOFMS)技术检测43例基线期胆石症患者和43名年龄配对健康对照者的血浆代谢物,采用主成分分析(PCA)、正交偏最小二乘判别分析(OPLS-DA)以及t检验筛选胆石症患者的特异性代谢物。通过Logistic回归分析构建胆石症预测模型,以ROC曲线对模型进行初步评价。结果:UPLC-QTOFMS和GC-TOFMS共检测出血浆代谢物618个,根据OPLSDA VIP 1. 0且t检验P 0. 05的筛选条件,筛选出差异代谢物30个,由其中的2-甲基-4-戊烯酸、磷脂酰乙醇胺PE(p-16∶0e/0∶0)、顺式-4,7,10,13,16,19-二十二碳六烯酸、2-氨基丁酸、尿酸、瓜氨酸、S-异戊烯基-L-半胱氨酸、羟环己脲和2,3-环氧甲基萘醌九个代谢物构建的胆石症预测模型ROC曲线下面积(AUC)为0. 97,敏感性和特异性分别为95. 4%和88. 4%。结论:胆石症患者的血浆代谢谱发生明显变化,由其中9个差异代谢物构建的预测模型能较好地区分胆石症患者与健康对照者。     

老年脑卒中后认知障碍伴失眠患者的血清代谢组学

目的 基于血清代谢组学分析老年脑卒中后认知障碍伴失眠患者差异代谢标志物。方法 对比老年缺血性脑卒中后认识障碍伴失眠患者和单纯脑卒中患者各9例,通过对这18例受试者进行血清液相色谱-质谱联用(LC-MS)非靶向代谢组学分析,筛选差异代谢物,探讨脑卒中后继发认知和失眠障碍的发生机制。结果 基于非靶向代谢组研究,发现老年脑卒中后认知障碍伴失眠患者的差异代谢中脂类占绝大多数,其最多的是甘油磷脂类,多数呈含量下调趋势,4种不饱和脂肪酸(二十二碳二烯酸、十八碳二烯酸、类花生酸、6-酮-前列腺素F1α)和两种饱和脂肪酸(二十二烷酸、棕榈酸)在观察组含量均上调,鞘磷脂代谢产物神经酰胺和神经鞘氨醇磷酸胆碱含量下调,而二氢神经鞘氨醇含量上调;氨基酸代谢中两种差异代谢物(半胱氨酸硫酸酯和左旋精氨酸)含量在观察组中上调;剩余的5种差异代谢物上调的次黄嘌呤、左旋肉碱、睾酮和异丙肾上腺素,下调的有尿苷二磷酸葡萄糖。结论 缺血性脑卒中后认知障碍伴失眠患者的血清代谢谱与单纯脑卒中者存在明显差别,多种代谢物及代谢通路可能参与老年脑卒中患者继发认知、失眠障碍的发病过程。     

基于血清代谢物的血管内皮功能障碍预测模型研究

目的:构建基于血清代谢物的血管内皮功能障碍预测模型并进行验证。方法:应用气相色谱-飞行时间质谱(GC-TOF-MS)检测建模组77例和外验证组26例研究对象的血清代谢物。采用正交偏最小二乘法-判别分析(OPLS-DA)和t检验,筛选建模组受试者血管内皮功能正常者和血管内皮功能障碍者的差异代谢物。运用Logistic回归方法优选差异代谢物与性别、年龄的组合并构建预测模型。以ROC曲线AUC和Hosmer-Lemeshow拟合优度检验分别评价模型的预测和校准能力,并对模型进行内部验证和外部验证。结果:共筛选出OPLS-DA变量投影重要度(VIP)值大于1且t检验中P0.05的差异代谢物16个。由性别、年龄、尿酸、色氨酸和尿素组成的预测模型最优,其AUC为0.974(95%CI:0.945～1.000),拟合优度检验差异无统计学意义(P0.05),模型表达式为:P=1/{1+exp[-(9.803-0.777×性别-0.204×年龄-0.343×ln尿酸+0.024×ln色氨酸+0.808×ln尿素)]}。该模型内部验证AUC为0.922 (95%CI:0.847～0.997),外部验证AUC为0.881 (95%CI:0.729～1.000),敏感度和特异度分别为91.7%和78.6%。结论:基于血清代谢物的血管内皮功能障碍预测模型具有良好的稳定性和预测能力。     

