激光捕获显微切割纯化的人正常支气管上皮与肺鳞癌组织的定量蛋白质组学研究

目的:建立肺鳞癌(human lung squamous carcinoma,HLSC)组织与正常支气管上皮(normal human bronchial epithelial,NHBE)组织的差异蛋白质表达谱.方法:运用激光捕获显微切割(laser capture microdissection,LCM)技术从HLS...     

miRNA-451 regulates rhesus choroid-retinal endothelial cell function and proteome profile

AIM: To evaluate the effect of miRNA-451 on rhesus macaque choroid-retinal endothelial (RF/6A) cell function and proteome profile. METHODS: The RF/6A cells were transfected with miRNA-451 mimic and inhibitor. The role of miRNA-451 on proliferation ability was evaluated by CCK-8 assay. Furthermore, iTRAQ quantitative proteomic analysis was applied to comprehensively illuminate the change of cellular proteins and biological function between different groups. RESULTS: In miRNA-451 overexpression group, cell proliferation of RF/6A decreased both at 24h and 48h; while in miRNA-451 inhibition group, on the contrary, RF/6A cell proliferation was increased at 48h. Based on iTRAQ quantitative proteomic analysis, 23 differentially expressed proteins (DEPs) were detected in the comparison of miRNA-451 mimic and mimic control-transfected RF/6A cells, and 30 DEPs were identified in the comparison of RF/6A cells transfected with miRNA-451 inhibitor and inhibitor control. DEPs such as GORASP2, KRT1, SLC7A2, RIC8A, DDX42, CAP1, PCBP2 might be closely related to the inhibitory effect of miRNA-451 on RF/6A cell proliferation, while PCYT1A, MGAT1, TUBB, MCU, SIL1, BID, MSH6 might account for the positive effect of miRNA-451 inhibitor on RF/6A cell growth. PTPN1, as the only protein exhibiting an opposite trend between miRNA-451 mimic and inhibitor-transfected cells, was most likely accountable for the inhibition of miRNA-451 mimic on RF/6A cell growth, and the promotion of miRNA-451 inhibitor on RF/6A cell proliferation. CONCLUSION: miRNA-451 overexpression can suppress the growth of RF/6A cells while knockdown of miRNA-451 can promote RF/6A cell viability. Among all DEPs, increased PTPN1 is most likely to account for the negative regulation of miRNA-451 on RF/6A proliferation. miRNA-451 can be a protective factor for neovascular disease of fundus via regulating choroid retinal endothelial cell function.     

基于iTRAQ的定量蛋白质组学技术探索 淡色库蚊越冬机制

目的　比较淡色库蚊越冬后与滞育准备阶段蛋白表达差异,探索淡色库蚊越冬休眠（滞育）的机制。方法　采用同位素标记相对和绝对定量（iTRAQ）技术,对越冬滞育及准备阶段淡色库蚊进行定量蛋白质组学分析。结果　淡色库蚊越冬滞育前后共鉴定出244种差异表达蛋白,其中上调蛋白126种、下调蛋白118种。生物信息学分析表明,这些差异表达蛋白与能量产生及转化、脂代谢、细胞骨架重塑、糖代谢、蛋白质转运、分子伴侣、应激耐受以及各种代谢酶等有关。结论　本研究首次采用现代蛋白质组学工具鉴定淡色库蚊越冬滞育相关蛋白,初步揭示了与淡色库蚊越冬滞育相关的KEGG通路及蛋白,进一步扩展了对蚊媒越冬滞育机制的理解。     

慢性自发性荨麻疹患者血清差异表达蛋白的筛选及C反应蛋白的测定

目的：探讨风团持续时间与慢性自发性荨麻疹（CSU）患者血清C反应蛋白（CRP）表达的相关性。方法：采用同位素标记相对与绝对定量(isobaric tag for relative absolute quantitation,iTRAQ)技术鉴定CSU患者血清差异蛋白,分析CRP的表达情况;通过免疫散射比浊法检测CSU患者CRP水平。结果：与正常人相比,CSU患者血清CRP表达上调（P<0.05）。风团持续时间长的CSU患者CRP表达水平高于风团持续时间短的CSU患者,差异有统计学意义（P<0.05）。结论：CSU患者血清CRP表达与风团持续时间相关。     

