WCX磁珠联合MALDI-TOF-MS技术在结直肠癌诊断中的应用

目的:比较结直肠癌与正常人血清蛋白质谱的变化,筛选特异性蛋白标志物,建立结直肠癌诊断分类树模型.方法:收集血清样本133例(其中结直肠癌67例,正常人66例),随机分为建模组和验证组.运用弱阳离子纳米磁珠(magnetic bead-weak cation exchange,MB-WCX)联合基质辅助激光解吸离子飞行质谱(matrix-assistedlaser desorption/ionization time-of-flight massspectrometry,MALDI-TOF-MS),建立结直肠癌与正常人血清蛋白质谱.用Flex Analysis2.4软件收集数据,应用ClinproTools2.2软件对建模组34例结直肠癌和33例正常人血清差异蛋白质谱进行定量分析,应用Genetic Algorithm算法建立结直肠癌诊断模型,应用所获取的诊断模型对验证组样本(33例结直肠癌和33例正常人)进行分类诊断,以评价诊断模型的诊断价值.结果:通过比较分析结直肠癌与正常人血清蛋白质谱,发现共有33个差异蛋白峰(P<0.05),其中在结直肠癌中表达上调25个,表达下调8个.利用其中5个差异峰(Mr分别为759,3316,4645,4248,2645Dr)建立诊断模型,获得了94.12%(32/34)敏感性和96.97%(32/33)的特异性,经独立样本双盲验证,其灵敏度为93.94%(31/33),特异度为96.97%(32/33).结论:基于磁珠分离和MALDI-TOF-MS技术能直接检测出结直肠癌患者血清差异表达蛋白,建立的诊断模型具有较高的敏感性和特异性,对提高结直肠癌的诊断具有一定的临床意义.     

胃癌与慢性胃炎唾液蛋白质组鉴别诊断模型

目的:探讨用蛋白质组学质谱技术筛选慢性胃炎患者与胃癌患者唾液蛋白质表达谱,寻找可用于胃癌与慢性胃炎鉴别诊断的特异性生物标志物.方法:采用蛋白质组学基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术,并运用弱阳离子磁珠(WCX)检测慢性胃炎患者和胃癌患者的唾液,得到相应的肽质量指纹图谱,建立鉴别诊断模型.结果:慢性胃炎患者与胃癌患者两组共得到蛋白质峰为77个,其中1个有统计差异显著的蛋白峰(P<0.05),质荷比为:6021.72 Da,通过分析差异蛋白峰表达谱,建立了分类预测模型,识别率为83.54%,预测能力60.23%.以此模型进行临床回代检验结果14例慢性胃炎,其中10例被准确检出,23例胃癌,22例被准确检出,灵敏度71.43%(10/14),特异度95.65%(22/23).结论:初步得到了慢性胃炎与胃癌唾液差异蛋白质表达谱,并初步建立了以6021.72 Da蛋白质区分慢性胃炎与胃癌的唾液蛋白鉴别诊断模型.     

应用蛋白质组学技术对脂肪肝患者血清标志物的筛选

目的:用纳米磁珠与基质辅助激光解析离子化飞行时间质谱(MALDI-TOF-MS)技术检测脂肪肝患者血清蛋白质组,筛选候选标志物并建立诊断模型,初步探讨所建立的诊断模型在脂肪肝早期诊断中的临床意义.方法:95例中度和76例轻度脂肪肝患者均经B超证实,99名健康人血清.用WCX(弱阳离子交换型)纳米磁珠检测各血清标本获得血清蛋白指纹图谱.用Biomarker Wizard软件分析所获得的蛋白图谱找出差异蛋白,再用Biomarker Patterns建立诊断模型.扩大样本量,通过盲法分析进一步验证诊断模型的可靠性.结果:初始建模时,建立了由m/z为7626.2、24147.7、6118.2这3个差异蛋白峰组成的中度脂肪肝诊断模型,其敏感性为90.2%,特异性为93.9%.扩大样本盲法验证的敏感性为90.9%,特异性为92.0%.另选取了m/z为7626.2、3286.0、2760.7、2543.1、2746.1这5个差异蛋白峰组成的模型,能把轻度从中度脂肪肝患者中区分出来:选取m/z为7626.2、11721.0、24141.7、8529.1、9042.7这5个差异蛋白峰组成的模型,能将轻度脂肪肝患者从健康对照中区分出来.结论:蛋白质组学技术可筛选出有意义的血清生物标志差异蛋白.由三个差异表达蛋白及其特定组合构成的诊断模型可以区分中度脂肪肝患者与健康人,由五个特定组合构成的差异表达蛋白的模型能区分轻度、中度脂肪肝患者及区分轻度脂肪肝患者与健康对照.     

