非酒精性脂肪性肝病患者血浆外泌体差异蛋白分析*

目的 筛选非酒精性脂肪性肝病(NAFLD)患者血浆外泌体差异蛋白,分析其功能及其生物学过程,为NAFLD患者临床诊断提供参考依据。方法 2020年7月～10月我院诊治的NAFLD患者3例和健康体检者3例,采用串联质谱标签(TMT)标记定量蛋白质组学技术对血浆外泌体蛋白进行鉴定和定量分析,筛选差异蛋白并进行功能富集分析,了解其参与的生物学过程。结果 经外泌体蛋白质组学分析,共鉴定出蛋白质387种,以倍数上调＞1.2倍或下调＞1.2倍,且P＜0.05为标准筛选出差异表达蛋白34种;健康人比,NAFLD患者上调蛋白25种,下调蛋白9种;生物信息学分析结果显示,这些蛋白主要参与了脂质的储存和代谢、免疫反应、纤维素形成等生物学过程,并与胰岛素抵抗、炎症反应、细胞损伤等信号通路密切相关。结论 采用TMT标记定量蛋白质组学技术筛选NAFLD患者差异蛋白可能为其诊治提供指引。     

酒精性肝病患者血浆外泌体差异蛋白的分析

目的筛选酒精性肝病(alcoholic liver disease, ALD)患者血浆外泌体中的差异蛋白,分析其功能及生物学过程,为ALD患者的治疗和诊断提供参考依据。方法选取2021年5月至2021年10月在首都医科大学附属北京佑安医院住院并确诊为ALD的患者和健康体检者各3例,超速离心分离提纯外泌体,提取蛋白,串联质谱标签(tandem mass tag, TMT)标记后,采用定量蛋白组学技术对两者血浆中的外泌体蛋白进行鉴定和定量分析,筛选出差异蛋白,并用生物信息学分析差异蛋白的功能及其参与的生物学过程。结果共鉴定到可定量蛋白质387种,以倍数上调>1.2倍或下调>1.2倍且P<0.05为标准筛选出差异蛋白27种,与健康对照组相比,ALD组上调蛋白15种、下调蛋白12种,生物信息学分析结果显示,这些蛋白主要参与了脂质的代谢、免疫反应和细胞的死亡等生物学过程,并与炎症反应、细胞的损伤和补体级联反应等信号通路密切相关。结论 TMT标记定量蛋白质组学技术筛选出的差异蛋白可能作为ALD早期诊断的血清学标志物及治疗靶点。     

类风湿关节炎合并干眼病患者血清中差异蛋白的分析

目的筛选类风湿关节炎(RA)合并干眼病(DED)患者血清中差异蛋白,分析其功能及生物学过程。方法选取RA患者20例纳入对照组,同时选取RA(病程1～20年,平均10年)合并DED患者20例纳入观察组,用非标记(Label-free)定量蛋白质组学技术对两组血清中蛋白进行鉴定和定量分析,筛选差异蛋白;通过生物信息学分析差异蛋白功能及参与RA合并DED发病的生物学过程。结果共鉴定到345种蛋白,筛选出13种差异表达蛋白,其中上调蛋白9种、下调蛋白4种。13种差异蛋白在细胞代谢、生物调节、生物学过程中发挥重要作用,并具有分子结合功能,在RA合并DED发病过程中参与氧化应激、免疫和炎症反应、脂类代谢等过程。结论用Label-free定量蛋白质组学技术筛选出的相关血清差异蛋白,可作为诊断为RA合并DED早期血清学标志物。     

