【摘要】：目的:初次研究CREPT基因在口腔鱗狀細胞癌(Oral squamous cell carcinoma, OSCC)組織樣本及正常組織樣本中的表達情況,對CREPT基因在OSCC中起到的作用及其臨床意義進行分析。初步研究分析CREPT shRNA轉(zhuǎn)染SCC25和CAL27細胞系后,對鱗癌細胞系生物學功能的影響及機制。通過小鼠成瘤實驗,研究CAL27細胞經(jīng)慢病毒攜帶CREPT shRNA轉(zhuǎn)染后,裸鼠腫瘤生長情況的變化。為探索口腔鱗狀細胞癌發(fā)病機制、預(yù)后以及治療方法提供新的線索及研究靶點。方法:采用免疫組化技術(shù)(Immunohistochemistry, IHC),蛋白印跡法(Western blot, WB)和實時定量聚合酶鏈反應(yīng)法(Quantitative Real time Polymerase Chain Reaction, qRT-PCR)檢測分析,在OSCC組織樣本和正常組織樣本中,CREPT的表達情況;利用慢病毒攜帶shCREPT序列,敲低CREPT表達,建立SCC25con、SCC25shCREPT和CAL27con、CAL27shCREPT穩(wěn)定轉(zhuǎn)染細胞系,進行細胞生長檢測實驗,單細胞克隆實驗,劃痕實驗,流式細胞術(shù)檢測細胞凋亡實驗,驗證CREPT在SCC25和CAL27細胞中的功能;小鼠成瘤實驗檢測抑制CREPT表達后,CAL27細胞在BALB/c裸鼠體內(nèi)成瘤情況。結(jié)果:第一部分:CREPT在口腔鱗狀細胞癌組織中的表達情況1、對SCC25與CAL27細胞系檢測發(fā)現(xiàn),CREPT的基因與蛋白高表達。2、對新鮮口腔鱗狀細胞癌組織檢測發(fā)現(xiàn),CREPT在腫瘤組織中的表達高于正常組織。3、對OSCC腫瘤石蠟組織樣本檢測發(fā)現(xiàn),CREPT呈陰性至強陽性表達;在正�？谇火つな灲M織樣本當中,CREPT表達呈陰性至陽性表達;腫瘤組與正常組組間差異顯著;4、臨床資料統(tǒng)計結(jié)果顯示:CREPT陽性表達與腫瘤大小及淋巴轉(zhuǎn)移具有顯著相關(guān)性。第二部分:CREPT對口腔鱗狀細胞癌細胞系體外生物學行為的影響1、下調(diào)CREPT的表達,SCC25和CAL27細胞的增殖能力能受到抑制;2、下調(diào)CREPT的表達,SCC25和CAL27細胞的單細胞克隆形成能力受到抑制;3、下調(diào)CREPT的表達,SCC25和CAL27細胞的遷移能力受到抑制;4、下調(diào)CREPT的表達,SCC25和CAL27細胞凋亡水平增高;5、下調(diào)CREPT的表達,SCC25和CAL27細胞的cyclin D1、 c-Myc的表達降低。第三部分:CREPT對口腔鱗狀細胞癌細胞系體內(nèi)生物學行為的影響1、CREPTshRNA轉(zhuǎn)染CAL27細胞后,裸鼠體內(nèi)實驗顯示,腫瘤的生長受到抑制,2、CREPTshRNA轉(zhuǎn)染CAL27細胞后,形成瘤體的CREPT、cyclinD1與c-Myc表達降低。結(jié)論:1、CREPT在OSCC細胞系及腫瘤組織中表達升高,其陽性表達與OSCC的T、N分期正相關(guān)。2、下調(diào)CREPT表達可以抑制SCC25和CAL27細胞增殖能力、細胞遷移能力,促進細胞系凋亡的發(fā)生,抑制cyclin D1、c-Myc的表達。3、下調(diào)CREPT表達可以抑制CAL27細胞在裸鼠體內(nèi)腫瘤的生長。
[Abstract]:Objective: to investigate the expression of CREPT gene in oral squamous cell carcinoma (OSCC) (Oral squamous cell carcinoma, OSCC) tissues and normal tissues for the first time, and to analyze the role of CREPT gene in OSCC and its clinical significance. The effect and mechanism of CREPT shRNA transfection on the biological function of SCC25 and CAL27 cell lines were studied. The changes of tumor growth in nude mice after CAL27 cells were transfected with lentivirus carrying CREPT shRNA were studied by mouse tumorigenesis test. To explore the pathogenesis, prognosis and treatment of oral squamous cell carcinoma (OSCC). Methods: the expression of crept in OSCC tissue samples and normal tissue samples was detected by immunohistochemical (Immunohistochemistry, IHC), Western blot (Western blot, WB) and real-time quantitative polymerase chain reaction (Quantitative Real time Polymerase Chain Reaction, qRT-PCR), and the shCREPT sequence was carried by lentivirus. The stable transfection cell lines of SCC25shCREPT and CAL27cong CAL27shCREPT were established. Cell growth assay, single cell clone assay, scratch assay, flow cytometry were used to detect the apoptosis of SCC25 and CAL27 cells, and the function of CREPT in SCC25 and CAL27 cells was verified. Tumorigenesis in BALB/c nude mice was detected by murine tumorigenesis assay after inhibiting the expression of CREPT. Results: the first part was the expression of crept in oral squamous cell carcinoma (1). The gene and protein expression of crept in SCC25 and CAL27 cell line was found to be higher than that in oral squamous cell carcinoma, and in the tissue of fresh oral squamous cell carcinoma, crept was found in tumor tissue. The expression of CREPT in paraffin tissue of OSCC was negative and strong. In the paraffin tissues of normal oral mucosa, the expression of crept was negative to positive, and the difference between tumor group and normal group was significant (4). The clinical data showed that the positive expression of crept was significantly correlated with tumor size and lymphatic metastasis. The second part: the effect of crept on the biological behavior of oral squamous cell carcinoma cell line in vitro. 1. Down-regulating the expression of CREPT and the proliferation ability of SCC25 and CAL27 cells can be inhibited, and down-regulating the expression of CREPT and the single cell clone formation ability of CAL27 cells. The down-regulation of CREPT expression and the migration ability of CAL27 cells were inhibited. The expression of CREPT was down-regulated and the apoptotic level of SCC25 and CAL27 cells was increased. The expression of CREPT and CAL27 cells was down-regulated. The cyclin D1 and c-Myc expression of SCC25 and CAL27 cells were decreased. Effect of: crept on the biological behavior of oral squamous cell carcinoma cell lines in vivo. 1 after transfection of CREPTshRNA into CAL27 cells, the results of nude mice experiments showed that the expression of cyclinD1 and c-Myc in the tumor formation decreased after the tumor growth was inhibited and CREPTshRNA was transfected into CAL27 cells. Conclusion the positive expression of crept in OSCC cell line and tumor tissue is increased, and its positive expression is positively correlated with the OSCC stage. Down-regulation of CREPT expression can inhibit the proliferation and migration of SCC25 and CAL27 cells, and promote the apoptosis of the cell line. [WT5 "HZ] [WT5" BZ] [WT5 "BZ] [WT5" BZ] [WT5BZ] Inhibition of the expression of cyclin D1Tc-Myc. 3 and down-regulation of CREPT expression could inhibit the growth of CAL27 cells in nude mice.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R739.8

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