早期与进展期胃癌蛋白组学差异研究

发布时间：2018-05-04 15:20

本文选题：胃癌 + 肿瘤标记物　；参考：《宁夏医科大学》2015年硕士论文

【摘要】：目的胃癌是目前全球最常见的恶性消化道肿瘤之一,筛查敏感性、特异性高的胃癌肿瘤标志物,能够达到早期发现、早期诊断、早期治疗胃癌的目的,进而明显改善患者的预后和提高其生存率。目前广泛用于临床的肿瘤标志物CEA、CA199、CA724等诊断胃癌的灵敏度和特异性均不理想,而近些年发现在胃癌中相对特异的标志物MG7-抗原、胃蛋白酶原在我国胃癌筛查中尚无大样本的证据支持。因此,本课题拟通过利用同位素标记相对和绝对定量技术(i TRAQ)联合液相色谱串联质谱(LC-MS/MS)蛋白组学技术筛查早期、进展期胃癌和健康人群血清中差异表达的蛋白,并经生物信息学分析后筛选出与胃癌发生、发展相关的特异蛋白,最后用Western blot和免疫组化方法共同验证,以期发现与胃癌分期及预后有关的蛋白标志物。方法用iTRAQ定量蛋白质组学方法分析具有明显差异的早期与进展期胃癌患者血清各3例以及2例正常体检者血清,登陆GO及KEGG数据库对鉴定蛋白质进行生物信息学分析。通过蛋白差异表达情况、生物信息学分析并结合文献查阅,选定与胃癌发生发展、信号转导通路、侵袭转移等相关的差异表达蛋白。然后从早期、进展期胃癌与正常对照各30例血清样本中各组随机抽取9例血清,利用Western blot技术对其中的2个差异蛋白进行验证。另收集与早期、进展期胃癌血清对应的术后石蜡包埋的胃癌组织及癌旁正常组织各30例,应用免疫组化方法对2个差异蛋白进行验证。最后,应用SPSS16.0统计软件分别采用t检验和Kaplan-Meier生存分析法,分析2个差异蛋白的表达与胃癌患者复发转移的关系及其对预后的影响。结果 iTRAQ定量蛋白质组学方法共鉴定得到119个差异蛋白质(53个上调,66个下调)。登陆GO数据库搜索发现,差异蛋白主要定位于细胞内、细胞浆、细胞器及细胞膜等处。其功能与结合、催化、酶调节等相关。参与的生理和病理生理过程有信号传导通路、代谢、单细胞生物过程、抗氧化、调节通道等。登陆KEGG pathway数据库搜索,它们主要参与MAPK信号通路、P53信号通路等。根据筛选条件共选出7个关键性差异表达蛋白:IGFBP-3、ARF-1、HRG、PON3、FN1、HGF和CETP,并对其中IGFBP-3与ARF-1蛋白进行相关验证。Western blot结果显示IGFBP-3蛋白在进展期胃癌血清中的表达低于早期胃癌与正常对照血清中的表达,ARF-1蛋白在进展期胃癌血清中的表达则高于早期胃癌与正常对照血清中的表达,差异均具有统计学意义(P值均0.05),两个差异蛋白在早期血清与正常对照血清中的表达,经比较差异无统计学意义(P0.05)。免疫组化结果显示IGFBP-3蛋白与ARF-1蛋白阳性表达均定位于细胞浆及部分细胞核。IGFBP-3在组织中的表达水平与Western blot结果一致,ARF-1在进展期胃癌组织中的表达与早期胃癌、正常对照组织中的表达比较结果与Western blot结果一致,且ARF-1在早期组表达水平也高于正常组表达水平,差异具有统计学意义(P值0.05)。IGFBP-3与胃癌的复发转移相关,ARF-1与胃癌的临床分期、复发转移均相关,差异均具有统计学意义(P值均0.05)。生存分析显示:IGFBP-3阴性表达患者的平均生存时间明显小于阳性表达患者的平均生存时间,而ARF-1阴性表达患者的平均生存时间明显大于阳性表达患者的平均生存时间,差异均具有统计学意义(P值均0.05)。结论本实验应用定量蛋白质组学i TRAQ技术研究初步筛选了早期胃癌、进展期胃癌与正常人血清中的差异蛋白,得到了胃癌血清差异表达蛋白质谱,共发现了119个差异蛋白。重点筛选出了7个关键性差异蛋白:IGFBP-3、ARF-1、HRG、PON3、FN1、HGF和CETP。对其中IGFBP-3与ARF-1两个差异蛋白进行相关验证,发现IGFBP-3蛋白在进展期胃癌患者的组织和血清中均表达下调,且与胃癌的复发转移和预后相关;ARF-1蛋白在胃癌患者的组织和血清中均表达上调,且与胃癌的临床分期、复发转移和预后相关。ARF-1可能是潜在的价值较大的胃癌肿瘤标志物,IGFBP-3虽为胃癌肿瘤标志物的研究提供了新的线索,但其价值尚需进一步扩大样本量研究证实。
[Abstract]:Objective gastric cancer is one of the most common malignant digestive tract tumors in the world at present. Screening sensitive and specific tumor markers for gastric cancer can achieve early detection, early diagnosis, early treatment of gastric cancer, and then obviously improve the prognosis and improve the survival rate of the patients. It is widely used in clinical tumor markers CEA, CA199, CA724. The sensitivity and specificity of the diagnosis of gastric cancer are not ideal, but in recent years, it is found that the relative specific marker MG7- antigen in gastric cancer is not supported by large samples in the screening of gastric cancer in our country. Therefore, we should use the relative and absolute quantitative technique of isotope labeling (I TRAQ) combined with liquid chromatography tandem mass spectrometry. LC-MS/MS) proteomics was used to screen differentially expressed proteins in the sera of early, progressive gastric cancer and healthy people. After bioinformatics analysis, specific proteins related to the development of gastric cancer were screened. Finally, Western blot and immunohistochemical method were used to verify the protein markers in order to detect the protein markers related to the stage and prognosis of gastric cancer. Methods the iTRAQ quantitative proteomics method was used to analyze the serum of 3 patients with early and progressive gastric cancer in early and progressive gastric cancer and 2 cases of normal physical examination. Bioinformatics analysis was carried out on the identification of protein by GO and KEGG database. The differential expression proteins related to the occurrence and development of gastric cancer, signal transduction pathway, invasion and metastasis, and then 9 serum samples were randomly selected from 30 serum samples of early, progressive gastric cancer and normal control, and 2 of them were verified by Western blot technique. 