發(fā)布時(shí)間：2018-03-16 23:33

  本文選題：喉腫瘤　切入點(diǎn)：小RNA干擾　出處：《廣州醫(yī)科大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：研究背景:喉癌是耳鼻咽喉科常見(jiàn)的惡性腫瘤之一,占頭頸腫瘤的30%~50%,近年發(fā)病有上升趨勢(shì)。美國(guó)癌癥協(xié)會(huì)根據(jù)近30年的數(shù)據(jù)指出喉癌是所有惡性腫瘤五年生存率唯一沒(méi)得到提高的腫瘤。隨著分子生物學(xué)的發(fā)展,喉癌的發(fā)生發(fā)展己被證實(shí)是多基因、多步驟漸進(jìn)發(fā)展的結(jié)果。尋找新的分子診斷標(biāo)記物和治療靶點(diǎn),實(shí)現(xiàn)喉癌的個(gè)體化治療已成為當(dāng)前喉癌研究的重要方向之一。Toll樣受體(Toll like receptors,TLRs)是一種模式識(shí)別受體(pattern recognition receptor,PRRs),能識(shí)別來(lái)源于宿主的病原體相關(guān)分子模式(athogen-associated molecular patterns,PAMPs)。其中Toll樣受體3(Toll-like receptor 3,TLR3)是TLRs家族中重要成員之一,是一類(lèi)最重要的PRRs[1],與其它TLRs信號(hào)轉(zhuǎn)導(dǎo)不同的是,TLR3不依賴(lài)轉(zhuǎn)接蛋白髓樣分化因子88(myeloid differentiation foctor 88,MyD88),而是依賴(lài)TRIF(TIR-domain-containing molecule1,TICAM1)轉(zhuǎn)接蛋白,即所謂TRIF途徑。TLR3激活后,通過(guò)TRIF途徑介導(dǎo)了細(xì)胞的凋亡,其凋亡過(guò)程是通過(guò)過(guò)受體相互作用(receptor-inferact-ing protein,RIP)介導(dǎo)的caspase-3激活誘導(dǎo)的途徑。眾多研究表明腫瘤的發(fā)生與凋亡受阻有關(guān),誘導(dǎo)細(xì)胞凋亡已成為腫瘤治療的重要思路。在多發(fā)性骨髓瘤細(xì)胞,乳腺癌細(xì)胞,黑色素瘤細(xì)胞中TLR3均參與于細(xì)胞凋亡過(guò)程。迄今為止,國(guó)內(nèi)外針對(duì)喉癌中TLR3表達(dá)情況及其可能作用機(jī)制少見(jiàn)報(bào)道。本研究首先檢測(cè)TLR3在喉鱗癌組織表達(dá)與喉癌臨床特征相關(guān)情況;并利用小RNA干擾技術(shù)沉默TLR3在Hep-2細(xì)胞的表達(dá)進(jìn)一步研究siTLR3誘導(dǎo)Hep-2細(xì)胞凋亡可能機(jī)制和siTLR3對(duì)Hep-2抑制遷移及侵襲能力生物學(xué)行為的影響;以期明確TLR3基因在喉癌中的地位及可能的作用機(jī)制,為喉癌的診治提供新的篩選基因。本文旨在證實(shí)TLR3在喉鱗癌組織表達(dá)與喉癌臨床特征相關(guān)情況;并初步探討沉默TLR3基因后誘導(dǎo)喉癌hep-2細(xì)胞凋亡的可能調(diào)控機(jī)制。方法:(1)用免疫組化法檢測(cè)TLR3蛋白在53例喉鱗癌組織和30例癌旁正常(對(duì)照組)組織中的表達(dá),分析其與臨床病理學(xué)特征的關(guān)系(2)設(shè)計(jì)并合成針對(duì)TLR3的小干擾RNA(small interference RNA,siRNA)3條序列及與TLR3無(wú)基因同源性的陰性對(duì)照(NC,negative control),在lipofectamine3000介導(dǎo)下轉(zhuǎn)染Hep-2細(xì)胞,應(yīng)用PCR及Western blot驗(yàn)證轉(zhuǎn)染效率;(3)采用細(xì)胞劃痕實(shí)驗(yàn)和transwell侵襲實(shí)驗(yàn)檢測(cè)沉默TLR3對(duì)Hep-2細(xì)胞遷移和侵襲能力的影響。(4)通過(guò)細(xì)胞TUNEL檢測(cè)法和流式細(xì)胞技術(shù)分別檢測(cè)沉默TLR3后Hep-2細(xì)胞活力和細(xì)胞周期、細(xì)胞凋亡的變化。(5)采用Western blot檢測(cè)沉默TLR3表達(dá)后caspase/bcl-2家族蛋白水平的表達(dá)。結(jié)論:(1)TLR3在喉癌腫瘤組織中的表達(dá)和68%(36/53)明顯高于癌旁組織中的表達(dá)11%(6/53),差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。TLR3的表達(dá)水平與患者年齡、性別及吸煙史無(wú)明顯相關(guān)性,但與腫瘤T分期及淋巴結(jié)轉(zhuǎn)移N分期顯著相關(guān),差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。(2)TLR3siRNA-3組的TLR3表達(dá)最低(P0.05)。(3)細(xì)胞劃痕實(shí)驗(yàn)和Transwell侵襲實(shí)驗(yàn)顯示,siTLR3組細(xì)胞遷移和侵襲細(xì)胞數(shù)明顯低于NC組(P0.05)。(4)與NC組相比,siTLR3組細(xì)胞的細(xì)胞周期被阻滯在G2/M期(P0.05),S期細(xì)胞明顯減少(P0.05)。與NC組比,siTLR3實(shí)驗(yàn)組細(xì)胞凋亡率為較NC組的增加(P0.05)。siTLR3組的72小時(shí)細(xì)胞活力明顯低于NC組(P0.05)(5)細(xì)胞TUNEL實(shí)驗(yàn):siTLR3實(shí)驗(yàn)組每視野性中TUNEL檢測(cè)陽(yáng)性的細(xì)胞占20.1%,NC(陰性對(duì)照組)每視野性中TUNEL檢測(cè)陽(yáng)性的細(xì)胞占5%。兩者相比具有統(tǒng)計(jì)學(xué)意義(P0.05)(6)Western blot結(jié)果顯示,沉默TLR3基因后細(xì)胞cleaved-Caspase3、cleaved-Caspase8和cleaved-Caspase9均有不同程度的升高,抗凋亡蛋白Bcl-2的表達(dá)水平下降。
[Abstract]:Background: laryngeal cancer is one of the most common malignant tumor in otolaryngology head and neck cancer, accounting for 30%~50%, the incidence is rising in recent years. According to the American Cancer Society nearly 30 years of data suggest that laryngeal cancer is the five year survival rate of all malignant tumors not only improved the tumor. With the development of molecular biology, the occurrence and development of laryngeal carcinoma has proved to be multi gene, multi - step development. Looking for molecular diagnostic markers and novel therapeutic targets, implementation of individualized treatment of laryngeal cancer has become an important direction of current research of.Toll like receptors in laryngeal carcinoma (Toll like receptors, TLRs) is a kind of pattern recognition receptors (pattern, recognition receptor, PRRs) that can identify the source of pathogen associated molecular patterns from host (athogen-associated molecular patterns, PAMPs). The Toll like receptor 3 (Toll-like receptor 3, TLR3) is important in the TLRs family One of the most important, is a kind of PRRs[1], unlike other TLRs signal transduction is TLR3 independent adaptor protein myeloid