基于iTRAQ技术分析联合用药作用大鼠肝星状细胞后差异表达的蛋白

目的 筛选联合用药作用大鼠肝星状细胞(HSC-T6)后差异表达的蛋白质,以探讨联合用药抑制肝星状细胞增殖的机制。方法 以牛磺酸、表没食子儿茶素没食子酸酯(EGCG)和三羟基异黄酮三者组成的联合用药作用HSC-T6后,提取细胞总蛋白,运用同位素标记的iTRAQ技术结合高效液相色谱-电喷雾串联质谱(LC-ESI-MS/MS)的定量蛋白质组学方法,分析和鉴定差异表达的蛋白质,运用生物信息学分析差异表达的蛋白质功能。结果 质谱共分析鉴定到727种蛋白质。3次实验分别鉴定到的差异表达蛋白质数量分别为85,102,94个,在3次重复均鉴定到的差异表达蛋白为52个,其中上调蛋白24个,下调蛋白28个。生物信息学分析表明,差异表达的蛋白参与翻译后修饰、转录、重组和信号传导等过程,在这些蛋白质中既有与肝纤维化发生、发展直接相关的蛋白质如基质金属蛋白酶抑制剂1、层黏连蛋白、前纤维蛋白2等,也有在蛋白质的相互作用中处于功能网络交叉点的蛋白质如细胞周期素依赖性激酶4、肝癌衍生生长因子、谷氨酸脱氢酶 1和丝氨酸/精氨酸剪接因子9等。结论 联合用药抑制肝星状细胞增殖的作用,可能是通过调控多种蛋白质的表达来实现,所获得的差异表达蛋白可能在肝纤维化发生发展和药物抗肝纤维化作用过程中扮演重要角色。     

NAD(P)‐dependent steroid dehydrogenase‐like protein and neutral cholesterol ester hydrolase 1 serve as novel markers for early detection of gastric cancer identified using quantitative proteomics

BackgroundGastric cancer (GC) is the third most common cause of cancer deaths worldwide. In the present study, we aimed to identify novel GC biomarkers by integrating isobaric tags of relative and absolute quantitation (iTRAQ) for aberrantly expressed proteins in GC patients.MethodsUsing stable isotope tags, we labeled an initial discovery group comprising four paired gastric cancer and adjacent gastric tissue samples, and subjected them to LC‐ESI‐MS/MS. We used a validation set comprising 129 paired gastric cancer and adjacent gastric tissues from patients and benign healthy controls to validate the candidate targets.ResultsWe identified two proteins, NAD(P)‐dependent steroid dehydrogenase‐like (NSDHL) and neutral cholesterol ester hydrolase 1 (NCEH1), that were significantly overexpressed in GC tissues. The sensitivity and specificity of NSDHL were 80.6% and 74.4%, respectively, in GC compared with a sensitivity of 25.6% in adjacent tissues and 24% in benign healthy controls. The area under the ROC curve (AUC) for NSDHL was 0.810 for GC detection. Overexpression of NSDHL in GC was significantly correlated with local tumor invasion. The sensitivity and specificity of NCEH1 were 77.5% and 73.6%, respectively, in GC compared with a sensitivity of 26.4% in adjacent tissues and 20% in benign controls. The AUC for NSDHL was 0.792. Overexpression of NCEH1 was significantly associated with tumor histological classification and local invasion. Moreover, a combined analysis of NSDHL and NCEH1 achieved a sensitivity and specificity of 85.7% and 83%, respectively, and the AUC was 0.872. The combined analysis of NSDHL and NCEH1 was significantly correlated with histological grade and TNM Ⅱ‐Ⅳ staging.ConclusionsiTRAQ‐labeled quantitative proteomics represents a powerful method to identify novel cancer biomarkers. The present study identified NSDHL and NCEH1 as useful biomarkers for screening, diagnosis, and prognosis of patients with gastric cancer.     

