食管鳞状细胞癌差异蛋白的筛选及其临床意义研究

发布时间：2018-07-04 07:09

本文选题：食管鳞状细胞癌 + 蛋白质组学　；参考：《新疆医科大学》2015年博士论文

【摘要】：目的:研究食管鳞状细胞癌差异蛋白的筛选及其临床意义。方法:(1)通过激光捕获显微切割技术分别从活检的新鲜肿瘤组织获取哈萨克族、汉族食管鳞癌细胞和癌旁远端食管正常上皮细胞的纯化细胞。(2)通过双向电泳联合基质辅助激光解吸电离飞行时间质谱检测获得表达差异蛋白。(3)使用蛋白质印迹和免疫组化技术对差异候选蛋白Bmi-1,PAI-1,Cofilin-1和Transgelin进行分析、验证。(4)对Bmi-1,PAI-1,Cofilin-1,Transgelin与食管鳞状细胞癌临床病理参数,如性别、年龄、民族、肿瘤大小、分化程度、病理分型、浸润深度、淋巴结转移、临床分期,及预后等方面的关联,初步探讨Bmi-1,PAI-1,Cofilin-1和Transgelin在食管癌发生发展中的作用。结果:(1)激光捕获显微切割技术捕获了较纯净的哈萨克族和汉族的食管鳞癌细胞及癌旁远端食管正常上皮细胞。(2)通过双向电泳联合基质辅助激光解吸电离飞行时间质谱鉴定出43个有意义的差异蛋白,其中在哈萨克族和汉族的食管鳞癌组织表达均上调的点有25个。在哈萨克族和汉族的食管鳞癌组织均下调的蛋白点有14个。还有4个蛋白在哈萨克族和汉族食管鳞癌组织中表达不一致。(3)在哈萨克族和汉族的食管鳞癌组织表达均上调的蛋白中,根据蛋白得分值,匹配肽段覆盖率,结合研究报道较少等因素,从中筛选出四个蛋白质Bmi-1,PAI-1,Cofilin-1和Transgelin。Bmi-1,PAI-1,Cofilin-1和Transgelin在哈萨克族、汉族食管鳞癌组织中的表达经过蛋白质印迹和免疫组化验证,表达明显高于食管正常组织。免疫组化结果:Bmi-1,PAI-1,Cofilin-1和Transgelin在食管鳞癌组织中表达的阳性率为78.75%(P0.05)、76.25%(P0.05)、60.00%(P0.05)、55.00%(P0.05),高于在正常食管组织的表达。(4)分析Bmi-1,PAI-1,Cofilin-1和Transgelin在食管鳞癌与性别、年龄、民族、肿瘤大小、分化程度、病理分型、浸润深度、淋巴结转移、临床分期的表达情况。Bmi-1的表达:在有淋巴结转移和无淋巴结转移食管鳞癌患者组织中,Bmi-1的阳性表达率分别为63.0%和86.8%,P0.05,两组之间差异有统计学意义。在不同浸润深度食管鳞癌患者组织中Bmi-1的阳性表达率,肿瘤细胞侵润食管粘膜或肌层为92.3%,浸润食管纤维膜及周围结构为72.2%,P0.05,差异有统计学意义,结果表明Bmi-1的表达可能与食管癌组织的浸润程度有关。PAI-1的表达:在食管鳞癌患者中,在有淋巴结转移和无淋巴结转移食管鳞癌患者组织中,PAI-1的阳性表达率分别为59.3%和84.9%,P0.05,两组之间差异有统计学意义,表明PAI-1的表达可能与食管癌组织的淋巴结转移有关。肿瘤细胞浸润食管粘膜或肌层为53.8%,肿瘤浸润食管纤维膜及周围结构阳性表达率87.0%,P0.05,差异有统计学意义,结果表明PAI-1的表达可能与食管癌组织的浸润迁移有关。Cofilin-1的表达:食管鳞癌患者中,在高分化、中分化和低分化食管鳞癌患者组织中Cofilin-1的阳性表达率分别为26.1%、78.3%和70.6%,P0.05,差异有统计学意义,表明Cofilin-1的表达可能与食管癌组织的分化程度有关,提示表达率高恶性程度高,与预后相关。在有淋巴结转移和无淋巴结转移食管鳞癌患者组织中,Cofilin-1的阳性表达率分别为88.9%和45.3%,P0.05,两组之间差异有统计学意义,表明Cofilin-1的表达与食管癌组织的淋巴结转移有关。在临床分期Ⅰ~Ⅱ期和Ⅲ~Ⅳ期食管鳞癌患者组织中Cofilin-1的阳性表达率分别为37.1%和77.8%,P0.05,两组之间差异有统计学意义,表明Cofilin-1的表达可能与食管癌的临床分期有关。Transgelin的表达:在高分化、中分化和低分化食管鳞癌患者组织中Transgelin的阳性表达率分别为34.8%、52.2%和70.6%,P0.05,差异有统计学意义,表明Transgelin的表达可能与食管癌组织的分化程度有关,提示表达率高恶性程度高,与预后相关。在有淋巴结转移和无淋巴结转移食管鳞癌患者组织中,Transgelin的阳性表达率分别为88.9%和37.7%,P0.05,两组之间差异有统计学意义,表明Transgelin的表达与食管癌组织的淋巴结转移有关。结论:(1)本研究提示激光捕获显微切割技术可以有效地解决组织异质性的问题,能较好地获取较为纯净的目的细胞。(2)食管鳞癌和正常食管上皮细胞的差异蛋白质图谱提示食管鳞癌的发生发展是多基因参与、多种蛋白质功能共同协同发挥作用的结果。(3)蛋白质印迹技术和免疫组化分析Bmi-1,PAI-1,Cofilin-1,Transgelin的验证结果与蛋白质组学技术的分析鉴定结果相一致。(4)Bmi-l、PAI-1、Cofilin-1和Transgelin这四个蛋白在食管鳞癌组织中表达上调,与食管鳞癌的分化程度、侵润、淋巴转移有关,在食管鳞状细胞癌的发生和发展中可能起一定作用,并且对食管鳞状细胞癌恶性程度及其预后有指导意义,为食管鳞癌分子靶向治疗和新的抗癌药物开发提供有价值的理论依据。
[Abstract]:Objective: To study the screening and clinical significance of differential protein in squamous cell carcinoma of the esophagus. Methods: (1) the pure cells of Kazak, Hans squamous cell carcinoma cells and normal epithelial cells near the distal esophagus were obtained by laser capture microdissection. (2) two dimensional electrophoresis combined with matrix assisted laser. The differential proteins were expressed by desorption ionization time of flight mass spectrometry. (3) the difference candidate proteins Bmi-1, PAI-1, Cofilin-1 and Transgelin were analyzed by Western blot and immunohistochemistry. (4) the clinicopathological parameters of Bmi-1, PAI-1, Cofilin-1, Transgelin and esophageal squamous cell carcinoma, such as sex, age, nationality, tumor size, were analyzed. The correlation of differentiation, pathological type, infiltration depth, lymph node metastasis, clinical stage, and prognosis, and the role of Bmi-1, PAI-1, Cofilin-1 and Transgelin in the development of esophageal cancer. Results: (1) laser