【作者】 赵辉；

【导师】 李惠翔； 吕新全；

【作者基本信息】 郑州大学 ， 病理学与病理生理学， 2010， 硕士

【摘要】 食管癌是一种较为常见的恶性程度极高的消化道肿瘤,是当今世界上对人类健康危害最大的恶性肿瘤之一,随着医疗水平的提高,食管癌的治疗现状较过去有明显的改善。早期食管癌的治疗最好的方法仍是手术切除；中晚期食管癌的治疗现状并不乐观,用局部的手术治疗难以达到满意的疗效,现阶段食管癌的主要的治疗方法为放疗、化疗及药物治疗,但目前术后复发率仍很高,病人五年生存率仍然十分低下,难以满足目前医疗发展的最终目标。当前兴起的靶向基因治疗已经成为对放、化疗效果不满意的肿瘤病人治疗的一种新型的替代手段。因而从分子水平上探讨食管鳞癌发生发展的分子机制,找到针对抑制食管鳞癌发生发展的有效的治疗靶点,对食管鳞癌的临床治疗、预防以及患者生存率和生存质量的提高都具有极其重要的意义。Pim-3最初在鼠嗜铬细胞瘤细胞系PC12细胞中发现,并被鉴定为诱导去极化的基因KID-1,因为KID-1与表达丝氨酸/苏氨酸激酶活性的原癌基因Pim家族具有高度的同源性,因而命名为Pim-3。近年来研究显示,激活的Pim激酶在恶性肿瘤的发病机制中发挥重要的作用,与肿瘤的发生发展关系十分密切,并且在肿瘤细胞凋亡中起着重要的作用,其在肿瘤细胞中的凋亡作用机制可能是原癌基因Pim-3主要磷酸化Bad蛋白112位的丝氨酸,进一步导致bcl-XL和bcl-2释放,阻止细胞凋亡。迄今为止,原癌基因Pim-3和抑凋亡基因bcl-2之间的相互关系在食管鳞癌发生发展中的作用在国内外尚未见报道,本研究采用RT-PCR、Western blotting、原位杂交和免疫组织化学4种不同的方法联合检测了食管癌高发区河南安阳的食管癌患者手术切除的癌组织中原癌基因Pim-3和抑凋亡基因bcl-2 mRNA和蛋白的表达情况,分析其与临床病理学特征的关系,进一步探讨Pim-3和bcl-2在食管鳞癌组织中表达的相关性,该研究旨在为进一步探讨Pim-3和bcl-2在食管鳞癌发生发展中的作用及寻找检测食管鳞癌的新的分了靶点奠定基础。方法1.采用半定量RT-PCR方法检测食管鳞癌组织、癌旁不典型增生组织以及正常食管粘膜上皮组织中Pim-3和bcl-2 mRNA的相对表达水平。2.采用Western blotting方法检测食管鳞癌组织、癌旁不典型增生组织以及正常食管粘膜上皮组织中Pim-3和bcl-2蛋白的相对表达水平。3.采用原位杂交技术检测45例食管鳞癌组织、22例癌旁不典型增生组织以及45例正常食管粘膜上皮组织中Pim-3和bcl-2 mRNA的表达,并分析其蛋白表达的相关性。4.采用免疫组织化学方法检测45例食管鳞癌组织、22例癌旁不典型增生组织以及45例正常食管粘膜上皮组织中Pim-3和bcl-2蛋白的表达,并分析其蛋白表达的相关性。5.统计学处理：应用SPSS 13.0软件分析,采用χ2检验、t检验和方差分析以及相关性分析。P<0.05表示具有统计学意义。结果1.Pim-3的半定量RT-PCR结果表明,在食管鳞癌组织中能成功扩增出500bp的Pim-3基因,而在正常食管粘膜上皮组织中未见相应的条带。食管鳞癌组织和癌旁不典型增生组织中Pim-3相对表达丰度分别为0.809±0.015和0.092±0.009,而正常组织中的表达为0,三者之间的表达具有显著性差异(P<0.05)。2. Bcl-2的半定量RT-PCR结果表明,在食管鳞癌组织中能成功扩增出595bp的bcl-2基因,而在正常食管粘膜上皮组织中未见相应的条带。食管鳞癌组织和癌旁不典型增生组织中bcl-2相对表达丰度分别为0.671±0.011和0.102±0.007,而正常组织中的表达为0,三者之问的表达具有显著性差异(P<0.05)。3. Western blotting结果表明,Pim-3和bcl-2蛋白在食管鳞癌组织中呈现高表达,而在正常食管粘膜组织中无表达。食管鳞癌组织中Pim-3和bcl-2蛋白的相对表达量分别为：0.796±0.018和0.757±0.014,显著高于癌旁边不典型增生组织中Pim-3和bcl-2蛋白的相对表达(分别为：0.299±0.011和0.225±0.009),正常食管粘膜组织中Pim-3和bcl-2的相对表达为0,三者之间的表达具有显著性差异(P<0.05)。4.原位杂交结果显示,Pim-3 mRNA主要定位于细胞质中,呈蓝紫色颗粒。Pim-3 mRNA在食管鳞癌组织中高表达,其阳性率为82.22%(37/45),显著高于癌旁不典型增生组织和正常食管上皮组织中Pim-3 mRNA的表达,二者的阳性率分别为22.73%(5/22)和0.00%(0/45),三组之间表达的差异具有统计学意义(χ2=82.487,P<0.05)。5.Pim-3蛋白定位于细胞胞质中,呈浅黄色至棕黄色颗粒。Pim-3蛋白高表达于食管鳞癌组织,阳性率高达75.56%(34/45),显著高于癌旁不典型增生组织和正常食管上皮组织中Pim-3蛋白的表达,其阳性率分别为：18.18%(4/22)和0.00%(0/45),三组之间表达的差异具有统计学意义(χ2=72.570,P<0.05)。6.原位杂交结果显示,bcl-2 mRNA主要定位于细胞质中,呈蓝紫色颗粒。在45例食管鳞癌组织中bcl-2 mRNA的阳性表达例数为35例,其阳性表达率为：77.78%(35/45),而在癌旁不典型增生组织为7例,其mRNA表达的阳性率为31.82%(7/22),在正常食管黏膜组织中尚未发现bcl-2mRNA的表达。Bcl-2 mRNA在食管鳞癌组织、癌旁不典型增生组织和正常食管黏膜组织中的阳性表达率比较差异具有统计学意义(χ2=58.451,P<0.05)。7. Bcl-2蛋白阳性染色定位于细胞胞质中,呈浅黄色至棕黄色颗粒。免疫组化检测结果表明,在45例食管鳞癌组织中bcl-2蛋白的阳性表达例数为32例,其阳性表达率为：71.11%(32/45),而在癌旁不典型增生组织为5例,其表达的阳性率为22.72%(5/22),在正常食管黏膜组织中尚未发现bcl-2蛋白的表达。Bcl-2蛋白在食管鳞癌组织、癌旁不典型增生组织和正常食管黏膜组织中的阳性表达率比较差异具有统计学意义(χ2=52.724,P<0.05)。8.利用SPSS 13.0统计学处理软件分析Pim-3 mRNA表达与食管鳞癌临床病理学特征的关系。结果表明,Pim-3 mRNA表达与淋巴结转移和TNM分期具有明显的相关性(P<0.05),但与组织学分级、浸润深度无关(P>0.05)。9.Pim-3和bcl-2的相关性分析结果表明,在45例食管鳞癌组织中,Pim-3蛋白阳性表达34例中,其bcl-2蛋白阳性表达占30例,而Pim-3蛋白阴性表达的11病例中,其bcl-2蛋白阴性表达占9例。Pim-3和bcl-2在食管鳞癌组织中的表达强度呈正相关关系(γp=0.664,P=-0.000)。结论1.食管鳞癌组织中Pim-3和bcl-2均呈现高表达,而正常食管黏膜上皮组织中Pim-3和bcl-2的表达均缺失。2.食管鳞癌组织中Pim-3和bcl-2的高表达与淋巴结转移和TNM分期有关,但与组织学分级、浸润深度无关,提示Pim-3和bcl-2在食管鳞癌的发生发展中起重要作用。3.食管鳞癌组织中Pim-3和bcl-2 mRNA和蛋白在食管鳞癌组织中的表达强度呈正相关关系,提示Pim-3在肿瘤中的抑制细胞凋亡可能与bcl-2的表达密切相关。更多还原

