本文關(guān)鍵詞： SACC PNI P53 EMT　出處：《第四軍醫(yī)大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：腺樣囊性癌(ACC)是一種臨床常見的口腔頜面部惡性腫瘤,發(fā)病率占所有唾液腺腫瘤的10%左右,占所有唾液腺惡性腫瘤22%,占所有的頭部和頸部惡性腫瘤約1%。小唾液腺及口腔是最常見的發(fā)病位點(diǎn)。ACC具有嗜神經(jīng)侵襲(PNI)的特性,該特性是臨床診斷和治療ACC的一個(gè)難點(diǎn)。上皮間質(zhì)轉(zhuǎn)化(EMT)是指細(xì)胞由上皮形態(tài)轉(zhuǎn)化為間質(zhì)形態(tài),是一個(gè)快速且通常可逆的細(xì)胞表型的改變,目前已被廣泛引入腫瘤的發(fā)生發(fā)展研究中來。p53基因是一個(gè)公認(rèn)的抑癌基因,國(guó)際癌癥研究機(jī)構(gòu)(IARC)p53數(shù)據(jù)庫(kù)中含有超過26,000個(gè)體細(xì)胞突變型p53基因。不同腫瘤中p53基因突變的發(fā)生率不同,造血系統(tǒng)惡性腫瘤p53突變率約為10%,而卵巢癌、大腸癌、頭頸部惡性腫瘤的突變率為50-70%。在本課題組前期的研究中發(fā)現(xiàn),涎腺腺樣囊性癌嗜神經(jīng)侵襲基因表達(dá)譜中p53基因存在表達(dá)下調(diào)的情況,另有文獻(xiàn)報(bào)道53基因可以調(diào)控乳腺癌細(xì)胞的EMT過程。因而揭示p53、EMT、涎腺腺樣囊性癌PNI之間的相互關(guān)系在ACC的PNI研究和臨床治療中就產(chǎn)生了重要意義。目的:1.通過RNA干擾實(shí)驗(yàn)探明P53基因是否可以調(diào)控ACC發(fā)生EMT。2.通過實(shí)驗(yàn)探索p53基因與涎腺腺樣囊性癌PNI之間的關(guān)系。方法:1.通過Real time-PCR、Western blot技術(shù)篩選出p53基因RNA干擾的最佳干擾片段。2.通過RNA干擾技術(shù)降低涎腺腺樣囊性癌細(xì)胞系SACC-83細(xì)胞中p53基因的表達(dá),并利用Western blot和流式細(xì)胞術(shù)檢測(cè)EMT典型細(xì)胞表面標(biāo)志物的表達(dá)情況,從而明確p53基因是否可以調(diào)控涎腺腺樣囊性癌細(xì)胞系SACC-83細(xì)胞發(fā)生EMT。3.使用MTT細(xì)胞活性檢測(cè)方法探明p53基因表達(dá)降低對(duì)SACC-83細(xì)胞活力的影響4.使用流式細(xì)胞術(shù)和Annexin V-FITCPI凋亡檢測(cè)試劑盒探明p53基因表達(dá)降低對(duì)SACC-83細(xì)胞抗凋亡能力的影響。5.通過Transwell小室細(xì)胞遷移實(shí)驗(yàn)和劃痕實(shí)驗(yàn)探明p53基因表達(dá)降低對(duì)SACC-83細(xì)胞體外嗜神經(jīng)遷移能力的影響。6.通過改良Transwell小室細(xì)胞侵襲實(shí)驗(yàn)探明p53基因表達(dá)降低對(duì)SACC-83細(xì)胞體外嗜神經(jīng)侵襲能力的影響。結(jié)果:1.成功篩選出p53基因RNA干擾最佳片段p53-homo270。2.RNA干擾降低p53基因表達(dá)可以誘導(dǎo)SACC-83發(fā)生EMT樣轉(zhuǎn)化。3.EMT樣轉(zhuǎn)化后的SACC-83細(xì)胞具有更強(qiáng)的細(xì)胞活力和抗凋亡性。4.EMT樣轉(zhuǎn)化后的SACC-83細(xì)胞具有更強(qiáng)的體外嗜神經(jīng)遷移和侵襲能力。結(jié)論:1.p53基因可能通過表達(dá)降低誘導(dǎo)涎腺腺樣囊性癌細(xì)胞系SACC-83發(fā)生上皮間質(zhì)樣轉(zhuǎn)化;2.p53基因表達(dá)降低誘導(dǎo)產(chǎn)生的EMT樣SACC-83細(xì)胞表型具有高表達(dá)間質(zhì)細(xì)胞表型標(biāo)志物Vimentin、N-Cadherin和C-Cadherin,低表達(dá)上皮細(xì)胞標(biāo)志物E-cadherin、EMA和CK5同時(shí)這保持上皮細(xì)胞極性形態(tài)特征的特點(diǎn);3.p53基因可以通過誘導(dǎo)EMT參與涎腺腺樣囊性癌細(xì)胞系SACC-83細(xì)胞的體外嗜神經(jīng)侵襲過程;4.p53基因可以通過誘導(dǎo)EMT增強(qiáng)涎腺腺樣囊性癌細(xì)胞系SACC-83細(xì)胞的體外嗜神經(jīng)侵襲能力;5.EMT樣SACC-83細(xì)胞具備很強(qiáng)的體外嗜神經(jīng)侵襲能力。
[Abstract]:Adenoid cystic carcinoma (ACCs) is a common malignant tumor in oral and maxillofacial region. The incidence of ACCs is about 10% of all salivary gland tumors. It accounts for 22 percent of all malignant tumors of the salivary glands, about 1 percent of all malignant tumors of the head and neck. The small salivary glands and the mouth are the most common sites for the disease. ACC has the characteristics of neurophilic invasion of PNIs. This characteristic is a difficult point in clinical diagnosis and treatment of ACC. Epithelial mesenchymal transformation (EMT) is a rapid and usually reversible phenotypic change of cells from epithelial to interstitial morphology. At present, p53 gene is a recognized tumor suppressor gene, which has been widely introduced into the study of tumorigenesis and development. More than 26,000 somatic mutant p53 genes are found in the IARCU p53 database of the International Agency for Research on Cancer. The incidence of p53 gene mutations varies from tumor to tumor. The mutation rate of p53 in malignant tumors of