慢性乙型肝炎与HBV相关慢加急性肝衰竭患者血浆外泌体差异miRNA的生物信息学分析

目的 探讨慢性乙型肝炎(CHB)患者与HBV相关慢加急性肝衰竭(HBV-ACLF)患者血浆外泌体microRNA(miRNA)表达谱差异，分析其功能及生物学过程，以期获得可用于HBV-ACLF临床诊断的参考依据。方法 选取2021年10月—2022年6月青岛市市立医院感染科住院的6例CHB患者及青岛市第六人民医院血液净化中心接受治疗的6例HBV-ACLF患者。运用Illumina高通量测序技术对这些患者的血浆外泌体miRNA进行检测，筛选差异miRNA并进行功能富集分析，分析其参与的生物学过程。检测得到的外泌体差异miRNA以倍数上调>2倍或下调>2倍且P<0.05为标准筛选。计量资料两组间比较采用Mann-Whitney U秩和检验。计数资料两组间比较采用χ²检验。结果 筛选差异miRNA共249种，与CHB组相比，HBV-ACLF组上调miRNA 126种，下调miRNA 123种。生物信息学分析结果显示，这些差异表达miRNA主要参与了性腺发育、调控蛋白质稳定性、细胞对外界刺激反应等生物学过程，并与乙型肝炎、蛋白多糖在癌症中的作用、调节干细...     

食管鳞状细胞癌患者血浆α-2-HS-糖蛋白和S100A9蛋白水平及意义

目的评估食管鳞状细胞癌(ESCC)血浆外泌体中表达上调的α-2-HS-糖蛋白(AHSG)和S100A9蛋白是否有潜力作为ESCC早期诊断的血浆外泌体肿瘤标志物。方法通过对46例ESCC血浆标本及46例健康对照组血浆标本差异蛋白进行Western印迹验证,如果结果与蛋白质组学结果一致,接着再利用酶联免疫吸附(ELISA)实验进行大样本的蛋白定量验证。结果 Western印迹实验结果表明,AHSG和S100A9在ESCC患者血浆外泌体中表达量与对照组相比明显升高。ELISA验证结果表明,ESCC组和对照组血浆外泌体中AGSH和S100A9浓度存在显著差异。结论两种蛋白可以作为ESCC早期诊断的血浆外泌体肿瘤标志物,但尚需进行更大批次临床样本的实验验证,同时还需要对这两种蛋白的特异性进行分析。     

急性心肌梗死患者血浆外泌体mRNA表达谱分析

目的 比较分析急性心肌梗死(AMI)患者和非心源性胸痛(NCCP)患者血浆外泌体中mRNA的表达差异,并探讨差异表达mRNA对AMI发生发展可能的影响。方法 入选北京朝阳医院和内蒙古包头市中心医院NCCP患者和AMI患者各15例。提取入选者血浆外泌体及外泌体总RNA,高通量测序技术检测AMI患者和NCCP患者血浆中外泌体信使RNA(mRNA)的表达谱,筛选出AMI患者中差异表达的mRNA。对差异表达mRNA进行基因本体(GO)分析和京都基因与基因组百科全书(KEGG)信号通路分析。结果 提取的外泌体为粒径在30~200 nm之间的双层膜小囊泡,表达外泌体标志分子分化簇63(CD63)和热休克蛋白70(HSP70),而不表达甘油醛-3-磷酸脱氢酶(GAPDH),符合外泌体的特征。对入选AMI患者和NCCP患者血浆外泌体中的mRNA表达量进行检测,AMI患者血浆中外泌体转录本数量为64 258条,NCCP患者血浆中外泌体转录本数量为64 374条。其中差异表达的mRNA共1 800条(上调951条,下调849条)。GO和KEGG分析表明上述差异表达的mRNA参与了AMI的生物学调节功能和通路。结论 AMI患者血浆外泌体中mRNA的表达水平与NCCP组比较存在明显的差异,这些差异表达的外泌体mRNA可能在AMI的发生发展过程中发挥重要的作用。     

iTRAQ联合LC-MS/MS技术在肺腺癌血浆生物标志物筛选中的应用

目的应用同位素标记相对和绝对定量(iTRAQ)联合液相色谱串联质谱(LC-MS/MS)技术检测正常者及肺腺癌患者血浆中的差异蛋白,从而筛选出潜在的肺腺癌血浆生物标志物,并探讨其临床意义。方法收集10例健康体检者血浆(正常组),10例肺腺癌患者血浆(腺癌组),应用iTRAQ标记联合LC-MS/MS技术对两组血浆进行差异蛋白组学分析。结果质谱共鉴定到蛋白369种,其中差异表达的蛋白有35种;腺癌组较正常组上调的蛋白有21种、下调的蛋白有14种,其中SCGB3A2、SFTPB蛋白表达显著上调。结论筛选出多种与肺腺癌相关的差异蛋白,其中SCGB3A2、SFTPB有望成为潜在的肺腺癌血浆生物标志物,提示iTRAQ联合LC-MS/MS技术可用于肺腺癌血浆肿瘤标志物的筛选。     