【Abstract】 Esophageal cancer is a more common malignant tumor of digestive tract, and is the one of the world’s most harmful to human health in all malignant tumors, with the improvement of medical standards; the status quo treatment of esophageal cancer has improved significantly over the past. The best treatment of early esophageal cancer is surgical excision. Up to now, the treatment of advanced esophageal cancer are not optimistic, local treatment of surgical treatment can hardly be a satisfactory outcome, the currently predominant treatment methods include radiotherapy, chemotherapy and drug treatment, but recurrence rates remain high, and five-year survival rate of patients is still is very low, it is difficult to meet the current medical development and the ultimate goal. The current rise of targeted gene therapy has been a new alternative for radiotherapy and chemotherapy in cancer patients. Therefore, a better understanding of the molecular mechanisms in carcinogenesis and progression of ESCC helps to improve therapy, prevention, the prognosis of patients with ESCC.Pim-3, a member of the proto-oncogene Pim family that expresses serine/ threonine kinase activity, was originally identified as a depolarization-induced gene, KID-1, in PC 12 cells (a rat pheochromocytoma cell line). Because KID-1 shows a high sequence similarity with the proto-oncogene Pim family that expresses serine/ threonine kinase activity, it was renamed as Pim-3. Accumulating evidence indicates activated Pim family members play an important role in various types of carcinogenesis, and thus associated with occurrence and development of malignant neoplasm, further play a key role in apoptosis of tumors, certain molecular mechanism of cell apoptosis mediated by Pim-3 is as following:Pim-3 may mainly phosphorylate Bad Ser 112, further phosphorylation of Bad resulted in the liberation of Bcl-XL and Bcl-2, and thus prevent apoptosis. To date, the relationships between Pim-3 and bcl-2 in the occurrence and development of esophageal squamous cell carcinoma have not been investigated worldwide. In the present study, expressions of Pim-3 and bcl-2 mRNAs and proteins were detected in the patients with ESCC from high-incidence area of esophageal cancer in Henan province by RT-PCR, Western blotting, in situ hybrid and immunohistochemistry methods, and the relationships between expressions of Pim-3 and bcl-2 and clinicopathological features were analyzed, further correlations of Pim-3 and bcl-2 expressions were explored in ESCC. The findings may lay a foundation for making further investigations on the role of Pim-3 and bcl-2 in the occurrence and development of ESCC and seeking a potential new molecular target for ESCC therapy.Methods(1) Relative expressions of Pim-3 and bcl-2 mRNAs were detected by semi-quantitative RT-PCR method in ESCC tissue, mucosa adjacent to cancer, and normal esophageal mucosa epithelial tissue.