hematopoietic system is about 10 percent, while that in ovarian cancer and colorectal cancer is about 10 percent. The mutation rate of malignant tumors of head and neck ranged from 50 to 70. In our previous study, we found that p53 gene expression was down-regulated in salivary adenoid cystic carcinoma gene expression profile. It has also been reported that the 53 gene can regulate the EMT process of breast cancer cells. Therefore, it is important to reveal the relationship between p53 EMT, salivary adenoid cystic carcinoma and PNI in the PNI research and clinical treatment of ACC. The interference experiment was conducted to find out whether p53 gene could regulate the occurrence of EMT.2. to explore the relationship between p53 gene and PNI in salivary adenoid cystic carcinoma. Methods: 1. The best interfering fragment of p53 gene RNA interference was screened by Real time-PCRN Western blot technique. The expression of p53 gene in salivary adenoid cystic carcinoma cell line SACC-83 was reduced by RNA interference. Western blot and flow cytometry were used to detect the expression of EMT typical cell surface markers. In order to determine whether p53 gene can regulate the development of EMT.3in salivary adenoid cystic carcinoma cell line SACC-83, the effect of decreased p53 gene expression on the viability of SACC-83 cells was detected by MTT assay. 4. Flow cytometry and Annexin V-FITCPI regulation were used to detect the effect of p53 gene expression on the viability of SACC-83 cells. 4. Flow cytometry and Annexin V-FITCPI regulation. The effect of decreased p53 gene expression on the anti-apoptotic ability of SACC-83 cells was detected by the assay kit. 5. The effect of decreased p53 gene expression on the neuronal migration ability of SACC-83 cells in vitro was detected by Transwell cell migration assay and scratch test. The effect of decreased p53 gene expression on the eosinophil invasiveness of SACC-83 cells in vitro was demonstrated by modified Transwell chamber cell invasion assay. Results: 1. The best fragment of p53 gene RNA interference p53-homo270.2.RNA interference can induce p53 gene expression. EMT like transformation of SACC-83. 3. EMT-like transformed SACC-83 cells have stronger cell viability and anti-apoptosis. 4. EMT-like transformed SACC-83 cells have stronger eosinophil migration and invasion ability in vitro. Conclusion: 1. P53 gene may be expressed downwards. Low induced epithelial interstitial transformation of salivary gland adenoid cystic carcinoma cell line 2.The expression of p53 gene reduced the induced phenotype of EMT like SACC-83 cells with high expression of interstitial cell phenotype markers Vimentin N-Cadherin and C-Cadherin. P53 gene can induce EMT to participate in the process of eosinophil invasion of salivary adenoid cystic carcinoma cell line SACC-83 cells in vitro. 4. P53 gene can enhance salivary gland gland by inducing EMT to enhance salivary gland gland. In vitro eosinophil invasion ability of SACC-83 cells. 5. EMT-like SACC-83 cells have strong in vitro eosinophil invasion ability.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.87

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