原发性胆汁性胆管炎血清代谢物组的氢谱核磁共振初步研究

背景:氢谱核磁共振(~1H-NMR)技术为代谢组学常用方法,用于肝炎肝硬化、肝癌等疾病的研究,但关于原发性胆汁性胆管炎(PBC)患者血清代谢物组的研究较为少见。目的:探讨~1H-NMR筛选PBC血清代谢物的能力。方法:采用~1H-NMR对20例PBC、20例乙型肝炎肝硬化和20例对照组进行检测并获得~1H-NMR图谱,结合主成分分析(PCA)、正交偏最小二乘法判别分析(OPLS-DA)法创建诊断模型。根据OPLS-DA模型的相关系数P(corr)、VIP值和非配对t检验,筛选出对照组与两组肝硬化组的血清差异性代谢物。结果:OPLS-DA模型能有效区分对照组与PBC患者,模型解释率和预测率分别为81.9%和44.8%(P=0.029 3),谷氨酰胺、叶酸、尿刊酸和4-乙基苯甲酸为两组的差异性代谢物。OPLS-DA模型能有效区分对照组与乙型肝炎肝硬化患者,尿刊酸、1-甲基组胺、1-甲基腺嘌呤、葡萄糖、L-乙酰肉毒碱为两组的差异性代谢物。OPLS-DA模型不能区分PBC和乙型肝炎肝硬化患者。结论:血清谷氨酰胺、叶酸可能为PBC潜在的特异性生物标记物,可能与PBC的免疫损伤机制和预后有关。~1H-NMR技术结合OPLS-DA诊断模型有望成为一种研究肝硬化的新方法。     

中国不同地域汉族健康人群血清代谢组学参照谱的差异

目的建立中国汉族健康人群血清代谢组参照谱, 探讨代谢组特征在不同地域、性别、年龄间的差异。方法基于国民健康调查的横断面研究数据和生物样品, 纳入7个省份的1 039名男性和1 032名女性汉族健康成年人(≥30岁)为研究对象, 采用超高效液相色谱-质谱串联(UPLC-MS)方法检测血清代谢组, 采用正交偏最小二乘-判别分析(OPLS-DA)比较不同地域、性别、年龄组间代谢组的差异。结果不同地域、性别、年龄人群血清代谢组存在显著性差异。共筛选并鉴定出地域差异性代谢物114种, 其中参与人体代谢通路的53种, 以肽类(20种)和甘油磷脂代谢相关(14种)代谢物为主。共鉴定出性别差异性代谢物59种, 其中可替宁在7个省份不同性别间均有显著性差异。在陕西和海南的研究对象中发现不同年龄间代谢组的差异, 共鉴定出差异性代谢物22种。结论血清代谢组受不同地域、性别和年龄的影响, 因此在利用代谢组学进行疾病诊断和筛选生物标志物时, 不同研究不能采用同一参照, 应遵循严格的实验设计和入选标准, 针对地域、性别、年龄和检测批次等因素选择差异性参照。     

细粒棘球蚴感染小鼠血清的代谢组学分析

目的 检测分析感染细粒棘球蚴的囊型包虫病(cystic echinococcosis, CE)小鼠与健康小鼠血清代谢产物的差异性,从代谢组学层面为CE的早期诊断和发病机制研究提供依据。方法 门静脉注射感染细粒棘球绦虫原头蚴,建立CE小鼠模型。用液相色谱质谱联用技术(1iquid chromatography-mass spectrometry, LC-MS)分别对CE小鼠和对照组小鼠血清进行检测,采用无监督的主成分分析(principal component analysis, PCA)、偏最小二乘判别分析(partial least squares-discriminant analysis, PLS-DA)、正交偏最小二乘法判别分析(orthogonal partial least squares discriminant analysis, OPLS-DA)及学生t检验筛选差异代谢物,并对其进行层次聚类和代谢通路富集分析。结果 PLS-DA和OPLS-DA分析两组血清共获得198个显著差异代谢产物,显著上调的代谢产物有88个,显著下调的代谢产物有110个。与对照组比较,CE组小鼠...     

