p53基因表达下调调控涎腺腺样囊性癌细胞SACC-83发生上皮间质样转化

发布时间：2018-02-20 05:56

本文关键词： SACC PNI P53 EMT　出处：《第四军医大学》2014年博士论文　论文类型：学位论文

【摘要】：腺样囊性癌(ACC)是一种临床常见的口腔颌面部恶性肿瘤,发病率占所有唾液腺肿瘤的10%左右,占所有唾液腺恶性肿瘤22%,占所有的头部和颈部恶性肿瘤约1%。小唾液腺及口腔是最常见的发病位点。ACC具有嗜神经侵袭(PNI)的特性,该特性是临床诊断和治疗ACC的一个难点。上皮间质转化(EMT)是指细胞由上皮形态转化为间质形态,是一个快速且通常可逆的细胞表型的改变,目前已被广泛引入肿瘤的发生发展研究中来。p53基因是一个公认的抑癌基因,国际癌症研究机构(IARC)p53数据库中含有超过26,000个体细胞突变型p53基因。不同肿瘤中p53基因突变的发生率不同,造血系统恶性肿瘤p53突变率约为10%,而卵巢癌、大肠癌、头颈部恶性肿瘤的突变率为50-70%。在本课题组前期的研究中发现,涎腺腺样囊性癌嗜神经侵袭基因表达谱中p53基因存在表达下调的情况,另有文献报道53基因可以调控乳腺癌细胞的EMT过程。因而揭示p53、EMT、涎腺腺样囊性癌PNI之间的相互关系在ACC的PNI研究和临床治疗中就产生了重要意义。目的:1.通过RNA干扰实验探明P53基因是否可以调控ACC发生EMT。2.通过实验探索p53基因与涎腺腺样囊性癌PNI之间的关系。方法:1.通过Real time-PCR、Western blot技术筛选出p53基因RNA干扰的最佳干扰片段。2.通过RNA干扰技术降低涎腺腺样囊性癌细胞系SACC-83细胞中p53基因的表达,并利用Western blot和流式细胞术检测EMT典型细胞表面标志物的表达情况,从而明确p53基因是否可以调控涎腺腺样囊性癌细胞系SACC-83细胞发生EMT。3.使用MTT细胞活性检测方法探明p53基因表达降低对SACC-83细胞活力的影响4.使用流式细胞术和Annexin V-FITCPI凋亡检测试剂盒探明p53基因表达降低对SACC-83细胞抗凋亡能力的影响。5.通过Transwell小室细胞迁移实验和划痕实验探明p53基因表达降低对SACC-83细胞体外嗜神经迁移能力的影响。6.通过改良Transwell小室细胞侵袭实验探明p53基因表达降低对SACC-83细胞体外嗜神经侵袭能力的影响。结果:1.成功筛选出p53基因RNA干扰最佳片段p53-homo270。2.RNA干扰降低p53基因表达可以诱导SACC-83发生EMT样转化。3.EMT样转化后的SACC-83细胞具有更强的细胞活力和抗凋亡性。4.EMT样转化后的SACC-83细胞具有更强的体外嗜神经迁移和侵袭能力。结论:1.p53基因可能通过表达降低诱导涎腺腺样囊性癌细胞系SACC-83发生上皮间质样转化;2.p53基因表达降低诱导产生的EMT样SACC-83细胞表型具有高表达间质细胞表型标志物Vimentin、N-Cadherin和C-Cadherin,低表达上皮细胞标志物E-cadherin、EMA和CK5同时这保持上皮细胞极性形态特征的特点;3.p53基因可以通过诱导EMT参与涎腺腺样囊性癌细胞系SACC-83细胞的体外嗜神经侵袭过程;4.p53基因可以通过诱导EMT增强涎腺腺样囊性癌细胞系SACC-83细胞的体外嗜神经侵袭能力;5.EMT样SACC-83细胞具备很强的体外嗜神经侵袭能力。
[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.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.87

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