巨大肝细胞癌根治术后nomogram预测模型的建立及复发相关蛋白质组学初步研究

发布时间：2018-07-28 06:34

【摘要】：第一部分巨大肝细胞癌术后生存分析及nomogram预测模型的建立目的:手术仍是目前肝癌最有效的治疗手段,在肝细胞癌亚型中,我们选择预后差、复发率高的巨大肝癌病人(直径≥10 cm)进行临床病理资料分析,建立术前和术后nomogram预测模型对病人的生存和复发进行分析。方法:回顾性分析2008年1月到2009年12月在东方肝胆外科医院行肝切除术的464例巨大肝癌病人以及2008年1月到2010年4月的在福建医科大学附属第一医院行肝切除术的90例巨大肝癌病人的临床病理资料。观察术后围手术期并发症、死亡率、复发间隔时间和总生存期。将东方肝胆外科医院病人随机分为建模组和内部验证组,全部的福建医科大学附属第一医院病人为外部验证组,Cox回归分析对术前和术后资料分别进行单因素和多因素分析,将多因素有意义(P0.05)的变量纳入最终的术前和术后nomogram预测模型中。通过C-index、校准曲线以及验证组验证该模型的预测性。结果:东方肝胆外科医院病人的术后并发症发病率为18.6%(n=95);术后1、3、5年累计复发率为43.8%,74.1%和82.1%;生存率为75.1%,49%和33.3%。福建医科大学附属第一医院病人的术后并发症发病率为30.5%(n=32);术后1、3、5年累计复发率为38.3%,69.2%和84.1%;生存率为77.7%,44.9%和21.5%。基于术前和术后资料预测生存的nomograms预测生存的C-index分别为0.75(95%CI,0.72-0.78)和0.78(0.75-0.81)。术后3年、5年生存的校准曲线nomograms预测的生存情况和实际生存情况有较高的吻合度。通过nomograms预测模型,将病人进行分层,将术后病人有效的分为生存率逐渐增加的四等分(P0.01)。术前临床病理资料多因素分析表明:AFP200ug/L、高HBV-DNA载量、多发肿瘤、较大的肿瘤直径,以及合并门脉癌栓为生存的独立危险因素;AFP200ug/L、高HBV-DNA载量、多发肿瘤、较大的肿瘤直径以及合并门脉癌栓为复发的独立危险因素。术后临床病理资料多因素分析表明:高HBV-DNA载量、多发肿瘤、较大的肿瘤直径、有血管侵犯、肿瘤包膜不完整、较差的Edmonson-Stenier分级为生存的独立危险因素;高HBV-DNA载量、多发肿瘤、较大的肿瘤直径、有血管侵犯、肿瘤包膜不完整为复发的独立危险因素。结论:术前和术后nomogram预测模型对预测巨大肝癌术后病人的长期生存和复发情况有良好的准确性。第二部分巨大肝细胞癌根治术后复发相关的差异蛋白质组学研究目的通过同位素标记的定量蛋白质组学技术(i TRAQ)鉴定与筛选巨大肝细胞癌根治术后复发相关的差异表达蛋白。方法选取巨大肝细胞癌手术切除术后复发病人的初次手术新鲜肝癌组织标本,根据术后复发时间分组。应用i TRAQ试剂标记的二维液相色谱-串联质谱(2DLC-MS/MS)联用技术进行分析得到差异表达蛋白,并进一步行生物信息学分析。结果6个月和2年复发组比较之间存在显著表达差异(大于1.5倍或小于0.67倍)的蛋白质分子共计409种;6-12个月和2年复发组比较之间存在显著表达差异的蛋白质分子共计518种。在本研究中,我们进一步筛选出随复发时间不同而变化较大的差异蛋白质10种,上调的有S100A9、MPO、APOA4、TMEM97、NR5A2,下调的有DHRS2、TTC4、CHD2、HPD、FTCD。GO分析显示,差异表达蛋白相关基因多参与类固醇代谢,氧化还原以及蛋白代谢。IPA分析显示,差异表达蛋白主要参与分别由NR5A2、IL1、ERK1/2、NFk B和IFNα调控的转录调控网络。结论iTRAQ标记的定量蛋白质组学技术从整体上了解肝细胞癌术后复发过程中蛋白质的表达差异,为筛选出有意义的生物标记物提供一个良好的平台。生物信息学的研究方法进一步了解差异表达蛋白的功能和以及其调控网络。第三部分巨大肝细胞癌根治术后复发的的差异蛋白表达验证目的从m RNA和蛋白水平对筛选的蛋白进行验证,筛选与肝细胞癌术后复发相关的差异蛋白。方法选取S100A9、DHRS2、TMEM97和NR5A2等四种蛋白进一步验证。不同复发时间巨大肝癌病人的新鲜肝癌组织标本进行荧光定量PCR(Q-PCR)、免疫印迹(Western blot)验证。不同复发时间巨大肝癌病人的肝癌石蜡切片标本行免疫组化验证。结果Q-PCR和Western blot验证结果显示,TMEM97高表达为术后复发的危险因素。免疫组化验证结果显示,TMEM97高表达(P=0.005,HR1.534)、术前高血清AFP(0.003,1.595)、肿瘤多发(0.000,2.422)、肿瘤直径较大(0.018,1.042)、有肉眼癌栓(0.002,1.776)、有MVI(0.019,1.504)、包膜不完整(0.004,0.595)、术中输血(0.028,1.590)为术后复发的独立危险因素。结论TMEM97在蛋白和m RNA水平的表达与蛋白组学结果一致,TMEM97可能作为肝细胞癌根治术后复发相关的预警蛋白。
[Abstract]:Part 1 Survival Analysis and nomogram prediction model for giant hepatocellular carcinoma (HCC) objective: surgery is still the most effective treatment for liver cancer. In the subtype of hepatocellular carcinoma, we choose a large liver cancer patient with a poor prognosis and a high recurrence rate (diameter more than 10 cm) to analyze the pathological data of the clinicopathology, and establish the preoperation and postoperative nomogram prediction The model was used to analyze the survival and recurrence of the patients. Methods: a retrospective analysis of the clinicopathological data of 464 huge hepatocellular carcinoma patients who underwent hepatectomy in the Eastern Department of hepatobiliary surgery from January 2008 to December 2009 and 90 cases of hepatectomy in the First Affiliated Hospital of Fujian Medical University from January 2008 to April 2010. The perioperative complications, mortality, recurrence interval and total survival time were observed. The patients in the Eastern Department of hepatobiliary surgery were randomly divided into the modeling group and the internal verification group. All the patients in the First Affiliated Hospital of Fujian Medical University were the external verification group. The Cox regression analysis was used to analyze the single factor and multi factor analysis of the preoperative and postoperative data respectively. The multivariate and meaningful (P0.05) variables were included in the final preoperation and postoperatively nomogram prediction models. The predictability of the model was verified by C-index, calibration curve and verification group. Results: the incidence of postoperative complications was 18.6% (n=95) in the Oriental Department of Hepatobiliary Surgery; the cumulative recurrence rate in 1,3,5 years after operation was 43.8%, 74.1% and 82.1%; survival was 74.1% and 82.1%. The incidence of postoperative complications of 75.1%, 49%, and 33.3%. Affiliated First hospitals of Fujian Medical University was 30.5% (n=32); the cumulative recurrence rate was 38.3%, 69.2% and 84.1% in 1,3,5 years after operation; the survival rate was 77.7%, 44.9% and 21.5%. predicted survival based on preoperative and postoperative data to predict survival C-index for 0.75 (95%CI, 0.72-0.78) and 0, respectively. .78 (0.75-0.81). 