抑制HES1、HEY1对涎腺腺样囊性癌细胞功能的影响
发布时间：2018-08-28 12:51
【摘要】：涎腺腺样囊性癌(SACC)是好发于腮腺、颌下腺及小涎腺的口腔颌面部恶性肿瘤之一,具有嗜神经侵袭以及远处转移两大生物学特性,其临床生物学特性表现为感觉和(或)运动神经功能障碍,易通过血行转移至肺,术后易复发,预后较差。本课题组通过前期大量的实验研究,发现Notch信号通路与肿瘤的发生发展密切相关,通路中的受体基因Notch1能够作为促癌基因促进涎腺腺样囊性癌的增殖、侵袭及迁移。国内外众多研究表明,HES1、HEY1基因是Notch1信号通路的下游基因,二者可能在多种肿瘤的发展演化中发挥着癌基因的作用。本研究在课题组前期研究的基础上,筛选出Notch1信号通路的下游基因HES1、HEY1,研究其与涎腺腺样囊性癌细胞增殖、侵袭、迁移的关系并对可能的机制进行探讨。本研究按照基因的筛选和细胞功能学实验分为两部分,分述如下:一、在SACC细胞中利用Real-time PCR技术筛选出与Notch1变化相关的下游基因,并在组织水平验证其表达。目的:筛选与Notch1表达变化相关的下游靶基因。方法:根据文献选取的常见的Notch1的下游基因设计引物。用Notch1的si RNA转染SACC-83细胞,通过Real-time PCR技术检测Notch1下游基因的表达量;构建Notch1基因重组腺病毒表达载体,感染SACC-83细胞,应用Real-time PCR技术研究其下游基因的表达变化。收集涎腺腺样囊性癌组织和癌旁组织的石蜡标本,利用筛选出的下游基因的相应抗体进行免疫组化,经2名病理科医师阅片评分后统计结果。结果:Real-time PCR检测结果显示Notch1表达下降后,HES1、HEY1的表达也相应降低;而Notch1表达升高后,HES1、HEY1的表达也随之上升。涎腺腺样囊性癌癌组织标本中HES1、HEY1的表达高于癌旁组织。结论:HES1、HEY1的表达变化与Notch1的表达变化呈正相关关系。二、利用si RNA干扰技术探讨HES1、HEY1对SACC细胞功能的影响。目的:研究HES1、HEY1在SACC细胞增殖、侵袭以及迁移中的作用。方法:设计合成HES1、HEY1的si RNA,转染SACC-83细胞,HES1、HEY1的表达量的变化通过Real-time PCR技术进行验证,筛选出两条干扰有效的si RNA,转染SACC-83细胞使其HES1、HEY1的表达显著下调后,采用CCK8法、平板克隆形成实验研究细胞增殖能力的变化;采用细胞划痕愈合法研究细胞迁移能力的变化;采用侵袭小室法研究细胞侵袭能力的变化;采用流式细胞术研究细胞周期和凋亡的变化。结果:Real-time PCR结果显示HES1、HEY1表达下降;CCK8和平板克隆形成实验证实HES1、HEY1转染后,细胞增殖能力弱于阴性对照组;侵袭小室、划痕愈合实验结果表明,HES1、HEY1表达下降后,SACC-83细胞的侵袭、迁移能力也随之下降;流式细胞术的结果发现转染HES1、HEY1后细胞的早期和晚期凋亡率升高,而细胞周期和对照组相比没有差异。结论:抑制HES1、HEY1基因的表达后SACC细胞的增殖、侵袭和迁移能力均下降,而细胞凋亡率升高。
[Abstract]:Salivary adenoid cystic carcinoma (SACC) is one of the most common oral and maxillofacial malignancies in parotid, submandibular and small salivary glands. Its clinical biological characteristics were sensory and / or motor nerve dysfunction, which were easily metastasized to the lung by blood, and were easy to recur after operation, and the prognosis was poor. Through a large number of experimental studies, we found that the Notch signaling pathway is closely related to the occurrence and development of the tumor. The receptor gene Notch1 in the pathway can promote the proliferation, invasion and migration of salivary adenoid cystic carcinoma (SCC) as a gene to promote the proliferation, invasion and migration of salivary adenoid cystic carcinoma. Many studies at home and abroad have shown that the HES1 gene is the downstream gene of Notch1 signaling pathway, and both of them may play an important role in the development and evolution of many kinds of tumors. On the basis of the previous study, the downstream gene HES1,HEY1, of Notch1 signaling pathway was selected to study its relationship with salivary adenoid cystic carcinoma cell proliferation, invasion and migration, and the possible mechanism was discussed. This study was divided into two parts according to gene screening and cell function experiment. Firstly, the downstream genes associated with Notch1 changes were screened by Real-time PCR technique in SACC cells, and their expression was verified at the tissue level. Objective: to screen the downstream target genes associated with the change of Notch1 expression. Methods: primers were designed according to the common downstream genes of Notch1. SACC-83 cells were transfected with si RNA of Notch1 and the expression of downstream gene of Notch1 was detected by Real-time PCR. The recombinant adenovirus expression vector of Notch1 gene was constructed and infected with SACC-83 cells. The expression of downstream gene was studied by Real-time PCR technique. Paraffin specimens of salivary adenoid cystic carcinoma and paracancerous tissues were collected. The corresponding antibodies of the downstream genes were used for immunohistochemistry. The results were evaluated by two pathologists. Results the expression of HES1 and HEY1 was also decreased after the decrease of Notch1 expression, and the expression of HES 1 + HEY1 was also increased after the increase of Notch1 expression. The expression of HES1,HEY1 in salivary adenoid cystic carcinoma tissues was higher than that in paracancerous tissues. Conclusion there is a positive correlation between the expression of Notch1 and the changes of the expression of HeS1 and HEY1. Secondly, the effect of HES1,HEY1 on the function of SACC cells was studied by si RNA interference technique. Aim: to investigate the role of HES1,HEY1 in the proliferation, invasion and migration of SACC cells. Methods: the changes of the expression level of HES1,HEY1 si RNA, transfected SACC-83 cells were verified by Real-time PCR technique. Two interfering si RNA, transfected SACC-83 cells were screened out and their HES1,HEY1 expression was significantly down-regulated. CCK8 assay was used. The changes of cell proliferation ability were studied by plate clone formation assay, cell migration ability was studied by cell scratch healing method, and cell invasion ability was studied by invasive chamber method. The changes of cell cycle and apoptosis were studied by flow cytometry. Results the cell proliferation ability after transfection of HES1,HEY1 was weaker than that of negative control group, the cell proliferation ability was weaker after transfection of HES1,HEY1, and the invasion of SACC-83 cells after HES1,HEY1 transfection and scratch healing test showed that the expression of HES 1 and HEY1 decreased. Flow cytometry showed that the early and late apoptosis rate of HES1,HEY1 transfected cells increased, but the cell cycle had no difference compared with the control group. Conclusion: after inhibiting the expression of HES1,HEY1 gene, the proliferation, invasion and migration of SACC cells decreased, while the rate of apoptosis increased.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R739.87
