摘要目的探讨卵巢上皮性癌（卵巢癌）铂类耐药患者血清中耐药相关的差异表达蛋白质的筛选及其临床价值。方法收集1998年8月—2013年9月在广西医科大学附属肿瘤医院治疗的卵巢肿瘤患者106例，其中卵巢癌铂类耐药患者44例（铂类耐药组），卵巢癌铂类敏感患者52例（铂类敏感组），卵巢良性上皮性肿瘤患者10例（良性肿瘤组）；以2008年在本院进行健康体检的妇女33例为对照组。采集4组患者的血清标本。（1）每组各取10份血清标本，采用基于同位素标记的相对和绝对定量（iTRAQ）技术的蛋白质组学方法筛选不同组间[即卵巢癌组（包括铂类敏感组和铂类耐药组）与对照组、卵巢癌组与良性肿瘤组、铂类耐药组与铂类敏感组]的差异表达蛋白质，结合生物信息学方法筛选出与卵巢癌铂类耐药相关的差异表达蛋白质。（2）收集铂类耐药组（44例）、铂类敏感组（52例）、对照组（33例）3组患者的血清，采用蛋白印迹（WB）法及ELISA法对筛选出的差异表达蛋白质的表达进行验证。（3）采用Pearson相关分析法，分析差异表达蛋白质的表达与铂类耐药卵巢癌患者临床病理指标的相关性；采用Kaplan-Meier法，分析差异表达蛋白质的表达及不同临床病理指标对铂类耐药卵巢癌患者预后的影响；采用受试者工作特征（ROC）曲线分析法，评价差异表达蛋白质对铂类耐药卵巢癌的诊断效能。结果（1）基于iTRAQ技术的蛋白质组学方法结合生物信息学方法分析显示，卵巢癌组与对照组间筛选出56个差异表达蛋白质，卵巢癌组与良性肿瘤组间筛选出39个差异表达蛋白质，铂类耐药组与铂类敏感组间筛选出62个差异表达蛋白质。通过检索Haplotter数据库对差异表达蛋白质进行正选择分析显示，C6、CNTN1蛋白在亚洲人群中以及BCHE蛋白在欧洲人群中均存在正选择作用（P值分别为0.031、0.010、0.040）。基于COREMINE数据库的文献挖掘及TCGA数据库基因芯片数据进行交集分析，进一步印证CRP、FN1、S100A9、TF、ALB、VWF、APOC2、APOE、CD44、F2、GPX3、ACTB蛋白与卵巢癌铂类耐药相关。（2）铂类耐药组、铂类敏感组、对照组患者血清中，SERPINA1蛋白的表达水平，WB法检测分别为41.7±9.2、32.8±6.6、14.2±3.6，ELISA法检测分别为（816±246）、（686±205）、（756 ±244）μg/μl；ORM1蛋白的表达水平，WB法检测分别为37.9±7.0、27.0 ±22.5、21.7 ±2.6， ELISA法检测分别为（221±35）、（174±23）、（157±18）μg/μl；FN1蛋白的表达水平，WB法检测分别为30.3±11.4、18.2±5.2、23.7±3.9，ELISA法检测分别为（71±13）、（62±13）、（69±13）ng/μl；GPX3蛋白的表达水平，WB法检测分别为1.2±0.3、2.2±0.3、1.6±0.3。使用WB法及ELISA法验证的各蛋白的表达趋势与iTRAQ技术检测的表达趋势一致。（3）Pearson相关分析显示，SERPINA1、FN1蛋白的表达与卵巢癌铂类耐药患者的复发、生存状态呈明显正相关（P<0.01，P<0.05），而与卵巢癌铂类耐药患者的无进展生存时间呈明显负相关（P<0.05）；ORM1蛋白的表达与卵巢癌铂类耐药患者的复发呈明显正相关（P<0.01）。Kaplan-Meier法分析显示，手术病理分期、初次治疗结局以及SERPINA1、FN1、ORM1蛋白的表达与卵巢癌铂类耐药患者的无进展生存时间明显有关（P<0.05）；初次治疗结局与卵巢癌铂类耐药患者的总生存时间明显有关（P=0.027）。ROC曲线分析显示，FN1、ORM1、SERPINA1蛋白的ROC曲线下面积（AUC）分别为0.679、0.910、0.666，ORM1蛋白对卵巢癌铂类耐药患者的诊断效能明显高于FN1、SERPINA1蛋白（P=0.000）。结论血清中筛选出的差异表达蛋白质FN1、SERPINA1及ORM1蛋白的表达水平可能对卵巢癌铂类耐药有预警及诊断的作用。ORM1蛋白诊断铂类耐药卵巢癌可获得较高的诊断效能，有望成为临床上筛选及诊断铂类耐药卵巢癌的潜在的血清学肿瘤标志物。

abstractsObjective To identified differentially expressed proteins associated with platinum resistance in platinum resistance epithelial oarian cancer (EOC) patients in serum and investigate their clinical value. Methods A total of 106 patients withoverian tumor in affiliated tumor hospital of Guangxi Medical University from August 1998 to September 2013 were enrolled in this study, which include 52 cases od platinum-sensitive (PTS), 44 cases of platinum-resistant (PTR) and 10 cases of benign ovarian cyst (BOC). Thirty-three cases of normal women proceeded physical examination in our hospital in 2008 were chosen as control group (NC). Four groups of patients serum samples of 4 groups were collected and preserved.(1) Differentially express level of serum proteins of 10 cases of every group (PTS&PTR vs NC, PTS&PTR vs BOC, PTS vs PTR) were identified with isobaric tags for relative and absolute quantitative (iTRAQ) based quantitative proteomic approach and then was subjected to bioinformatics analysis.(2)Proteins that played a important role in multidrug resistance were validated by western blot (WB) and ELISA in 44 PTR patients, 52 PRS patients and 33 NC women.