capture microdissection has captured the purified esophageal squamous cell carcinoma cells and the paracancerous distal end of the Kazak and Han nationality Normal epithelial cells of the esophagus (2) 43 significant differential proteins were identified by two-dimensional electrophoresis combined with matrix assisted laser desorption ionization time of flight mass spectrometry. There were 25 up-regulated points for esophageal squamous cell carcinoma in Kazak and Han people. There were 14 down regulated protein points in the Hazakh and Han squamous cell carcinoma tissues. The expression of 4 proteins in the Kazak and Han esophageal squamous cell carcinoma tissues is inconsistent. (3) in the Hazakh and Han population, the expression of esophageal squamous cell carcinoma is up to up protein, according to the value of the protein score, the coverage rate of the matched peptide segment, and less research reports, four proteins, Bmi-1, PAI-1, Cofilin-1 and Transgelin.Bmi-1, are screened. The expression of PAI-1, Cofilin-1 and Transgelin in the Hazakh and Han esophageal squamous cell carcinoma tissues was verified by Western blot and immunohistochemistry. The expression was significantly higher than that of the normal esophagus. The positive rate of Bmi-1, PAI-1, Cofilin-1 and Transgelin in the tissues of esophageal squamous cell carcinoma was 78.75% (P0.05), 76.25% (P0.05), and 60% (P0.05). 55% (P0.05), higher than the expression of normal esophageal tissue. (4) analysis of the expression of Bmi-1, PAI-1, Cofilin-1 and Transgelin in esophageal squamous cell carcinoma with sex, age, nationality, tumor size, differentiation, pathological classification, infiltration depth, lymph node metastasis, and clinical stage expression of.Bmi-1: lymph node metastasis and lymphatic metastasis of esophageal squamous cell carcinoma. The positive rates of Bmi-1 were 63% and 86.8% in the patients' tissues, respectively, and 86.8%, respectively. The difference between the two groups was statistically significant. The positive rate of Bmi-1 in the tissues of the patients with different infiltrating depth of esophageal squamous cell carcinoma, the tumor cells infiltrating the esophageal mucosa or the myometrium were 92.3%, the infiltrating esophagus membrane and the surrounding structure were 72.2%, and the difference was statistically significant. The results show that the expression of Bmi-1 may be related to the expression of.PAI-1 in the invasive degree of esophageal carcinoma: in the patients with esophageal squamous cell carcinoma, the positive expression rate of PAI-1 is 59.3% and 84.9% in the tissues of the patients with lymph node metastasis and no lymph node metastasis, respectively, and the difference between the two groups is statistically significant, indicating that the expression of PAI-1 can be expressed. It was related to lymph node metastasis of esophageal carcinoma tissue. The tumor cells infiltrated the esophagus mucosa or myometrium 53.8%, the positive expression rate of the tumor infiltrating esophageal fibrous membrane and surrounding structure was 87%, P0.05, the difference was statistically significant. The results showed that the expression of PAI-1 may be related to the expression of.Cofilin-1 in the infiltration and migration of esophageal cancer tissue: the patients with esophageal