胰腺癌患者血清代谢组学研究

目的检测胰腺癌患者与健康者血清的代谢组学,筛选能够鉴别胰腺癌与健康者的差异代谢物,寻找潜在的胰腺癌早期诊断标志物。方法采用病例对照成组分析方法,以病理诊断为胰腺导管腺癌患者作为病例组,健康人群为对照组。应用高效液相色谱单级四级杆飞行时间串联质谱联用仪检测胰腺癌及健康者的血清代谢组学。通过单维及多维统计方法分析两组间代谢组学的差异,根据分子质量、代谢产物数据库和质谱／质谱（MS／MS）信息筛选差异代谢物。通过差异代谢物的受试者工作特征曲线下面积（AUC）评价它们鉴别胰腺癌及健康者的价值。结果共纳入胰腺导管腺癌患者及健康者各54例,年龄及性别相匹配。最终筛选出12种差异代谢物,其中胰腺癌患者血清中羟基花生四烯酸（HETE）、苯丙氨酸、乙酰肉碱、亚油酸、棕榈酰肉碱、亚油醇肉碱、尿嘧啶脱氧核苷酸（dUMP）和甘氨鹅脱氧胆酸8种代谢产物水平显著高于健康者;溶血磷脂酰胆碱[LysoPC（18：0）]、LysoPC（P-16：0）、溶血磷脂酸[LPA（182／0：0）]、LysoPC（14：0）4种代谢产物水平显著低于健康者（P值均〈0．05）。HETE、LysoPC（18：0）、苯丙氨酸、LPA（18：2／0：0）、dUMP的AUC分别为0．98、0．96、0．92、0．91,均大于CA19-9的AUC（0．90）。结论胰腺导管腺癌患者及健康者的血清代谢组共筛选出12种差异代谢物,其中HETE、LysoPC（18：0）、苯丙氨酸、LPA（18：2／0：0）、dUMP有可能是潜在的诊断标志物。     

基于“同病异证”理念的HIV/AIDS患者代谢组学初步分析

目的探讨代谢组学技术在同病异证中的诊断价值。方法收集40例艾滋病脾气虚和脾肾亏虚证患者血清,采用超高效液相色谱—四极杆飞行时间串联质谱(UHPLC-Q-TOF-MS)对其代谢产物进行检测,应用主成分分析(PCA)及正交偏最小二乘法-判别分析(OPLS-DA)的方法探讨两组代谢物的差异。结果脾气虚和脾肾亏虚证患者血清代谢物经PCA分析得到7个主成分的模型(R~2=0.521,Q~2=0.151),模型经OPLS-DA置换检验,R~2Y=0.944,Q2=0.892,能够明显将两种证型区分,并确定了14种差异代谢物并涉及花生四烯酸、糖基化磷脂代谢、甘油磷脂代谢、类脂物、嘌呤代谢通路。结论运用代谢组学技术可以将艾滋病脾气虚和脾肾亏虚证明显区分,为艾滋病中医证候辨证分型提供初步参考,同时为"同病异证"及"同病异治"提供实验依据。     