3 years after the operation, the 5 year survival calibration curve nomograms predicted survival and the actual survival of a higher degree of anastomosis. Through the nomograms prediction model, the patients were stratified, and the postoperative patients were effectively divided into four equal fraction (P0.01) with increasing survival rate (P0.01). Preoperative clinicopathological data analysis showed that: AFP200ug /L, high HBV-DNA load, multiple tumor, larger tumor diameter, and an independent risk factor for survival with portal vein tumor thrombus; AFP200ug/L, high HBV-DNA load, multiple tumor, larger tumor diameter and combined portal vein thrombus as independent risk factors for recurrence. Multivariate analysis of postoperative clinicopathological data showed high HBV-DNA load and multiple swelling. Tumor, large tumor diameter, vascular invasion, tumor capsule incomplete, poor Edmonson-Stenier classification as independent risk factors for survival; high HBV-DNA load, multiple tumor, larger tumor diameter, vascular invasion, and incomplete tumor capsule as an independent risk factor for recurrence. Conclusion: preoperation and postoperative nomogram prediction model is predicted. The long-term survival and recurrence of huge hepatocellular carcinoma patients have good accuracy. Second the difference of proteomics of the recurrence related after radical hepatocarcinoma radical resection for large hepatocellular carcinoma (I TRAQ) identification and screening of differential expression related to recurrence of giant hepatocellular carcinoma after radical resection Methods the primary hepatocarcinoma tissue specimens of the patients with recurrent hepatocellular carcinoma (HCC) after surgical excision were selected to be grouped according to the postoperative recurrence time. Two dimensional liquid chromatography tandem mass spectrometry (2DLC-MS/MS) combined with I TRAQ reagent was used to analyze the differential expression of protein, and further bioinformatics analysis was performed. There were 409 protein molecules with significant differences (more than 1.5 times or less than 0.67 times) in the 6 month and 2 year recurrence groups, and 518 protein molecules with significant differences in the 6-12 and 2 year recurrence groups. In this study, we further screened the differences that varied with the recurrence time. 10 different proteins, up regulated by S100A9, MPO, APOA4, TMEM97, NR5A2, and down regulated DHRS2, TTC4, CHD2, HPD, and FTCD.GO analysis showed that the differentially expressed protein related genes were involved in steroid metabolism, redox and protein metabolism.IPA analysis. Conclusion the iTRAQ tagged quantitative proteomics technology provides a good platform for the screening of significant biomarkers in the process of recurrence of hepatocellular carcinoma. The bioinformatics research method further understands the function of differentially expressed proteins and its regulatory network. The differential protein expression in third parts of the recurrence of giant hepatocellular carcinoma (HCC) after radical resection aims to verify the selected proteins from m RNA and protein levels and to screen the differential proteins associated with the recurrence of hepatocellular carcinoma. Methods such as four proteins, such as S100A9, DHRS2, TMEM97 and NR5A2, were further verified. Fluorescence quantitative PCR (Q-PCR) and Western blot (Western blot) were tested for human fresh liver cancer tissue specimens. The paraffin section specimens of hepatoma cancer patients with different recurrence time were verified by immunohistochemistry. Results the results of Q-PCR and Western blot showed that the high expression of TMEM97 was a risk factor for postoperative recurrence. The immunohistochemical verification results showed that TMEM97 high expression (P=0.005, HR1.534), preoperative high serum AFP (0.003,1.595), tumor multiple (0.000,2.422), tumor diameter (0.018,1.042), naked eye cancer thrombus (0.002,1.776), MVI (0.019,1.504), incomplete capsule (0.004,0.595), and intraoperative blood transfusion (0.028,1.590) as an independent risk factor for postoperative recurrence The expression is consistent with the proteomics results. TMEM97 may be used as a warning protein for recurrence after radical resection of hepatocellular carcinoma.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7

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