心力衰竭患者血浆外泌体microRNA检测及其功能分析

目的明确心力衰竭(心衰)患者血浆外泌体microRNA(miRNA)表达谱的差别,寻找其在心衰早期诊断和预后评估中的价值。方法应用外泌体提取试剂盒抽提10例心衰患者及10例非心衰对照者外周血浆外泌体,对血浆外泌体miRNA进行高通量测序,初步筛选差异表达miRNA,并通过实时荧光定量PCR方法验证,分析差异表达miRNA的功能及其与NT-proBNP的相关性。结果 miRNA高通量测序共筛选出24条显著差异表达的血浆外泌体miRNA。其中14条miRNA表达显著上调,10条miRNA表达明显下调。定量PCR验证和高通量测序筛选结果一致。差异表达miRNA的功能涉及炎性反应、细胞凋亡、增值、自噬等。相关性分析提示miR-122-5p与NT-proBNP具有相关性(r=0.537,P=0.015)。结论血浆外泌体来源miRNA可能成为潜在的辅助诊断心力衰竭和评估预后的一类新型生物标志物。     

再灌注治疗对急性心肌梗死患者血浆外泌体微小RNA差异表达的影响

目的 观察冠状动脉再灌注治疗对急性心肌梗死(AMI)患者血浆外泌体微小RNA(miRNA,miR)差异表达的影响。方法 选取2022年10月至11月于深圳市龙岗区第三人民医院确诊的3例老年男性AMI患者,进行冠状动脉再灌注治疗,留取患者入院时、术后2 h和术后24 h静脉血,应用miRNA测序技术筛选3例患者血浆外泌体中共同差异表达miRNA后,对其靶基因进行生信分析,利用定量聚合酶链反应验证其中差异表达的miR-499a-5p。结果 与入院时比较,术后2 h血浆外泌体miRNA有418个上调和406个下调,术后24 h血浆外泌体miRNA有320个上调和225个下调(P<0.05);与术后2 h比较,术后24 h血浆外泌体miRNA有344个上调和350个下调(P<0.05)。Kyoto Encyclopedia of Genes and Genomes富集分析显示,差异表达的miRNA富集在磷脂酰肌醇-3-激酶-蛋白激酶B、缺氧诱导因子1和血管平滑肌收缩等通路。Gene Ontology富集分析显示,差异表达的miRNA靶基因分子功能方面主要富集于蛋白结合和DNA结合;...     

酒精性肝病患者脂代谢相关差异蛋白的定量分析

目的应用串联质谱标记(TMT)技术筛选和鉴定酒精性肝病(ALD)患者肝脏组织中的差异蛋白, 对脂代谢相关蛋白和通路进行分析, 探索其功能及生物学过程。方法收集符合纳入标准的肝脏组织, 筛选出酒精性肝硬化患者样本8例, 正常对照组样本3例。采用TMT技术筛选差异蛋白并进行富集信号通路分析和蛋白质网络互作分析, 探索其参与的生物学过程。结果蛋白质组学分析鉴定出2组资料中有2 741种差异蛋白具有统计学意义(P < 0.05), 以P<0.05且|log2(foldchange)| >1的标准筛选出差异表达蛋白106种, 与对照组相比, ALD组上调蛋白12种, 下调蛋白94种。其中, 与脂代谢相关的上调差异蛋白有2种, 下调差异蛋白有14种。生物信息学分析结果显示这些蛋白在脂代谢中主要参与了脂质转运, 脂肪酶活性的调节, 脂肪酸结合以及胆固醇代谢等生物学过程, 并与过氧化物酶体增殖物激活受体信号通路、胆固醇代谢、甘油三酯代谢及脂肪细胞内脂解的调节等脂代谢相关信号通路密切相关。结论 16种脂代谢相关差异蛋白可能是ALD发病机制中的关键蛋白。     