squamous cell carcinoma The positive expression rates of Cofilin-1 in the tissues of highly differentiated, medium and low differentiated esophageal squamous cell carcinoma were 26.1%, 78.3% and 70.6%, and P0.05, respectively. The difference was statistically significant, indicating that the expression of Cofilin-1 may be related to the degree of differentiation of esophageal cancer tissue, suggesting that the expression rate is high and is associated with the prognosis. There are lymph node metastasis and no The positive rates of Cofilin-1 were 88.9% and 45.3% in the lymph node metastasis of esophageal squamous cell carcinoma, P0.05, respectively, and the difference between the two groups was statistically significant, indicating that the expression of Cofilin-1 was related to the lymph node metastasis of the esophageal carcinoma. The positive expression rate of Cofilin-1 in the tissues of the patients with esophageal squamous cell carcinoma in stage I and II stage and in stage III to IV stage were divided. The difference between 37.1% and 77.8%, P0.05, and two groups was statistically significant, indicating that the expression of Cofilin-1 may be related to the expression of.Transgelin in the clinical stages of esophageal cancer: the positive expression rate of Transgelin in highly differentiated, moderately differentiated and poorly differentiated esophageal squamous cell carcinoma tissues is 34.8%, 52.2% and 70.6%, and P0.05, respectively, with statistical significance. The expression of Transgelin may be related to the degree of differentiation of esophageal cancer tissue, suggesting that the expression rate is high and is associated with the prognosis. In the tissues of patients with lymph node metastasis and non lymph node metastasis of esophageal squamous cell carcinoma, the positive expression rate of Transgelin is 88.9% and 37.7% respectively, P0.05, and the difference between the two groups is statistically significant, indicating that Transgelin The expression is related to lymph node metastasis of esophageal carcinoma. Conclusion: (1) this study suggests that laser capture microdissection can effectively solve the problem of tissue heterogeneity and can better obtain more purified target cells. (2) the differential protein atlas of esophageal squamous cell carcinoma and normal esophageal epithelial cells suggests the development and development of esophageal squamous cell carcinoma. (3) the results of Western blot and immunohistochemical analysis Bmi-1, PAI-1, Cofilin-1, Transgelin are consistent with the analysis and identification of proteomics technology. (4) Bmi-l, PAI-1, Cofilin-1, and Transgelin are the four proteins in the squamous cell carcinoma of the esophagus The up-regulated expression is related to the degree of differentiation, invasion and lymphatic metastasis of esophageal squamous cell carcinoma. It may play a role in the occurrence and development of squamous cell carcinoma of the esophagus, and has a guiding significance for the malignancy and prognosis of esophageal squamous cell carcinoma, which provides a valuable theoretical basis for the molecular targeting therapy of esophageal squamous cell carcinoma and the development of new anticancer drugs.
【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.1

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