丙型肝炎病毒自发清除者的血清代谢组学研究

目的 探索HCV自发清除者、慢性丙型肝炎(丙肝)及健康人血清代谢组学的差异.方法 纳入HCV自发清除者、慢性丙肝患者和健康对照各30例,采用快速液相色谱-串联质谱联用(LC-MS/MS)技术,应用主成分分析(PCA)、正交偏最小二乘法-判别分析(OPLS-DA)进行模式识别,然后通过变量重要性因子(VIP)、非参数检验,结合数据库检索筛选鉴定有差异的代谢物.结果 25个变量被确认为存在显著差异的代谢物,其中7个变量被鉴定为花生四烯酸、棕榈油酸、葵酰基肉碱、溶血磷脂酰胆碱(20∶5,16∶0)、溶血磷脂酰乙醇胺(16∶0,18∶0),涉及脂肪酸、磷脂等代谢.其中花生四烯酸以及未鉴定出明确结构的m/z 179.0719、m/z 382.1360、m/z 548.3475、m/z680.4281、m/z 303.2323等物质与自发清除组的相关性较好,受试者工作特征曲线下面积为0.887～0.977,具有较好的特异度和敏感度.结论 HCV自发清除者、慢性丙肝感染者、健康人在血清代谢水平上存在明显差异,这些差异的意义有待进一步探索.     

人参皂苷Re对阿尔茨海默病模型小鼠脑组织生物标记物调控作用的研究

目的通过超高速液相色谱-质谱联用(UPLC-MS)技术研究人参皂苷Re(G-Re)对阿尔茨海默病(AD)模型小鼠生物标记物的调控作用以及机制,为AD的中药治疗提供新思路。方法将小鼠随机分为对照组、AD模型组和G-Re治疗组,通过免疫组化实验观察小鼠脑部海马区细胞的病理学改变,运用以UPLC-MS为基础的代谢组学技术研究G-Re对AD模型小鼠脑组织生物标记物的调控作用。结果免疫组化实验发现,对照组小鼠海马区无Aβ沉积,AD模型组小鼠海马区可见大量Aβ沉积,经G-Re治疗后小鼠海马区Aβ沉积明显减少。代谢组学分析发现,AD模型组小鼠脑组织中存在次黄嘌呤、次黄嘌呤核苷、苯丙氨酸、16碳鞘氨醇、植物鞘氨醇、溶血磷脂酰胆碱C16:0、溶血磷脂酰胆碱C18:1、溶血磷脂酰胆碱C18:0等9种生物标记物。与对照组比较,AD组次黄嘌呤、次黄嘌呤核苷和苯丙氨酸的含量明显升高(P0.05),16碳鞘氨醇、植物鞘氨醇、溶血磷脂酰胆碱(C16:0、C18:1、C18:0)的含量明显降低(P0.05)。经G-Re干预后,AD组小鼠脑组织中次黄嘌呤、次黄嘌呤核苷和苯丙氨酸的含量明显降低(P0.05),16碳鞘氨醇、植物鞘氨醇、溶血磷脂酰胆碱(C16:0、C18:1、C18:0)的含量明显升高(P0.05)。结论 G-Re可以干预AD小鼠体内氨基酸、核酸及脂质等代谢途径,减少小鼠海马区的Aβ沉积,从而对AD起到治疗作用。     

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.     

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.     

Changes in connexin43, the gap junction protein of astrocytes, during development of the rat pineal gland

Abstract: The abundance of gap junctions between rat pineal astrocytes formed by connexin43 (Cx43) was studied during development. Levels and distribution of Cx43 were measured by immunoblotting and indirect immunofluorescence, respectively. The amount of Cx43 in cells located within the gland was low until about the 7th postnatal day and increased to adult values between the 14th and 21st days postpartum. Although astrocytes, recognized by their vimentin immunoreactivity, were scarce before birth, they were abundant by the 7th postnatal day suggesting that the low levels of Cx43 found at this age corresponded to a low expression of this protein. Localization of the immunoreactivity to Cx43 and vimentin showed a close correlation, indicating that mature or immature pineal astrocytes form gap junctions made of Cx43. Since Cx43 levels attained their adult values at about the time the innervation and the functional state of the gland reached maturity (2–3 weeks after birth), it is proposed that astrocyte gap junctions are involved in the function of the adult rat pineal gland.     

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.     

