本文選題：蛋白酪氨酸磷酸酶Shp2 + 口腔鱗狀細胞癌�。� 參考：《山東大學(xué)》2014年博士論文

【摘要】：目的： 頭頸部鱗狀細胞癌(Head and neck squamous cell carcinoma, HNSCC)主要影響口腔、舌咽、口咽、喉部及涎腺等,在常見的惡性腫瘤類型中位居第六,而口腔頜面部惡性腫瘤中約80%以上為口腔鱗狀細胞癌(oral squamous cell carcinoma, OSCC)。盡管近幾年我們在OSCC的治療以及發(fā)病機制研究方面取得了一定的進展,但是在過去的幾十年中其存活率仍沒有明顯改善,平均每年確診的病例中仍有約50%的死亡率。蛋白酪氨酸磷酸酶Shp2(Src homology phosphotyrosyl phosphatase2)由PTPN11基因編碼,廣泛地表達于機體各種組織和細胞中,其含有2個SH2結(jié)構(gòu)區(qū)域,分別為N-SH2、C-SH2,及一個PTP結(jié)構(gòu)區(qū)域,其作為細胞因子、生長因子及其他胞外刺激因素的下游信號分子,參與細胞增殖、分化、遷移等諸多細胞重要的生命活動。研究表明,Shp2的編碼基因PTPN11存在大量與人類疾病相關(guān)的遺傳多態(tài)性與突變位點,作為重要的節(jié)點分子,Shp2在血液系統(tǒng)惡性腫瘤如白血病及實體腫瘤如乳腺癌、肺癌、胃癌、肝癌等的發(fā)生發(fā)展過程中發(fā)揮著重要的調(diào)控作用,是其腫瘤治療的潛在分子靶點。目前國內(nèi)外關(guān)于Shp2在口腔鱗癌中的表達及作用的研究鮮見報道。因此,本實驗從臨床標(biāo)本、體外細胞和分子機制三個層面研究Shp2在口腔鱗癌中的表達及對其生物學(xué)行為的影響。首先通過臨床病例標(biāo)本檢測口腔鱗癌組織及癌旁組織中Shp2的表達差異,并進一步分析其表達水平與患者的臨床資料之間的關(guān)系,探討Shp2在口腔鱗癌中的臨床意義。其次,應(yīng)用RNA干擾(RNA interference, RNAi)技術(shù),構(gòu)建Shp2慢病毒干擾載體,抑制舌鱗狀細胞癌細胞SCC-4的Shp2基因表達,探討Shp2基因沉默后對SCC-4細胞增殖、凋亡及侵襲能力的作用。同時研究Shp2基因沉默后對細胞凋亡相關(guān)蛋白的影響,初步闡明Shp2在口腔鱗癌細胞凋亡發(fā)生中的分子機制。本研究將為口腔鱗癌臨床診斷、預(yù)后及治療的新靶點提供新的思路和理論基礎(chǔ)。 方法： 1.收集臨床口腔鱗癌組織蠟塊及臨床有關(guān)資料,采用免疫組織化學(xué)染色方法觀察Shp2蛋白在口腔鱗癌組織中的表達,并對免疫組化結(jié)果進行評分,評估Shp2的表達與患者的年齡、性別、煙酒暴露、腫瘤部位、腫瘤大小、臨床分期、淋巴轉(zhuǎn)移及病理分級等的相關(guān)性。 2.收集18例臨床手術(shù)中新鮮的口腔鱗癌及相應(yīng)的癌旁正常組織,采用WesternBlot方法,進一步驗證Shp2蛋白在口腔鱗癌組織及癌旁正常組織中的表達差異。 3.于Shp2PTPN11基因編碼區(qū)選擇4個siRNA靶點合成4對shRNA干擾序列,分別與慢病毒載體連接,構(gòu)建Shp2基因RNA干擾慢病毒載體,篩選出最佳干擾組,顯微鏡下觀察Shp2慢病毒干擾載體感染目的細胞人舌鱗狀細胞癌細胞SCC-4的效率。應(yīng)用Real Time PCR及Western blot檢測其干擾效率,篩選出最佳干擾組,建立穩(wěn)定抑制Shp2基因表達的SCC-4細胞株,為探討Shp2在口腔鱗癌發(fā)生中的作用提供細胞模型。 4.采用四甲基偶氮唑鹽(MTT)比色法觀察Shp2基因沉默后對SCC-4細胞24h、48h、72h及96h增殖率的影響。5.應(yīng)用流式細胞技術(shù)(Annexin V-APC/7-ADD染色法)及Westen Blot方法研究Shp2基因沉默后對SCC-4細胞凋亡的影響及其凋亡相關(guān)蛋白p53、Bax及Bcl-2的變化。6.采用Transwell小室侵襲實驗,通過蘇木精染色和細胞計數(shù)觀察Shp2基因沉默后對SCC-4細胞體外侵襲能力的影響。結(jié)果：1.免疫組化結(jié)果發(fā)現(xiàn),在口腔正常黏膜組織中,Shp2表達陰性或者呈弱表達,而在口腔鱗癌臨床樣本中高表達；進一步臨床資料統(tǒng)計分析表明,在口腔鱗癌中Shp2蛋白的表達與患者的性別、年齡、是否吸煙、飲酒無關(guān),與腫瘤的發(fā)生部位和體積大小亦無關(guān)。然而,臨床分期在III/IV期的腫瘤Shp2的表達較Ⅰ/Ⅱ期的強,在有淋巴轉(zhuǎn)移的病例中,Shp2的表達顯著增加,統(tǒng)計學(xué)上均有顯著性差異(p0.05)。 2.臨床新鮮口腔鱗癌組織進一步證明,除一例以外,Shp2在口腔鱗癌組織中的表達明顯高于癌旁正常組織中的表達(p0.05),進一步驗證了免疫組化結(jié)果。 3. Real-time PCR和Western blot檢測結(jié)果顯示Shp2-shRNA3干擾效率最佳,SCC-4細胞中Shp2基因及蛋白表達明顯降低,應(yīng)用RNAi設(shè)計的慢病毒干擾載體具有較好的干擾效果。 4.MTT法測定發(fā)現(xiàn),Shp2基因沉默后,96h內(nèi)SCC-4細胞的生長明顯慢于對照組,出現(xiàn)明顯的增殖抑制。 5.采用nnexin-V-APC/7-ADD雙熒光標(biāo)記,流式細胞儀檢測細胞凋亡發(fā)現(xiàn),轉(zhuǎn)染Shp2慢病毒于擾載體72h后的SCC-4細胞中,細胞凋亡率明顯增加,同對照組正常細胞相比,差異具有顯著性(p0.05)。Western Blot結(jié)果顯示,凋亡相關(guān)通路蛋白p53、Bax表達增加,Bcl-2表達下降。 6.[ranswell小室侵襲實驗結(jié)果顯示,轉(zhuǎn)染Shp2慢病毒干擾載體72h后的SCC-4細胞的侵襲能力與對照組相比較明顯下降,差異具有顯著性(p0.05)。 結(jié)論： Shp2在口腔鱗癌組織中的表達明顯高于癌旁正常組織,過表達的Shp2與口腔鱗癌的臨床分期及淋巴轉(zhuǎn)移密切相關(guān)；Shp2基因?qū)谇击[癌細胞的生長起促進作用,同時可提高腫瘤細胞的侵襲能力；Shp2能抑制口腔鱗癌細胞凋亡,凋亡相關(guān)蛋白p53、Bax及bcl-2可能參與其凋亡調(diào)控機制。以上結(jié)果提示,Shp2與口腔鱗癌的進展和轉(zhuǎn)移有關(guān),在口腔鱗癌可能發(fā)揮重要的促癌基因作用,是口腔鱗癌淋巴結(jié)轉(zhuǎn)移的危險因子,可作為口腔鱗癌臨床診斷、判斷預(yù)后、診療方案制定及腫瘤靶向治療的潛在生物標(biāo)記物和分子指標(biāo)。
