【作者】 连晓雯；

【导师】 崔燕；

【作者基本信息】 甘肃农业大学 ， 基础兽医学， 2017， 博士

【摘要】 背景:胃癌(Gastric Cancer,GC)是危害人类健康的常见恶性肿瘤,也是全球癌症相关死亡的第二大原因。由于胃癌的浸润转移是一个多基因参与的复杂过程,不仅与癌细胞自身基因结构改变与表达异常有关,还受到组织微环境的影响,这一过程中细胞黏附分子的变化直接影响胃癌的转移侵袭过程。亮氨酸拉链转录因子样1(LZTFL1)是近年来新发现的肿瘤抑制因子,自发现以来,其与BBS综合征的研究较多,但作为肿瘤抑制基因,其分子生物学功能的相关研究较少。现有的研究表明,LZTFLl在多种恶性肿瘤中低表达,与胃癌、非小细胞肺癌、肝癌均存在相关性。胃癌组织中LZTFL1与E-cadherin介导的黏附连接有关,提示LZTFL1可能通过细胞粘附蛋白来影响细胞的迁移运动。但目前还未见到LZTFL1与不同粘附蛋白相关性的研究报道。目的:对肿瘤抑制基因LZTFLl在胃癌组织中的表达及其与细胞粘附因子的相关性进行研究。方法:1.应用免疫组化法检测133例胃癌样本、40例癌旁组织样本和40例胃炎样本中LZTFLl,E-cadherin,β-catenin,ICAM-1和VCAM-1的表达情况,分析与临床病理特征和预后的关系;2.采用实时荧光定量聚合酶链反应(Real Time PCR)检测59对胃癌及癌旁组织中LZTFLl,E-cadherin,β-catenin,ICAM-1和VCAM-1的mRNA水平,分析与临床病理特征和预后的关系;3.分析LZTFL1与E-cadherin,β-catenin,ICAM-1和VCAM-1在蛋白水平和mRNA水平上的相关性;4.在已经建立的裸鼠人胃癌高转移模型上,采用免疫组化法和Real Time PCR分别对接种原代胃癌患者和传代裸鼠胃癌组织中LZTFLl,E-cadherin,β-catenin,ICAM-1和VCAM-1的蛋白和mRNA水平进行检测比较,分析其变化规律。结果:1.与癌旁组织样本和胃炎标本相比,胃癌组织中LZTFL1蛋白水平明显下调(P<0.01),其LZTFL1阳性表达率与TNM分期、肿瘤分化程度及浸润深度显著相关(P<0.05);与E-cadherin呈正相关(P<0.05),与β-catenin和ICAM-1负相关(P<0.05),与VCAM-1无关(P<0.05)。2.与癌旁组织相比,LZTFL1在胃癌组织中mRNA水平下调(P<0.05),但在不同临床病理特征分组中LZTFL1 mRNA表达水平均无明显差异;且LZTFL1的mRNA表达与E-cadherin,β-catenin,ICAM-1和VCAM-1均不相关(P>0.05)。3.接种原代胃癌患者组织中LZTFL1,β-catenin,ICAM-1和VCAM-1蛋白均为阳性表达,E-cadherin为阴性表达;传代裸鼠胃癌组织中LZTFL1为弱阳性表达,E-cadherin,β-catenin,ICAM-1和VCAM-1均为阴性表达。传代裸鼠胃癌组织中LZTFL1mRNA表达水平无明显变化,β-catenin的mRNA表达水平下降,E-cadherin、ICAM-1和VCAM-1的mRNA表达无法测出。结论:通过对133例胃癌样本、40例癌旁组织样本和40例胃炎样本的免疫组化检测发现,与胃炎组织相比,LZTFLl在胃癌组织中呈低水平表达,其蛋白表达与细胞粘附蛋白E-cadherin,β-catenin和ICAM-1蛋白表达有关。通过对59对胃癌和癌旁组织的mRNA检测发现,胃癌组织较正常癌旁组织LZTFL1 mRNA水平下调,但未发现LZTFL1 mRNA水平与E-cadherin,β-catenin,ICAM-1和VCAM-1 mRN A水平的关系。更多还原