(2) Relative expressions of Pim-3 and bcl-2 proteins were detected by Western blotting method in ESCC tissue, mucosa adjacent to cancer, and normal esophageal mucosa epithelial tissue.(3) Expressions of Pim-3 and bcl-2 mRNAs were detected by in situ hybrid method in 45 cases of ESCC tissue,22 cases of mucosa adjacent to cancer, and 45 cases of normal esophageal mucosa, and correlation between expressions of Pim-3 and bcl-2 mRNAs was analyzed.(4) Expressions of Pim-3 and bcl-2 proteins were detected by immunohisto chemistry method in 45 cases of ESCC tissue,22 cases of mucosa adjacent to cancer, and 45 cases of normal esophageal mucosa, and correlation between expressions of Pim-3 and bcl-2 proteins was investigated.(5) Statistical analysis:The data were carried out with chi square test, t test, analysis of variance and correlation using SPSS version 13.0. In all statistical analyses, a P-value<0.05 was considered statistically significant.Results(1) The results of semi-quantitative RT-PCR demonstrated that Pim-3 gene, about 500bp, was successfully amplified in ESCC tissue, but not in normal esophageal mucosa epithelial tissue. Relative expression levels of Pim-3 in ESCC tissue and mucosa adjacent to cancer were 0.809±0.015 and 0.092±0.009, respectively, while there was no expression in normal esophageal mucosa epithelial tissue, there were significant differences among three groups (P<0.05).(2) The results of semi-quantitative RT-PCR demonstrated that bcl-2 gene, about 595bp, was successfully amplified in ESCC tissue, but not in normal esophageal mucosa epithelial tissue. Relative expression levels of bcl-2 mRNA in ESCC tissue and mucosa adjacent to cancer were 0.671±0.011 and 0.102±0.007, respectively, while there was no expression in normal esophageal mucosa epithelial tissue, there were significant differences among three groups (P<0.05).(3) The results of Western blotting indicated that Pim-3 and bcl-2 proteins appeared high expressions in ESCC, but there was no expression in normal esophageal mucosa epithelial tissue. The relative expressions of Pim-3 and bcl-2 proteins (0.796±0.018 and 0.757±0.014, respectively) in ESCC tissue were significantly higher than those in mucosa adjacent to cancer (0.299±0.011 and 0.225±0.009, respectively), while there was no expression in normal esophageal mucosa epithelial tissue, there were significant differences among three groups (P<0.05).(4) The results of in situ hybrid showed that Pim-3 mRNA was mainly localized in cytoplasm, appearing ianthinus granules. There was high expression of Pim-3 mRNA in ESCC, and positive ratio was 82.22%(37/45), significantly higher than those of mucosa adjacent to cancer and normal esophageal mucosa epithelial tissue, and positive ratios were 22.73%(5/22) and 0.00%(0/45), respectively, there were significant differences among three groups (P<0.05).