胃癌与慢性胃炎的唾液蛋白指纹图谱鉴别诊断模型

目的:筛选胃癌患者与慢性胃炎患者及健康志愿者的唾液蛋白质组差异表达谱,并建立胃癌患者与慢性胃炎唾液无创伤分子诊断(判别)模型.方法:采集57例胃癌患者(术前且无放化疗)和28例慢性胃炎患者的唾液标本,用WCX(弱阳离子交换型)纳米磁珠联合基质辅助激光解析离子化飞行时间质谱(matrix assisted laser desorption ionization time-offlight mass spectrometry,MALDI-TOF-MS)技术进行检测,获得各标本的蛋白指纹图谱.用Biomarker Wizard软件分析所获得的蛋白指纹图谱找出差异蛋白,再用Biomarker Patterns 5.0.2建立鉴别诊断模型.结果:优化筛选建立了由m/z为4267.09、6564.85、2138.14等3个差异蛋白峰组成的胃癌与慢性胃炎的鉴别诊断模型,经临床回代检验该分子诊断模型对鉴别胃癌与慢性胃炎的灵敏度和特异度分别为96%(55/57)和86%(24/28),通过交叉验证法进一步验证诊断模型的可靠性,结果该模型的灵敏度和特异度分别为89%(51/57)和75%(21/28).结论:基于WCX结合MALDI-TOF-MS技术建立的唾液蛋白组分子诊断模型为胃癌与慢性胃炎的鉴别诊断提供了一种敏感无创的新方法.     

质谱技术在诊断急淋中枢神经系统白血病中的应用研究

目的研究质谱技术在诊断急淋中枢神经系统白血病中的应用价值。方法选取我院2015年1月~12月收治的急性淋巴白细胞(ALL)患者20例作为研究对象,及健康体检者10名为研究对象,应用SELDI-TOF-MS质谱技术检测其脑脊液建立蛋白质指纹模型图,并分析差异蛋白。结果与对照组比较,单纯ALL组发现有8个差异显著的蛋白峰,差异有统计学意义(P0.05);与单纯ALL组比较,ALL合并CNSL组发现有11个差异显著的蛋白峰,差异有统计学意义(P0.05)。对差异蛋白建立决策树模型,用此模型诊断ALL合并CNSL,敏感性85.71%(6/7),特异性为91.30%(21/23)。结论应用SELDI-TOF-MS质谱技术建立急淋中枢神经系统白血病患者脑脊液蛋白质指纹模型图,发现差异蛋白,有助于疾病诊断。     

胰腺癌血清蛋白质波谱模型的构建及诊断价值研究

目的 建立胰腺癌诊断模型,寻找与胰腺癌分期相关的蛋白峰并鉴定出新型的肿瘤标志物.方法 应用表面增强激光解吸离子化飞行时间质谱(SELDI-TOF-MS)技术,用SAX2蛋白芯片,检测胰腺癌患者、胰腺良性疾病患者和正常对照者的血清蛋白指纹谱,统计建立决策树诊断模型和Logistic回归模型并评估诊断价值,蛋白芯片免疫法鉴定差异蛋白峰,ELISA法测定其在血清中的浓度.结果 胰腺癌组和正常对照组指纹谱比较.26个蛋白峰差异有统计学意义(P＜0.01),胰腺癌组和胰腺良性疾病组指纹谱比较,16个蛋白峰差异有统计学意义(P＜0.05).建立的决策树诊断模型对胰腺癌的敏感性为83.3%,特异性为100.0%,经ROC曲线评估,该模型优于糖链抗原(CA)19-9.有6个差异蛋白峰在不同分期咦腺癌中差异有统计学意义(P＜0.01),建立Logistic回归模型诊断早期胰腺癌.敏感性为81.6%,特异性为80.6%.鉴定出差异蛋白峰质荷比(M/Z)28068为C140H166,其诊断胰腺癌的敏感性＞82%,特异性＞88%.结论 应用SELDI-TOF-MS技术建立的诊断模型对胰腺癌的诊断有较大价值,明显优于CA19-9.鉴定出的差异蛋白C140rf166有望在胰腺癌诊断中发挥作用.     