Presence of immunoreactive growth hormone and prolactin in the ovine pineal gland

Abstract: The use of antisera raised against bovine growth hormone (GH) and ovine prolactin (PRL) enabled the detection of related immunoreactive (ir) sequences of proteins in ovine pineal tissue. The isolation of PRL-like ir-material was accomplished using a 0.25 M ammonium sulphate (pH 5.5) extraction followed by ethanol precipitation, whereas the resulting 2.0 M ammonium sulphate (pH 7.0) precipitate contained a GH-like immunoreactivity. Gel chromatography of the GH-like immunoreactivity (Sephadex G-100) indicated the presence of several GH-like fragments ranging in the M_r range of 7,000 to 55,000. Analyses of the PRL-like ir-material found in pineal tissue on HPLC using a TSK 545-DEAE column led to the resolution into a single peak of immunoreactivity. A single peak of activity was also observed following chromatofocusing and hydrophobic interaction chromatography of the ir-peak from the TSK 545-DEAE column. The PRL-like ir-material inhibited the binding of [¹²⁵I]ovine PRL-S14 to anti-ovine PRL antibodies without showing an affinity for binding to anti-rat PRL or anti-bovine GH antibodies. Scatchard analysis of the binding of pineal PRL-like ir-material and pituitary ovine PRL-S14 to liver membranes from day-20 pregnant rats revealed similar affinity constants (K_a of 4.7 ± 0.2 × 10⁹ M^-1). In addition, the replication of Nb 2 Node rat lymphoma cells was stimulated by pineal PRL-like ir-material, an effect known to be specific for lactogenic hormones. The pineal PRL-like immunoreactivity appeared on sodium dodecyl sulfate polyacrylamide gels as a single major band of M_r 24,000. The functional status of PRL-and GH-like ir-material in the ovine pineal remains to be determined, but evidence is presented that the overall protein synthesis rate of the rat pineal responded to circulating concentrations of PRL.     

Microbiology of human immunodeficiency virus anorectal disease

PURPOSE: Individuals who are seropositive for the human immunodeficiency virus are at high risk for opportunistic infection and anorectal disorders. Little prospective information is available regarding anorectal pathogens in these patients. METHODS: One hundred sixty-three HIV-seropositive patients presented to the colorectal clinic between 1989 and 1992. Forty-seven (29 percent) patients were thought to have an infectious process and were prospectively studied using a standardized multiculture protocol. RESULTS: Mean age was 33 (range, 19–59) years. All were male; high-risk behavior accounted for 87 percent of HIV transmissions. Presenting complaints included anorectal pain (79 percent), pus per anum (28 percent), and blood per anum (26 percent). Examination revealed perianal tenderness (60 percent), condyloma (38 percent), perianal ulcers (38 percent), and anal fissures (34 percent). Sixty-six sets of cultures were performed; 28 patients had one set, 15 had two sets, and 4 had three sets. Thirty-two of these 47 patients (68 percent) had positive cultures including herpes (50 percent), cytomegalovirus (25 percent),Neisseria gonorrhoeae (16 percent), chlamydia (16 percent), acidfast bacilli (2 percent), and others (9 percent). Six of 32 patients with positive cultures had more than one organism cultured. Sixteen (50 percent) patients with positive cultures were treated medically, 8 (25 percent) were treated surgically and 8 (25 percent) were treated with both modalities. Sixty-one procedures were performed on 17 patients for condylomata. Eighteen patients had 20 procedures for abscesses, 50 percent of whom had positive cultures for other than common bowel flora; all improved. Fourteen patients underwent 33 procedures for perianal fistulas.Mycobacterium fortuitum was cultured from one patient who required 13 procedures for abscesses and fistulas. Forty-five (96 percent) patients were followed for an average of 12.5 months ±2.9 SEM (range, 1–94 months). Symptoms were improved or resolved in 22 of 32 (69 percent) patients with positive cultures and in 11 of 13 (84 percent) with negative cultures. CONCLUSIONS: Specific pathogens may often be identified in human immunodeficiency virus-seropositive patients with anorectal disorders if aggressively sought. Although patients without specific pathogens identified may be expected to improve with planned empiric treatment, positive identification allows more directed therapy.