30 cases of paraffin embedded gastric carcinoma and 30 normal tissues adjacent to cancer were used to verify 2 differential proteins by immunohistochemical method. Finally, t test and Kaplan-Meier survival analysis were used to analyze the relationship between the expression of 2 differential proteins and the recurrence and metastasis of gastric cancer patients and the effect on prognosis. Results iTRA A total of 119 different proteins (53 up-regulated and 66 down-regulated) were identified by the Q quantitative proteomics method. The GO database search found that the differential proteins were located mainly in cells, cytoplasm, organelles and cell membranes. Their functions were related to binding, catalysis, enzyme regulation and so on. The physiological and pathophysiological processes involved were signaling pathways. Metabolism, single cell biological processes, antioxidation, regulation channels, etc.. KEGG pathway database search, they are mainly involved in the MAPK signaling pathway, P53 signaling pathway and so on. According to the screening conditions, 7 key differentially expressed proteins are selected: IGFBP-3, ARF-1, HRG, PON3, FN1, HGF and CETP. The blot results showed that the expression of IGFBP-3 protein in the serum of advanced gastric cancer was lower than that of early gastric cancer and normal control sera, and the expression of ARF-1 protein in the serum of advanced gastric cancer was higher than that in the early gastric cancer and normal control sera, and the difference was statistically significant (the value of P was 0.05), and the two differential proteins were in the early sera. The expression of serum in the normal control serum was not statistically significant (P0.05). The immunohistochemical results showed that the expression of IGFBP-3 protein and ARF-1 protein expressed in the cytoplasm and part of the nucleus.IGFBP-3 in the tissue was consistent with the Western blot results. The expression of ARF-1 in the advanced gastric cancer tissues and the early gastric cancer, The results of expression comparison in normal control tissues were consistent with the results of Western blot, and the expression level of ARF-1 in early group was also higher than that of normal group. The difference was statistically significant (P 0.05) and.IGFBP-3 was related to the recurrence and metastasis of gastric cancer. ARF-1 was correlated with the clinical stage and relapse of gastric cancer, and the difference was statistically significant (P values were all). 0.05). The survival analysis showed that the average survival time of IGFBP-3 negative expression patients was significantly less than the mean survival time of positive expression patients, while the average survival time of ARF-1 negative expression patients was significantly greater than that of positive expression patients, and the difference was statistically significant (P value was 0.05). Conclusion this experiment applied quantitative eggs. The white matter group study I TRAQ technology preliminarily screened the differential proteins in the serum of the early gastric cancer, the advanced gastric cancer and the normal human, and obtained the differential expression protein mass spectrum of the gastric cancer serum, and found 119 different proteins. The emphasis was on screening 7 key differential proteins: IGFBP-3, ARF-1, HRG, PON3, FN1, HGF and CETP., of which IGFBP-3 and ARF-1 were two. The differential protein was proved to be down regulated in the tissues and serum of the patients with advanced gastric cancer, and it was associated with the recurrence and metastasis of gastric cancer and the prognosis of the gastric cancer. The expression of ARF-1 protein in the tissues and serum of gastric cancer patients was up-regulated, and.ARF-1 may be the potential price associated with the clinical stage of gastric cancer, the recurrence and metastasis and prognosis of.ARF-1. Although the value of the tumor marker, IGFBP-3 provides a new clue for the research of tumor markers for gastric cancer, its value still needs to be further expanded to confirm the sample size.

【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.2

【参考文献】