【Abstract】 Backgroud:Gastric cancer(GC)is one of the most common malignant tumors and the second leading cause of cancer-related death worldwide.The invasion and metastasis of gastric cancer is a complicated process that multiple genes participate in.It is not only related to structural change and abnormal expression of genes of cancer cells,but also affected by micro-environment of tissues.Meanwhile,the change of cell adhesion molecules directly impacts the invasion and metastasis of gastric cancer.LZTFL1 is a novel tumor suppressor in recent years.Since the discovery of LZTFL1,its connection with BBS syndrome has been studied by many researc hers,but the molecular biological function of LZTFL1,when regarded as a tumor suppressor gene,has been rarely reported.The existing studies indicate that the low expression of LZTFLl is found in multiple malignant tumors and is correlated with gastric cancer,non-small cell lung cancer,and liver cancer.In gastric cancer tissues,LZTFL1 is related to E-cadherin-mediated adherens junction,suggesting that LZTFL1 may influence cell migration through cell adhesion protein.However,the correlation of LZTF L1 with different adhesion proteins has not been reported.Purpose: This study focuses on the expression of LZTFL1 mRNA and protein in gastric cancer tissue,and the possible correlationship between LZTFL1 and cell adhesion proteins.Methods:1.The immunohistochemical method was applied to detect the expression of LZTFLl,E-cadherin,β-catenin,ICAM-1 and VCAM-1 in 133 samples of gastric cancer,40 tissue samples of paracancerous non-tumor gastric mucosa,and 40 samples of gastritis.The relation of the ir expression to clinicopathologic feature and prognosis was analyzed;2.Real Time PC R was used to detect the mRNA level of LZTFLl,E-cadherin,β-catenin,ICAM-1 and VCAM-1 in 59 pairs of gastric cancer and para-carcinoma tissues.The relation of their mRNA level to clinicopathologic feature and prognosis was analyzed;3.The correlation of LZTFLl with E-cadherin,β-catenin,ICAM-1 and VC AM-1 in terms of protein level and mRN A level was analyzed;4.A high-metastasis model of nude mice bearing human gastric carcinoma was established.Immunohistochemical method and Real Time PCR were used to detect and compare the protein level and mRNA level of LZTFLl,E-cadherin,β-catenin,ICAM-1 and VCAM-1 in primarily cultured tissues of patients with gastric cancer and sub-cultured gastric cancer tissues from nude mice.Their varying pattern was analyzed.Results:1.The protein level of LZTFL1 in gastric cancer tissue was significantly decreased(P<0.01),compared with that in tissue samples of paracancerous non-tumor gastric mucosa and gastritis samples.The positive rate of LZTFL1 was significantly correlated to TNM staging,degree of tumor differentiation and depth of infiltration(P<0.05);It was positively correlated to E-cadherin(P<0.05),negatively correlated to β-catenin and ICAM-1(P<0.05),and not correlated to VCAM-1(P<0.05).2.The mRNA level of LZTFL1 in gastric cancer tissue was decreased(P<0.05),compared with that in para-carcinoma tissue,but did not present any significant difference between different clinicopathologic feature groups;the mRN A expression of LZTFL1 was uncorrelated to E-cadherin,β-catenin,ICAM-1 and VCAM-1(P>0.05).3.In primarily cultured tissues of patients with gastric cancer,LZTFLl,β-catenin,ICAM-1 and VC AM-1 were positively expressed,while E-cadherin was negatively expressed;in sub-cultured gastric cancer tissues from nude mice,LZTFL1 presented a weakly positive expression,while the expression of E-cadherin,β-catenin,ICAM-1 and VCAM-1 was negative.In sub-cultured gastric cancer tissues from nude mice,the mRNA expression of LZTFL1 did not show any significant change,the mRNA expression of β-catenin decreased,and the mRNA expression of E-cadherin,ICAM-1 and VCAM-1 cannot be measured.Conclusion:Immunohistochemical analysis of 133 cases of gastric cancer,40 cases of paracancerous tissue samples and 40 cases of gastritis showed that LZTFLl presented a lower expression level in gastric cancer tissue,compared with its expression in gastritis tissues.Its protein expression was correlated to the expression of E-cadherin,β-catenin and ICAM-1.In 59 pairs of gastric cancer and para-carcinoma tissues,the mRNA level of LZTFL1 was decreased in gastric cancer tissue,compared with that in normal para-carcinoma tissues.However,the correlation of mRN A level of LZTFL1 with mRNA level of E-cadherin,β-catenin,ICAM-1 and VCAM-1 was not found.更多还原