[Abstract]:Salivary adenoid cystic carcinoma (SACC) is one of the most common oral and maxillofacial malignancies in parotid, submandibular and small salivary glands. Its clinical biological characteristics were sensory and / or motor nerve dysfunction, which were easily metastasized to the lung by blood, and were easy to recur after operation, and the prognosis was poor. Through a large number of experimental studies, we found that the Notch signaling pathway is closely related to the occurrence and development of the tumor. The receptor gene Notch1 in the pathway can promote the proliferation, invasion and migration of salivary adenoid cystic carcinoma (SCC) as a gene to promote the proliferation, invasion and migration of salivary adenoid cystic carcinoma. Many studies at home and abroad have shown that the HES1 gene is the downstream gene of Notch1 signaling pathway, and both of them may play an important role in the development and evolution of many kinds of tumors. On the basis of the previous study, the downstream gene HES1,HEY1, of Notch1 signaling pathway was selected to study its relationship with salivary adenoid cystic carcinoma cell proliferation, invasion and migration, and the possible mechanism was discussed. This study was divided into two parts according to gene screening and cell function experiment. Firstly, the downstream genes associated with Notch1 changes were screened by Real-time PCR technique in SACC cells, and their expression was verified at the tissue level. Objective: to screen the downstream target genes associated with the change of Notch1 expression. Methods: primers were designed according to the common downstream genes of Notch1. SACC-83 cells were transfected with si RNA of Notch1 and the expression of downstream gene of Notch1 was detected by Real-time PCR. The recombinant adenovirus expression vector of Notch1 gene was constructed and infected with SACC-83 cells. The expression of downstream gene was studied by Real-time PCR technique. Paraffin specimens of salivary adenoid cystic carcinoma and paracancerous tissues were collected. The corresponding antibodies of the downstream genes were used for immunohistochemistry. The results were evaluated by two pathologists. Results the expression of HES1 and HEY1 was also decreased after the decrease of Notch1 expression, and the expression of HES 1 + HEY1 was also increased after the increase of Notch1 expression. The expression of HES1,HEY1 in salivary adenoid cystic carcinoma tissues was higher than that in paracancerous tissues. Conclusion there is a positive correlation between the expression of Notch1 and the changes of the expression of HeS1 and HEY1. Secondly, the effect of HES1,HEY1 on the function of SACC cells was studied by si RNA interference technique. Aim: to investigate the role of HES1,HEY1 in the proliferation, invasion and migration of SACC cells. Methods: the changes of the expression level of HES1,HEY1 si RNA, transfected SACC-83 cells were verified by Real-time PCR technique. Two interfering si RNA, transfected SACC-83 cells were screened out and their HES1,HEY1 expression was significantly down-regulated. CCK8 assay was used. The changes of cell proliferation ability were studied by plate clone formation assay, cell migration ability was studied by cell scratch healing method, and cell invasion ability was studied by invasive chamber method. The changes of cell cycle and apoptosis were studied by flow cytometry. Results the cell proliferation ability after transfection of HES1,HEY1 was weaker than that of negative control group, the cell proliferation ability was weaker after transfection of HES1,HEY1, and the invasion of SACC-83 cells after HES1,HEY1 transfection and scratch healing test showed that the expression of HES 1 and HEY1 decreased. Flow cytometry showed that the early and late apoptosis rate of HES1,HEY1 transfected cells increased, but the cell cycle had no difference compared with the control group. Conclusion: after inhibiting the expression of HES1,HEY1 gene, the proliferation, invasion and migration of SACC cells decreased, while the rate of apoptosis increased.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R739.87
【参考文献】
相关期刊论文 前10条
1 王春华;郑志z，
本文编号：2209447
本文链接：https://www.wllwen.com/yixuelunwen/kouq/2209447.html
最近更新
教材专著