[Abstract]:Objective:
Head and neck squamous cell carcinoma (HNSCC) mainly affects oral, glossopharynx, oropharynx, larynx, and salivary glands. It ranks sixth in common malignant tumor types, while more than 80% of oral and maxillofacial malignant tumors are oral squamous cell carcinoma (oral squamous cell carcinoma, OSCC). Some progress has been made in the treatment and pathogenesis of SCC, but the survival rate has not improved significantly over the past few decades, with an average of about 50% of the mortality in the confirmed cases each year. The protein tyrosine phosphatase Shp2 (Src homology phosphotyrosyl phosphatase2) is encoded by the PTPN11 gene and is widely expressed in the machine. In various tissues and cells, it contains 2 SH2 structure regions, which are N-SH2, C-SH2, and a PTP structure region. They are downstream signal molecules of cytokines, growth factors and other extracellular stimuli, participating in cell proliferation, differentiation, migration and many other vital activities. The study shows that the Shp2 encoding gene PTPN11 exists. A large number of genetic polymorphisms and mutation sites associated with human disease, as an important node molecule, play an important regulatory role in the development of hematological malignancies such as leukemia and solid tumors such as breast cancer, lung cancer, gastric cancer, and liver cancer. It is a potential molecular target for the treatment of cancer, and Shp2 is currently in the world. The study of the expression and role of Shp2 in oral squamous cell carcinoma is rarely reported. Therefore, this experiment studies the expression of Shp2 in oral squamous cell carcinoma and its effect on its biological behavior from three levels of clinical specimens, in vitro cell and molecular mechanism. First, the difference of Shp2 expression in oral squamous cell carcinoma and para cancerous tissue is detected by clinical case specimens, and The relationship between the expression level and the clinical data of the patients was further analyzed, and the clinical significance of Shp2 in oral squamous cell carcinoma was discussed. Secondly, the RNA interference (RNA interference, RNAi) technology was used to construct the Shp2 lentivirus interference carrier, to inhibit the Shp2 gene expression of SCC-4 in the squamous cell carcinoma cell of the tongue and to explore the proliferation of SCC-4 cells after the Shp2 gene was silenced. The effect of apoptosis and invasion ability. At the same time, the effect of Shp2 gene silencing on apoptosis related proteins is studied, and the molecular mechanism of Shp2 in the apoptosis of oral squamous cell carcinoma is preliminarily clarified. This study will provide new ideas and theoretical basis for the clinical diagnosis of oral squamous cell carcinoma, and the new target of prognosis and treatment.
Method錛，

本文編號：1905415