Altered expression of metabolic pathways in CLL detected by unlabelled quantitative mass spectrometry analysis

Chronic lymphocytic leukaemia (CLL) remains the most common incurable malignancy of B cells in the western world. Patient outcomes are heterogeneous and can be difficult to predict with current prognostic markers. Here, we used a quantitative label-free proteomic technique to ascertain differences in the B-cell proteome from healthy donors and CLL patients with either mutated (M-CLL) or unmutated (UM-CLL) IGHV to identify new prognostic markers. In peripheral B-CLL cells, 349 (22%) proteins were differentially expressed between normal B cells and B-CLL cells and 189 (12%) were differentially expressed between M-CLL and UM-CLL. We also examined the proteome of proliferating CLL cells in the lymph nodes, and identified 76 (~8%) differentially expressed proteins between healthy and CLL lymph nodes. B-CLL cells show over-expression of proteins involved in lipid and cholesterol metabolism. A comprehensive lipidomic analysis highlighted large differences in glycolipids and sphingolipids. A shift was observed from the pro-apoptotic lipid ceramide towards the anti-apoptotic/chemoresistant lipid, glucosylceramide, which was more evident in patients with aggressive disease (UM-CLL). This study details a novel quantitative proteomic technique applied for the first time to primary patient samples in CLL and highlights that primary CLL lymphocytes display markers of a metabolic shift towards lipid synthesis and breakdown.     

Upregulation of miRNA-130a Represents Good Prognosis in Patients With HBV-Related Acute-on-Chronic Liver Failure: A Prospective Study

Prompt and accurate prediction of the outcome is the key to make correct medical decision and to reduce the mortality in patients with HBV-related acute-on-chronic liver failure (ACLF). Increasing evidence have certified that small, noncoding microRNAs (miRNAs) play critically regulatory roles in the pathogenesis of liver diseases. However, it remains unclear whether and how miRNAs involve in the prognosis of ACLF.Microarray analysis was performed to characterize the miRNA expression profiles in liver tissues from 1 HBV-related ACLF patient and 1 matched healthy control. Nine miRNAs with at least 5 folds difference between these 2 persons were picked out. The present prospective study involving 39 HBV-related ACLF patients including 20 recovered and 19 nonrecovered patients, which include death (n = 9) and liver transplantation (n = 10). The serum expression of these miRNAs detected by quantitative real-time Polymerase Chain Reaction (qRT-RCR) was then compared between the 2 groups. Moreover, the correlation between the serum miRNAs and the prognostic indexes for ACLF was analyzed.The result of microarray analysis showed 9 miRNAs had different expression in liver tissues of ACLF patient compared with healthy control (upregulated: miRNA-130a, −21, −143, and −200a; downregulated: miRNA-486–5p, −192, −148a, −122, and −194). Unlike the expression profiles in liver tissue, 8 serum miRNAs except miRNA-194 were markedly upregulated in ACLF patients (P < 0.05). Remarkably, the serum expression of miRNA-130a and miRNA-486–5p was higher in recovered than nonrecovered ACLF patients (P < 0.05). Especially, the serum miRNA-130a was negatively correlated with international normalized ratio, prothrombin time, Model for End-Stage Liver Disease score, and positively correlated with prothrombin time activity. The AUC for recovered versus nonrecovered patients of miRNA-130a was 0.741 (P = 0.02).miRNA-130a might be a useful prognosis biomarker in patients with HBV-related ACLF.     