(5) Pim-3 protein was mainly localized in cytoplasm, showing pale yellow to brown granules. There was high expression of Pim-3 protein in ESCC, and positive ratio was 75.56%(34/45), significantly higher than those of mucosa adjacent to cancer and normal esophageal mucosa epithelial tissue, and positive ratios were 18.18%(4/22) and 0.00%(0/45), respectively, there were significant differences among three groups (P<0.05).(6) The results of in situ hybrid showed that bcl-2 mRNA was mainly localized in cytoplasm, appearing ianthinus granules. There was high expression of bcl-2 mRNA in ESCC, and positive ratio was 77.78%(35/45), significantly higher than those of mucosa adjacent to cancer and normal esophageal mucosa epithelial tissue, and positive ratios were 31.82%(7/22) and 0.00%(0/45), respectively, there were significant differences among three groups (χ2=58.451, P<0.05).(7) Bcl-2 protein was mainly localized in cytoplasm, showing pale yellow to brown granules. The results of immunohistochemistry demonstrated that there was high expression of bcl-2 protein in ESCC, and positive ratio was 71.11% (32/45), significantly higher than those of mucosa adjacent to cancer and normal esophageal mucosa epithelial tissue, and positive ratios were 22.72% (5/22) and 0.00%(0/45), respectively, there were significant differences among three groups (χ2=52.724, P<0.05).(8) Relationship between expressions of Pim-3 and bcl-2 mRNAs and proteins and clinicopathological features were analyzed using SPSS 13.0 software. The results revealed that expressions of Pim-3 and bcl-2 were obviously related to lymph node metastasis and TNM staging (P<0.05), but not related to histo grade and invasion depth (P<0.05).(9) The results of correlation analysis of Pim-3 and bcl-2 revealed that 34 cases of Pim-3 positive expression included 30 cases of bcl-2 positive expression in the 45 cases of ESCC tissues, while 11 cases of Pim-3 negative expression included 9 cases of bcl-2 negative expression. Pim-3 expression and bcl-2 expression displayed positive correlation (γp=0.664, P=0.000).Conclusion1. Pim-3 and bcl-2 appear high expressions in esophageal squamous cell carcinoma, while there are no expressions in the normal esophageal mucosal epithelial tissue.2. High expressions of Pim-3 and bcl-2 are tightly associated with lymph metastasis and TNM staging, but not related to histological grade and depth of infiltration, suggesting Pim-3 and bcl-2 play an important role in the occurrence and development of esophageal squamous cell carcinoma.3. Expression of Pim-3 is positively correlated with the expression of bcl-2, indicating cell apoptosis induced by Pim-3 may be closely associated with bcl-2 expression.更多还原