differentiation factor 88 (myeloid differentiation 88 foctor, MyD88), but on TRIF (TIR-domain-containing molecule1, TICAM1) adaptor protein called TRIF to activate the.TLR3 pathway, mediated by TRIF pathway the cell apoptosis, the apoptosis process is through receptor interaction (receptor-inferact-ing protein RIP) mediated caspase-3 activation induced pathway. Many studies have shown that apoptosis and tumor blocked related apoptosis has become an important way for tumor therapy. In multiple myeloma cells, breast cancer cells, TLR3 melanoma cells were involved in the apoptotic process. So far, both at home and abroad for the expression of TLR3 in laryngeal carcinoma and its possible mechanism is rarely reported. This study firstly detected The expression and clinical features of laryngeal carcinoma TLR3 in laryngeal squamous cell carcinoma; and the use of small RNA further study on siTLR3 induced apoptosis in Hep-2 cells may influence the biological behavior of inhibition of migration and invasion mechanism and siTLR3 on the expression of Hep-2 silencing TLR3 in Hep-2 cells of interference; in order to clear the status of TLR3 gene in laryngeal carcinoma and its possible mechanism provide new genes for screening, diagnosis and treatment of laryngeal carcinoma. The purpose of this paper is to confirm the expression and clinical features of laryngeal carcinoma TLR3 in laryngeal squamous cell carcinoma; and to explore the possible regulatory mechanism of TLR3 gene silencing induced apoptosis of HEp-2 cells. Methods: (1) immunohistochemistry was used to detect the expression of TLR3 in 53 cases of laryngeal squamous cell carcinoma and 30 cases of adjacent normal (control group) in tissues, and analyze its relationship with clinical pathological feature (2) design and synthesis of small interfering RNA targeting TLR3 (small interference RNA, SiRNA) 3 sequences and TLR3 gene homologous negative control (NC, negative, control) in lipofectamine3000 mediated transfection of Hep-2 cells, the transfection efficiency of PCR Western blot and application verification; (3) the effect of silencing TLR3 by cell scratch assay and Transwell invasion assay on migration and invasion of Hep-2 cells. (4) Hep-2 after silencing TLR3 cell viability and cell cycle were detected by cell TUNEL assay and flow cytometry, changes of cell apoptosis. (5) using Western blot detection after silencing TLR3 expression of caspase/bcl-2 family protein expression. Conclusion: (1) the expression of TLR3 in laryngeal carcinoma and 68% tumor tissues (36/53 11%) was significantly higher than that in the adjacent tissues (6/53), the difference was statistically significant (P0.01) the expression level of.TLR3 with age, sex and smoking were significantly associated with the tumor, but T staging and lymph node metastases Shift N stage were significantly correlated, the difference was statistically significant (P0.01). (2) TLR3siRNA-3 group TLR3 was the lowest (P0.05). (3) showed cell scratch assay and Transwell invasion assay, cell migration and invasion of siTLR3 cells was significantly lower than that in NC group (P0.05). (4) compared with the NC group, cells group siTLR3 cell cycle arrest in G2/M phase (P0.05), S phase cells decreased significantly (P0.05). Compared with group NC, the apoptosis rate of siTLR3 cells in experimental group increased than those of group NC (P0.05).SiTLR3 group of 72 hours the cell viability was significantly lower than in group NC (P0.05) (5) TUNEL cell experiment siTLR3: each experimental group as the wild TUNEL detection in positive cells accounted for 20.1%, NC (negative control group) as per TUNEL compared to wild positive cells accounted for 5%. of the two has statistical significance (P0.05) (6) Western blot showed that TLR3 gene silencing cells after cleaved-Caspase3, cleaved-Caspase8 and cleaved-Caspase9 The expression of anti apoptotic protein Bcl-2 decreased in varying degrees.

【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R739.65

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