四种评分模型对慢加急性乙型肝炎肝衰竭患者短期预后的评价

目的比较终末期肝病模型（MELD）、MELD-Na、慢性重型肝炎预后指数（PI）和肝移植标准（LTS）模型对慢加急性乙型肝炎肝衰竭患者短期预后的预测价值.方法在138例慢加急性乙型肝炎肝衰竭患者入院24小时内进行MELD、MELD-Na、PI和LTS评分,并随访3个月.应用受试者工作特征曲线（ROC）下面积（AUC）判断四个模型的预测能力.结果在观察期内与肝病有关的死亡患者72例,生存者66例.死亡组LTS、MELD-Na、MELD和PI平均值明显高于生存组（P〈0.01）,四个模型的AUC分别为0.860、0.801、0.749、和0.749,差异无统计学意义;四个模型预测的正确率分别为82.61%、76.81%、75.36%和73.91%,差异无统计学意义.结论4种模型对慢加急性乙型肝炎肝衰竭患者短期预后均有较好的预测价值.     

慢加急性肝衰竭与失代偿性肝硬化患者合并急性肾损伤的临床特点比较

目的比较慢加急性肝衰竭(ACLF)与失代偿性肝硬化(DC)患者急性肾损伤(AKI)的临床特点。方法回顾性收集ACLF和DC患者的人口学资料、临床检查结果、诊疗经过等信息。比较ACLF合并AKI与DC合并AKI的临床特点及其对90 d死亡风险的影响。结果比较ACLF-AKI和DC-AKI患者的临床特点,结果显示ACLF-AKI患者白细胞计数、中性粒细胞绝对值、丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、总胆红素(TBil)均高于DC-AKI患者,凝血酶原活动度(PTA)、白蛋白低于DC-AKI患者,差异有统计学意义(P<0.05);ACLF-AKI患者合并感染的比例显著高于DC-AKI组(96.9%对比39.5%)(P<0.05);在诊断AKI时,ACLF患者的血肌酐中位值为147μmol/L(IQR:122~189),而DC组患者的血肌酐中位值为123.5μmol/L(IQR:103.8~155.5),两组差异有统计学意义(P<0.05);按照肝硬化HRS-AKI诊断标准,在ACLF-AKI患者中44例(68.8%)符合HRS-AKI诊断,显著高于DC-AKI患者中HRS-AKI的比例[18例(47.4%)](P<0.05)。DC-AKI患者30 d内死亡或肝移植4例(10.5%)、90 d内死亡或肝移植8例(21.1%),而在ACLF-AKI患者中,22例患者(34.4%)30 d内死亡或肝移植、35例(54.7%)90 d内死亡或肝移植;显著高于DC-AKI患者,χ2值分别为7.140、11.062;P<0.05。多因素回归分析结果提示影响DC患者90 d死亡的独立危险因素有肝性脑病、消化道出血、TBil;而影响ACLF患者90 d死亡风险的独立危险因素包括AKI、PTA、TBil。结论与DC-AKI患者相比,ACLF-AKI患者中感染比例更高,诊断AKI时的血肌酐水平更高,病情进展更快,造成的死亡风险更大。     

Plasma exchange in patients with acute and acute-on-chronic liver failure: A systematic review

BACKGROUND Acute liver failure(ALF)and acute-on-chronic liver(ACLF)carry high short-term mortality rate,and may result from a wide variety of causes.Plasma exchange has been shown in a randomized control trial to improve survival in ALF especially in patients who did not receive a liver transplant.Other cohort studies demonstrated potential improvement in survival in patients with ACLF.AIM To assess utility of plasma exchange in liver failure and its effect on mortality in patients who do not undergo liver transplantation.METHODS Databases MEDLINE via PubMed,and EMBASE were searched and relevant publications up to 30 March,2019 were assessed.Studies were included if they involved human participants diagnosed with liver failure who underwent plasma exchange,with or without another alternative non-bioartificial liver assist device.RESULTS Three hundred twenty four records were reviewed,of which 62 studies were found to be duplicates.Of the 262 records screened,211 studies were excluded.Fifty-one articles were assessed for eligibility,for which 7 were excluded.Twenty-nine studies were included for ALF only,and 9 studies for ACLF only.Six studies included both ALF and ACLF patients.A total of 44 publications were included.Of the included publications,2 were randomized controlled trials,14 cohort studies,12 case series,16 case reports.All of three ALF studies which looked at survival rate or survival days reported improvement in outcome with plasma exchange.In two out of four studies where plasma exchange-based liver support systems were compared to standard medical treatment(SMT)for ACLF,a biochemical improvement was seen.Survival in the non-transplanted patients was improved in all four studies in patients with ACLF comparing plasma exchange vs SMT.Using the aforementioned studies,plasma exchange based therapy in ACLF compared to SMT improved survival in non-transplanted patients at 30 and 90-d with a pooled OR of 0.60(95%CI 0.46-0.77,P<0.01).CONCLUSION The level of evidence for use of high volume plasma exchange in selected ALF cases is high.Plasma exchange in ACLF improves survival at 30-and 90-d in nontransplanted patients.Further well-designed randomized control trials will need to be carried out to ascertain the optimal duration and amount of plasma exchange required and assess if the use of high volume plasma exchange can be extrapolated to patients with ACLF.     

