本文關(guān)鍵詞： 喉癌 自噬 Beclin1 凋亡 順鉑　出處：《山西醫(yī)科大學(xué)》2013年博士論文　論文類型：學(xué)位論文

【摘要】：背景與研究目的 喉癌是頭頸部常見(jiàn)的惡性腫瘤之一,嚴(yán)重威脅著患者的健康,尋找有效的治療方法一直是臨床的迫切需求,在喉癌的治療中,基因治療已成為手術(shù)、化療放療三大治療外新的治療手段。 自噬是細(xì)胞內(nèi)成分被溶酶體降解過(guò)程的統(tǒng)稱,在清除損傷或多余的細(xì)胞器,維持細(xì)胞內(nèi)環(huán)境穩(wěn)定方面起著重要作用。自噬可清除損傷的細(xì)胞器,避免如自由基等有毒性物質(zhì)的產(chǎn)生,從而使突變率降低,抑制腫瘤的形成,而自噬的缺陷可導(dǎo)致腫瘤的發(fā)生。Beclin1基因是第一個(gè)被發(fā)現(xiàn)的哺乳動(dòng)物的自噬基因,在多種腫瘤中表現(xiàn)為表達(dá)降低和缺失,Beclin1表達(dá)缺失明顯增加肝癌、肺癌和淋巴瘤的發(fā)生率,但自噬基因Beclin1與喉癌的關(guān)系尚不清楚。 本研究通過(guò)檢測(cè)喉癌組織中自噬基因Beclin1的表達(dá),探討自噬基因Beclin1與喉癌發(fā)生發(fā)展的關(guān)系；研究自噬基因Beclin1在喉癌Hep-2細(xì)胞中表達(dá)增強(qiáng)和缺失對(duì)細(xì)胞體外增殖、自噬、凋亡的作用及對(duì)喉癌Hep-2細(xì)胞順鉑敏感性的影響,探討自噬誘導(dǎo)在喉癌治療中的可行性,為喉癌的基因治療提供新的思路。 方法： 1.采用免疫組化SP法對(duì)77例喉癌組織和22例正常喉組織進(jìn)行Beclin1蛋白表達(dá)水平進(jìn)行檢測(cè),分析Beclin1與喉癌臨床分型、T分期、組織分化程度、淋巴結(jié)轉(zhuǎn)移等臨床病理因素的相關(guān)性； 2.通過(guò)RT-PCR法從人正常喉組織中獲得目的基因Beclin1,將其插入真核表達(dá)載體pcDNA3.1中,構(gòu)建真核表達(dá)載體pcDNA3.1/Beclin1;設(shè)計(jì)針對(duì)Beclin1的特異性RNA干擾序列,構(gòu)建Beclin1基因的小發(fā)夾狀RNA (shRNA)真核表達(dá)質(zhì)粒pSUPER/Beclin1; 3.脂質(zhì)體法將重組質(zhì)粒pcDNA3.1/Beclinl和pSUPER/Beclin1轉(zhuǎn)染人喉癌細(xì)胞株Hep-2,篩選穩(wěn)定表達(dá)株,采用熒光定量RT-PCR及Western Blot分別在mRNA和蛋白質(zhì)水平檢測(cè)轉(zhuǎn)染前后Beclin1、LC3和p62蛋白的表達(dá)變化；流式細(xì)胞儀檢測(cè)自噬情況；Hoechs檢測(cè)細(xì)胞凋亡；MTT法檢測(cè)細(xì)胞的增殖變化； 4.順鉑處理Hep-2細(xì)胞后,MDC染色檢測(cè)自噬情況；將重組質(zhì)粒pcDNA3.1/Beclin1和pSUPER/Beclin1轉(zhuǎn)染人喉癌細(xì)胞株Hep-2后,順鉑處理,MTT法檢測(cè)細(xì)胞的增殖變化。 結(jié)果： 1.喉癌組織中Beclin1的蛋白表達(dá)陽(yáng)性率低于正常喉組織(P0.05),喉癌中常見(jiàn)Beclin1表達(dá)下調(diào)和缺失；Beclin1表達(dá)與喉癌的臨床分型無(wú)關(guān)(P0.05),與喉癌的T分期、分化程度及淋巴結(jié)轉(zhuǎn)移相關(guān)(P0.05) 2.利用PCR分析,酶切鑒定及DNA測(cè)序證實(shí)Beclin1基因的表達(dá)質(zhì)粒pcDNA3.1/Beclin1(?)PRNA干擾質(zhì)粒pSUPER/Beclin1構(gòu)建成功； 3. pcDNA3.1/Beclin1轉(zhuǎn)染Hep-2細(xì)胞后,Beclin1、LC3Ⅱ mRNA和蛋白的表達(dá)顯著提高,p62mRNA和蛋白的表達(dá)顯著降低(P0.05),與未轉(zhuǎn)染組相比,凋亡細(xì)胞和自噬細(xì)胞明顯增加,細(xì)胞生長(zhǎng)活性降低； 4. pSUPER/Beclin1轉(zhuǎn)染Hep-2細(xì)胞后,Beclin1 LC3Ⅱ mRNA和蛋白的表達(dá)明顯降低,p62mRNA和蛋白的表達(dá)顯著增高(P0.05),與未轉(zhuǎn)染組相比,凋亡細(xì)胞和自噬細(xì)胞明顯減少,細(xì)胞生長(zhǎng)活性增強(qiáng)； 5.順鉑處理Hep-2細(xì)胞后,細(xì)胞內(nèi)自噬增加,且呈濃度依賴關(guān)系,重組質(zhì)粒pSUPER/Beclin1可以顯著提高Hep-2細(xì)胞對(duì)順鉑的敏感性。 結(jié)論： 自噬基因Beclin1與喉癌的發(fā)生、發(fā)展有關(guān),自噬基因Beclin1可有效抑制喉癌細(xì)胞的增殖,提高腫瘤細(xì)胞的自噬和凋亡能力,同時(shí)抑制自噬基因Beclin1表達(dá)能提高喉癌Hep-2細(xì)胞對(duì)順鉑的敏感性。
[Abstract]:Background and research purpose
Laryngeal cancer is one of the most common malignant tumors in head and neck. It seriously threatens the health of patients. Finding effective treatment is always an urgent need. In the treatment of laryngeal cancer, gene therapy has become a new treatment for surgery, chemotherapy and radiotherapy, the three major treatment.
Autophagy is a cellular constituents known lysosomal degradation process, in the clearance of damaged or superfluous organelles, plays an important role in maintaining cell homeostasis. Autophagy may remove cell injury, to avoid such as free radicals, toxic substances, so that the mutation rate, inhibit the formation of tumors,.Beclin1 gene and autophagy defects can lead to tumor is the autophagy gene first discovered in mammals, in a variety of tumors in reduced expression and deletion, loss of Beclin1 expression significantly increased the incidence of liver cancer, lung cancer and lymphoma, but the relationship between autophagy gene Beclin1 and laryngeal cancer is not clear.
This study through the detection of expression of autophagy gene Beclin1 in laryngeal squamous cell carcinoma, and explore the relationship between autophagy gene Beclin1 and the occurrence and development of laryngeal carcinoma; and the lack of enhanced expression of autophagy on cell proliferation in vitro, study of autophagy gene Beclin1 in laryngeal carcinoma Hep-2 cells, apoptosis and sensitivity to cisplatin of Hep-2 laryngeal carcinoma cells, to explore the feasibility of autophagy induction in the treatment of laryngeal cancer in, to provide new ideas for gene therapy of laryngeal carcinoma.
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本文編號(hào)：1528239