(3)Pearson correlation analysis was used to explain the relationship between proteins and clinical pathological parameters of PTR individuals. Kaplan-Meier method was supposed to explore serum biomarkers associated with clinical prognosis data. Receiver operating characteristic (ROC) curves were used to determine the diagnostic value of the markers. Results (1) Based on the result of bioinformatics analysis, 56 proteins, 39 proteins and 62 proteins were identified respectively among PTS & PTR vs NC, PTS & PTR vs BOC, PTS vs PTR. It showed that C6 and CNTN1 have a positive seletion effect among Asians and BCHE among Europeans through searching Haplotter database. CRP, FN1, S100A9, TF, ALB, VWF, APOC2, APOE, CD44, F2, GPX3 and ACTB proein were further verified related with platinum resistance by taking intersection analysis in the COREMINE database and TCGA.(2)The expression level of SERPINA1 protein in serum of PTR group, PTS group and NC groupwere 41.7±9.2, 32.8±6.6 and 14.2±3.6 respectively using WB assay, and(816±246),(686±205)and (756 ± 244)μg/μl respectively using ELISA; the expression level of ORM1 protein in PTR, PTS and NC serum were 37.9±7.0, 27.0±22.5 and 21.7±2.6 respectively using WB assay, and(221±35),(174±23)and (157±18)μg/μl respectively using ELISA;the expression level of FN1 protein in PTR, PTS and NC serum were 30.3 ± 11.4, 18.2 ± 5.2, 23.7 ± 3.9 respectively by WB assay, and(71 ± 13),(62 ± 13),(69 ± 13)ng/μl respectively by ELISA;the expression level of GPX3 protein in PTR, PTS and NC serum were 1.2±0.3, 2.2± 0.3, 1.6±0.3 respectively WB assay. The expression of each protein by using western blot method and ELISA method had the same trend as that using iTRAQ technology.(3)Pearson correlation analysis showed, the expression of SERPINA1, FN1 and ORM1 had a positive correlation with recurrence and death of PTR patients (P<0.01, P<0.05), but was negatively correlated with progress free survival of PTR patients (P<0.05). Kaplan-Meier analysis indicated that clinical stage, initial treatment outcomes, the express level of SERPINA1, FN1 and ORM1 were significantly related with progression-free survival (P<0.05), the initial treatment outcomes was related with overall survival (P=0.027). The overall predictive accuracy of each protein was reflected by the area under the ROC curve (AUC), FN1 ORM1 and SERPINA with ROC areas of 0.679, 0.910 and 0.666 respectively. The diagnosis value of ORM1 protein in ovarian cancer patients with platinum resistance performance is significantly higher than that of FN1 and SERPINA1 protein (P=0.000) Conclusions The differentially express level of FN1, SERPINA1 and ORM1 between PTS and PTR play a essential role in measuring subtle changes in response to platinum-based chemotherapy and may be involved in biological processes of platinum resistance. ORM1 has higher diagnostic efficiency of platinum resistance in ovarian cancer patients. It may be a promising candidate biomarker for screening and diagnosis of ovarian cancer patients with platinum resistance.