Aberrant DNA-PKcs and ERGIC1 expression may be involved in initiation of gastric cancer

AIM to investigate the molecular mechanisms of gastric carcinogenesis.METHODS We used label-free quantification technology integrated with liquid chromatography-tandem mass spectrometry(Lc-m S/m S) analysis to identify differentially expressed proteins in 160 specimens of normal gastric mucosa,gastric mucosa with mild dysplasia,moderate dysplasia,severe dysplasia,and early mucosal gastric cancer(Gc) collected at the Second Hospital of Lanzhou University from 2010 to 2015. Immunohistochemistry was used to verify the differentially expressed proteins detected by Lc-m S/m S.RESULTS With a threshold of a 1.2-fold change and a P-value 0.05 between mild dysplasia,moderate dysplasia,severe dysplasia or early mucosal Gc and matched normal gastric mucosa tissues,proteomic analysis identified 365 significantly differentially expressed proteins. Er GIc1 expression decreased,while DNAPKcs expression increased gradually along with different stages of Gc initiation based on the tendency of fold change. the expression patterns of Er GIc1 and DNA-PKcs revealed by immunohistochemistry were consistent with the Lc-m S/m S results.CONCLUSION the results suggest that aberrant Er GIc1 and DNAPKcs expression may be involved in Gc initiation.     

Bacterial infection triggers and complicates acute-on-chronic liver failure in patients with hepatitis B virus-decompensated cirrhosis: A retrospective cohort study

BACKGROUND Reports on bacterial infection(BI)in decompensated cirrhosis(DC)is mainly from alcoholic cirrhosis.The role of BI as a trigger or complication of acute-onchronic liver failure(ACLF)in patients with hepatitis B virus decompensated cirrhosis(HBV-DC)remains to be investigated.AIM To investigate the impact of BI on the outcomes of the patients with HBV-DC admitted into the hospital with or without ACLF.METHODS This retrospective study included patients with HBV-DC admitted to two tertiary centers in China.In-hospital overall survival,90-d transplant-free survival,5-year post-discharge survival,and cumulative incidence of ACLF were evaluated.Risk factors for death were analyzed considering liver transplantation as a competing event.RESULTS A total of 1281 hospitalized HBV-DC patients were included;284 had ACLF at admission.The overall prevalence of BI was 28.1%.The patients with BI had a significantly lower in-hospital survival and transplant-free 90-d survival than those without,in both the patients admitted with and without ACLF.The presence of BI significantly increased the risk of developing ACLF[subdistribution hazard ratio(sHR)=2.52,95%CI:1.75-3.61,P<0.001]in the patients without ACLF.In the patients discharged alive,those who had an episode of BI had a significantly lower 5-year transplant-free survival.BI was an independent risk factor for death in the patients admitted without ACLF(sHR=3.28,95%CI:1.93-5.57),while in ACLF admissions,the presence of pneumonia,but not other type of BI,independently increased the risk of death(sHR=1.87,95%CI:1.24-2.82).CONCLUSION BI triggers ACLF in patients with HBV-DC and significantly impairs short-term survival.HBV-DC patients should be monitored carefully for the development of BI,especially pneumonia,to avoid an adverse outcome.