本文關(guān)鍵詞： 口腔黏膜鱗狀細(xì)胞癌 生長(zhǎng)分化因子15 TPF誘導(dǎo)化療 ERK1/2 AKT　出處：《上海交通大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：目的:在課題組的前期研究中，我們建立了人口腔黏膜上皮細(xì)胞體外癌變模型。采用寡核苷酸芯片檢測(cè)癌變模型中各階段細(xì)胞基因表達(dá)譜，GDF15基因表達(dá)水平升高最為顯著。GDF15在人體多種腫瘤的發(fā)生發(fā)展中發(fā)揮重要作用，而在口腔鱗癌中少見(jiàn)報(bào)道。本課題旨在探討GDF15在口腔鱗癌患者血清和組織標(biāo)本中的表達(dá)及增強(qiáng)或抑制GDF15表達(dá)對(duì)口腔鱗癌細(xì)胞系生物學(xué)行為的影響，從而綜合評(píng)價(jià)GDF15在口腔鱗癌發(fā)生發(fā)展中的作用。 方法： 1、應(yīng)用Western blotting和Real-time PCR方法在口腔上皮細(xì)胞癌變模型及口腔鱗癌細(xì)胞系中驗(yàn)證GDF15表達(dá)。 2、應(yīng)用ELISA的方法檢測(cè)正常人、白斑患者及口腔鱗狀細(xì)胞癌患者血清樣本中GDF15的表達(dá)，探討其血清水平與口腔鱗癌基線資料、預(yù)后及化療效果的關(guān)系。 3、應(yīng)用免疫組化方法檢測(cè)230例口腔鱗癌組織標(biāo)本和癌旁組織中GDF15蛋白的表達(dá)，探討該基因的表達(dá)與基線資料、預(yù)后及化療效果的關(guān)系。 4、構(gòu)建包含針對(duì)GDF15的干擾序列或GDF15全長(zhǎng)序列的慢病毒載體分別轉(zhuǎn)染HB96、HIOEC和HN30細(xì)胞，研究GDF15表達(dá)水平的變化對(duì)口腔鱗狀細(xì)胞癌細(xì)胞生物學(xué)行為的影響。并檢測(cè)與細(xì)胞生長(zhǎng)和凋亡相關(guān)的信號(hào)通路和蛋白的變化，從而探討GDF15對(duì)口腔鱗狀細(xì)胞癌細(xì)胞系的 作用以及相關(guān)分子機(jī)制。 結(jié)果： 1、在HB96及另外四種口腔鱗狀細(xì)胞癌細(xì)胞系中GDF15基因的mRNA和蛋白表達(dá)水平均高于HIOEC細(xì)胞，驗(yàn)證了基因芯片的結(jié)果 2、在正常人、白斑患者及口腔鱗癌患者血清中GDF15的水平依次顯著升高。繪制ROC曲線并以346.9ng/L作為界限值，低表達(dá)GDF15患者的預(yù)后及化療療效略好于高表達(dá)組，但兩組間無(wú)統(tǒng)計(jì)學(xué)差異。 3、GDF15蛋白在口腔鱗癌組織標(biāo)本表達(dá)升高，以免疫組化評(píng)分劃分高、低表達(dá)組。高表達(dá)者預(yù)后較差并易產(chǎn)生化療抵抗；統(tǒng)計(jì)分析表明GDF15是口腔鱗狀細(xì)胞癌不良預(yù)后的獨(dú)立危險(xiǎn)因素。 4、在HB96和HN30中，，干擾GDF15的表達(dá)AKT和ERK1/2的磷酸化水平下降，同時(shí)細(xì)胞增殖能力、遷移能力、侵襲能力、克隆形成能力和成瘤能力均受到抑制，應(yīng)用流式細(xì)胞術(shù)并應(yīng)用Western blot檢測(cè)PRP剪切體和BAX的表達(dá)均證明細(xì)胞凋亡現(xiàn)象的發(fā)生。而在HIOEC和HB96細(xì)胞中過(guò)表達(dá)GDF15則可引起AKT和ERK1/2磷酸化水平的上升，并可增強(qiáng)細(xì)胞增殖能力、遷移能力、侵襲能力和克隆形成能力。 結(jié)論： 1、GDF15在口腔鱗癌患者血清中表達(dá)升高，可作為口腔鱗癌的候選診斷標(biāo)記物。 2、在口腔鱗癌中GDF15表達(dá)與預(yù)后及化療抵抗相關(guān)。可作為預(yù)后及化療療效預(yù)測(cè)的候選生物標(biāo)記物。 3、GDF15作為促癌基因可通過(guò)AKT及ERK1/2信號(hào)通路影響口腔鱗狀細(xì)胞癌細(xì)胞生物學(xué)行為。
[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.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.8

【共引文獻(xiàn)】

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