GDF15在口腔鳞癌中的作用机制和临床意义

发布时间：2018-02-15 07:17

本文关键词： 口腔黏膜鳞状细胞癌生长分化因子15 TPF诱导化疗 ERK1/2 AKT　出处：《上海交通大学》2014年博士论文　论文类型：学位论文

【摘要】：目的:在课题组的前期研究中，我们建立了人口腔黏膜上皮细胞体外癌变模型。采用寡核苷酸芯片检测癌变模型中各阶段细胞基因表达谱，GDF15基因表达水平升高最为显著。GDF15在人体多种肿瘤的发生发展中发挥重要作用，而在口腔鳞癌中少见报道。本课题旨在探讨GDF15在口腔鳞癌患者血清和组织标本中的表达及增强或抑制GDF15表达对口腔鳞癌细胞系生物学行为的影响，从而综合评价GDF15在口腔鳞癌发生发展中的作用。方法： 1、应用Western blotting和Real-time PCR方法在口腔上皮细胞癌变模型及口腔鳞癌细胞系中验证GDF15表达。 2、应用ELISA的方法检测正常人、白斑患者及口腔鳞状细胞癌患者血清样本中GDF15的表达，探讨其血清水平与口腔鳞癌基线资料、预后及化疗效果的关系。 3、应用免疫组化方法检测230例口腔鳞癌组织标本和癌旁组织中GDF15蛋白的表达，探讨该基因的表达与基线资料、预后及化疗效果的关系。 4、构建包含针对GDF15的干扰序列或GDF15全长序列的慢病毒载体分别转染HB96、HIOEC和HN30细胞，研究GDF15表达水平的变化对口腔鳞状细胞癌细胞生物学行为的影响。并检测与细胞生长和凋亡相关的信号通路和蛋白的变化，从而探讨GDF15对口腔鳞状细胞癌细胞系的作用以及相关分子机制。结果： 1、在HB96及另外四种口腔鳞状细胞癌细胞系中GDF15基因的mRNA和蛋白表达水平均高于HIOEC细胞，验证了基因芯片的结果 2、在正常人、白斑患者及口腔鳞癌患者血清中GDF15的水平依次显著升高。绘制ROC曲线并以346.9ng/L作为界限值，低表达GDF15患者的预后及化疗疗效略好于高表达组，但两组间无统计学差异。 3、GDF15蛋白在口腔鳞癌组织标本表达升高，以免疫组化评分划分高、低表达组。高表达者预后较差并易产生化疗抵抗；统计分析表明GDF15是口腔鳞状细胞癌不良预后的独立危险因素。 4、在HB96和HN30中，，干扰GDF15的表达AKT和ERK1/2的磷酸化水平下降，同时细胞增殖能力、迁移能力、侵袭能力、克隆形成能力和成瘤能力均受到抑制，应用流式细胞术并应用Western blot检测PRP剪切体和BAX的表达均证明细胞凋亡现象的发生。而在HIOEC和HB96细胞中过表达GDF15则可引起AKT和ERK1/2磷酸化水平的上升，并可增强细胞增殖能力、迁移能力、侵袭能力和克隆形成能力。结论： 1、GDF15在口腔鳞癌患者血清中表达升高，可作为口腔鳞癌的候选诊断标记物。 2、在口腔鳞癌中GDF15表达与预后及化疗抵抗相关。可作为预后及化疗疗效预测的候选生物标记物。 3、GDF15作为促癌基因可通过AKT及ERK1/2信号通路影响口腔鳞状细胞癌细胞生物学行为。
[Abstract]:Objective: in the preliminary study of the research group, We established an in vitro carcinogenesis model of human oral mucosal epithelial cells, and used oligonucleotide microarray to detect the expression of GDF15 gene in different stages of carcinogenesis model. Play an important role in development, The purpose of this study was to investigate the expression of GDF15 in serum and tissue of patients with oral squamous cell carcinoma (OSCC) and the effect of increasing or inhibiting the expression of GDF15 on the biological behavior of oral squamous cell carcinoma cell line. Therefore, the role of GDF15 in the occurrence and development of oral squamous cell carcinoma (OSCC) was evaluated. Methods:. 1. Western blotting and Real-time PCR were used to verify the expression of GDF15 in oral epithelial cell carcinogenesis model and oral squamous cell carcinoma cell line. 2. ELISA was used to detect the expression of GDF15 in serum samples of normal subjects, patients with leukoplakia and oral squamous cell carcinoma (OSCC). The relationship between serum level and baseline data, prognosis and chemotherapeutic effect of oral squamous cell carcinoma (OSCC) was investigated. 3. Immunohistochemical method was used to detect the expression of GDF15 protein in 230 cases of oral squamous cell carcinoma tissues and adjacent tissues, and to investigate the relationship between the expression of GDF15 protein and baseline data, prognosis and the effect of chemotherapy. 4. The lentivirus vector containing interference sequence or full-length GDF15 sequence for GDF15 was constructed and transfected into HB96 HIOEC and HN30 cells, respectively. To study the effect of GDF15 expression on the biological behavior of oral squamous cell carcinoma cells, and to detect the changes of signal pathways and proteins related to cell growth and apoptosis, so as to explore the effect of GDF15 on oral squamous cell carcinoma cell lines. Action and related molecular mechanisms. Results:. 1. The mRNA and protein expression levels of GDF15 gene in HB96 and other four oral squamous cell carcinoma cell lines were higher than those in HIOEC cells, which confirmed the results of gene chip. 2. In normal subjects, patients with leukoplakia and oral squamous cell carcinoma, the level of serum GDF15 increased significantly in turn. Drawing ROC curve and taking 346.9 ng / L as the limit value, the prognosis and chemotherapeutic effect of low expression GDF15 patients were slightly better than those of high expression group. But there was no statistical difference between the two groups. 3The expression of GDF15 protein in oral squamous cell carcinoma tissues was increased, which was divided into high and low expression groups by immunohistochemical score. The prognosis of the patients with high expression was poor and the resistance to chemotherapy was easy. Statistical analysis showed that GDF15 was an independent risk factor for poor prognosis of oral squamous cell carcinoma. 4. In HB96 and HN30, the phosphorylation level of AKT and ERK1/2, which interfered with the expression of GDF15, was decreased, while the ability of cell proliferation, migration, invasion, clone formation and tumorigenesis were inhibited. Flow cytometry and Western blot were used to detect the expression of PRP shearing bodies and BAX. The expression of GDF15 in HIOEC and HB96 cells increased the phosphorylation level of AKT and ERK1/2, and enhanced the proliferation of the cells. Migration, invasion, and clone formation. Conclusion:. 1the expression of GDF15 in serum of patients with oral squamous cell carcinoma was increased and could be used as a candidate diagnostic marker for oral squamous cell carcinoma. 2. The expression of GDF15 in oral squamous cell carcinoma is associated with prognosis and chemotherapeutic resistance, and can be used as a candidate biomarker for predicting the prognosis and therapeutic effect of chemotherapy. 3GDF15 can affect the biological behavior of oral squamous cell carcinoma cells through AKT and ERK1/2 signaling pathway.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.8

【共引文献】