神经相关性自身抗体检测在神经精神性狼疮中的意义

目的 探讨神经相关性自身抗体在系统性红斑狼疮(SLE)和神经精神性狼疮(NPSLE)患者血清或脑脊液中的敏感性和特异性,评价其在NPSLE诊断中的意义.方法 利用间接免疫荧光法分别以成神经瘤细胞株(SK-N-SH)、神经胶质瘤细胞株(U251)以及C57BL/10小鼠的大脑及脊髓切片为底物检测121例SLE患者、34例疾病对照组和34名健康对照组的血清及24例NPSLE患者和22例疾病对照组的脑脊液中抗神经元细胞抗体、抗神经胶质细胞抗体、抗脑抗体及抗脊髓抗体.结果 SLE患者血清中抗神经元细胞抗体、抗脑抗体及抗脊髓抗体的阳性率(17.4%、25.6%和29.8%)明显高于类风湿关节炎(RA)患者以及正常对照组(P＜0.05或P＜0.01),在SLE诊断中的特异性分别为98.5%、95.6%和100%;抗神经元细胞抗体、抗脑抗体及抗脊髓抗体均与SLE疾病活动性指数(SLEDAI)积分呈正相关,并与NPSLE患者的癫痫发作、头痛、急性意识障碍以及情绪失调、焦虑等症状的发生有关;NPSLE患者血清的抗神经元细胞抗体、抗脑抗体和抗脊髓抗体的阳性率(27.8%、38.9%和61.1%)明显高于无中枢神经系统受累的SEE患者(P＜0.05或P＜0.01);NPSLE患者的脑脊液中抗脑抗体、抗脊髓抗体的阳性率均为29.2%,与疾病对照组比较,差异具有统计学意义(P＜0.05或P＜0.01);在NPSLE诊断中的特异性分别为100%和95.5%.结论 抗神经元细胞抗体、抗脑抗体及抗脊髓抗体对SLE诊断的特异性较高,但敏感性偏低;3种抗体均与SLE疾病活动性相关;血清中抗神经元细胞抗体、抗脑抗体及抗脊髓抗体是NPSLE诊断的较特异性抗体,抗脊髓抗体可能是诊断NPSLE较敏感的指标之一;脑脊液中的抗脑抗体、抗脊髓抗体对NPSLE的诊断更具有特异性.     

应用基质辅助激光解析电离飞行时间质谱法研究原发性胆汁性肝硬化的血清多肽谱

目的应用弱阳离子交换磁珠提取多肽结合基质辅助激光解析电离飞行时间质谱系统(MALDI-TOF-MS)分析原发性胆汁性肝硬化(PBC)患者的血清多肽谱,寻找具有潜在意义的血清标志物。方法收集105例血清样本,包括55例PBC患者、25例其他肝病患者和25例正常对照者血清样本,经弱阳离子交换磁珠纯化,MALDI-TOF-MS分析及ClinProTools生物信息学方法研究其血清多肽表达谱。结果在质荷比(m/z)1000～10 000 Da的范围内3组间共检测到158个血清多肽峰,其中83个有统计学意义(P<0.001),在PBC中表达上调的多肽有31个,表达下调的多肽有52个。组合m/z 1062.47、5356.13、4268.63、846.18、1945.31和6649.53Da 6个峰建立多肽诊断指纹图谱模型,该模型盲样验证的准确率在PBC组、正常对照组和其他肝病组分别为100%、81.8%和72.7%。结论可以利用MALDI-TOF-MS技术和ClinProTools软件来筛选PBC血清标志物,其在PBC诊断方面具有一定的价值。     

SELDI-TOF-MS技术诊断贲门癌高发区慢性萎缩性贲门炎及贲门黏膜不典型增生

目的探讨贲门癌高发区人群贲门黏膜不典型增生(DYS)、慢性萎缩性贲门炎(CAG)的血清学诊断方法,为贲门癌高危人群的筛查提供新手段。方法采用弱阳离子结合芯片(WCX2)及表面增强激光解吸/电离飞行时间质谱仪(SELDI-TOF-MS)检测食管癌高发区无症状普查人群143人(其中活检组织诊断为正常63人,CAG57人,DYS23人)的血清蛋白质质谱,对原始信号的总离子强度及相对分子质量做均一化校正及噪声滤过,并对同一质荷比蛋白质质谱峰平均强度值做组间t检验。应用BiomarkerPattern软件建立决策树分类模型,经10倍交叉验证得到该分类模型对测试组病变人群的诊断率和排除率。结果采用DYS和正常组质荷比为M3894·0的一种蛋白质建立的决策树分类模型,其对测试组DYS诊断率为87%,排除率为86%;CAG和正常组则有质荷比为M2942·15和M33316·6的2种蛋白质建立决策树分类模型,对测试组CAG诊断率为93%,排除率为92%。结论SELDI-TOF-MS蛋白质芯片技术检测血清蛋白质质谱法诊断贲门癌高发区DYS和CAG有较高的敏感性和特异性,为贲门癌高危人群筛查提供了新的血清学手段。     

非小细胞肺癌患者血清蛋白质标记物的检测

目的 检测非小细胞肺癌患者(NSCLC)血清蛋白质,筛选特异的蛋白质标记物,构建用于NSCLC早期诊断的血清蛋白质指纹图谱模型.方法 应用表面增强激光解析电离飞行时间质谱(SELDI-TOF-MS)技术检测235例血清标本的蛋白质质谱,并结合生物信息学方法(支持向量机)分析数据.结果 筛选出3个质荷比(m/z)位于6628,9191和11412的蛋白质标记物,构建NSCLC早期诊断模型.联合3种潜在蛋白质标记物,经留一法交叉验证,区分NSCLC和正常健康对照的敏感性为98%,特异性为96%.盲法验证显示,该模型诊断NSCLC的敏感性为96.56%,特异性为94.79%.结论 SELDI-TOF-MS结合支持向量机建立NSCLC血清蛋白质指纹图谱模型是早期诊断NSCLC的一种敏感性高、特异性强的新方法,值得进一步研究与应用.     

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.     

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.     

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.     

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.