Identification of differentially expressed proteins associated with recurrence in ovarian endometriotic cysts

ABSTRACT

The objective of this study was to identify proteins that are differentially expressed in the cystic wall tissues of ovarian endometriotic cysts, simple ovarian cysts, and in normal ovarian tissues. Specimens of ovarian endometriotic cyst wall tissue, simple ovarian cyst wall tissue, and normal ovarian tissue (six specimens per group) were collected from patients who received gynecologic surgery, respectively. Differentially expressed proteins related to the ovarian endometriotic cysts were screened by use of isobaric tags for relative and absolute quantitation (iTRAQ) combined with functional annotation and bioinformatics analyses. All differentially expressed proteins related to cysts were validated using immunohistochemistry methods in recurrent and non-recurrent ovarian endometriotic cyst. A total of 359 proteins were identified as up-regulated in ovarian endometriotic cyst groups when compared with both the normal ovary and simple ovarian cyst groups. The levels of 27 proteins were >two-fold higher in the ovarian endometriotic cyst group than that in the other two groups. Of note, the five most significantly upregulated proteins were Charcot-Leyden Crystal Galectin (CLC), Defensin, alpha 1 (DEFA1), S100 calcium-binding protein A9 (S100A9), S100 calcium-binding protein A8 (S100A8), and Ferritin Light Chain (FTL). Immunohistochemistry results showed that the changes of S100A9 and S100A8 were consistent with the results shown by iTRAQ. However, no similarity of CLC, DEFA1, and FTL proteins was found between iTRAQ and immunohistochemistry. The ratio of patients with abnormally high S100A9 and S100A8 expression in the recurrent ovarian endometriotic cyst group was significantly higher than that in the non-recurrent group (P < 0.05). Our data identify differentially expressed proteins S100A9 and S100A8, and suggest they may serve as novel molecular markers to predict postoperative recurrence of an ovarian endometriotic cysts.

Abbreviations: iTRAQ: isobaric tags for relative and absolute quantitation; HPRD: Human Protein Reference Database; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; EM: Endometriosis; COX-2: cyclooxyenase-2; NF-kB: nuclear factor kappa-B; PR-B: progesterone receptor type B.     

基于两种蛋白质组学技术筛选不同分期白癜风的差异表达蛋白

目的 采用两种不同的蛋白质组学技术鉴定和筛选白癜风不同分期的血清标志物，并研究它们之间的网络关系。 方法 收集白癜风稳定期和进展期以及健康人各15例血清样本，采用双向凝胶电泳技术（2-DE）和同位素标记相对和绝对定量技术（iTRAQ）两种蛋白质组学技术对所有样本进行检测并筛选出差异蛋白，并利用软件分析其可能作用的通路之间关系。结果 通过2-DE鉴定出稳定期白癜风患者共得11个差异蛋白，其中7个上调，4个下调；进展期白癜风患者共得20个差异蛋白，其中13个上调，15个下调。而iTRAQ鉴定出稳定期患者共得66个差异蛋白，其中30个上调和36个下调，进展期患者共得53个差异蛋白，其中32个上调和21个下调。两种蛋白组学鉴定出相同的差异蛋白包括稳定期的3个分别是α-胰蛋白酶抑制剂重链H4、补体C4-A和角蛋白,Ⅱ型细胞骨架,其表达上调；进展期鉴定出重合的3个差异蛋白分别为补体C4-B、载脂蛋白A下调和α-胰蛋白酶抑制剂重链H4上调，通过Go注释分析其差异蛋白作用通路主要为参与CCKR通路、纤溶酶原激活级联通路、p53通路、趋化炎症因子调控通路等。结论 基于2-DE与iTRAQ两种不同的蛋白质组学方法筛选出白癜风不同分期的差异表达蛋白,为进一步研究白癜风的发生机制奠定了基础。