【作者】 程勇；

【导师】 余优成；

【作者基本信息】 复旦大学 ， 口腔临床医学（专业学位）， 2013， 硕士

【摘要】 目的：口腔鳞状细胞癌是口腔颌面部常见的恶性肿瘤之一,其治疗主要采取以手术、放疗、化疗、生物治疗等为主的综合序列治疗方法。但近二十年来,口腔恶性肿瘤患者的五年生存率仍然维持在50%左右。晚期患者的生存率更低。根据口腔鳞状细胞癌的发生和发展的机理,发现或者发明新的治疗方法或治疗药物十分迫切。组蛋白去乙酰化酶(HDAC)与组蛋白乙酰化酶动态调控组蛋白与DNA的亲和性而调节染色质的状态,从而调控基因表达。目前研究发现多种肿瘤的发生发展与HDAC的调控异常有关。丙戊酸是一种经典的抗癫痫药物,近年来因为被发现可以抑制HDAC,从而影响肿瘤的生物学行为如增殖、凋亡、生长周期、分化等,可能成为一种新型的抗肿瘤药物而倍受瞩目。但是丙戊酸对口腔肿瘤的作用研究较少,其中的作用机制尚不清楚,本文主要研究其对口腔鳞状细胞癌细胞系CAL-27生物学行为的影响以及可能的作用机制进行探讨。方法：1.相差显微镜下,观察不同丙戊酸对CAL-27细胞形态的影响。2.甲基噻唑基四唑盐比色法,观察丙戊酸对CAL-27细胞的毒性和生长抑制情况。3.流式细胞技术检测丙戊酸对CAL-27细胞凋亡和细胞周期的影响。4.荧光定量PCR法检测在丙戊酸诱导CAL-27分化和凋亡过程中,小泛素样修饰物(SUMO)相关基因的mRNA变化。结果：1.随着丙戊酸浓度的升高,丙戊酸对CAL-27的生长抑制逐渐增加,细胞凋亡率亦逐渐增加,在浓度为2mM时,影响最为显著。丙戊酸作用于CAL-27后,细胞周期发生阻滞,G1期增加,S期减少,G2期无明显变化。2.丙戊酸作用于CAL-27细胞24小时后,分化指标外皮蛋白mRNA转录水平明显增加,细胞分化。3.在丙戊酸诱导CAL-27细胞凋亡和分化过程中,小泛素样修饰物的部分相关基因发生改变,其中SENP1表达下调,SENP3、SUMO1、SUM02表达上调。结论：1.丙戊酸可以抑制口腔鳞状细胞癌细胞系CAL-27细胞的生长,细胞周期阻滞,诱导细胞的凋亡,促进细胞的分化。2.在丙戊酸诱导口腔鳞状细胞癌细胞CAL-27凋亡和分化过程中,小泛素样修饰物相关的部分基因mRNA水平发生改变,其可能参与了丙戊酸诱导CAL-27细胞凋亡和分化。更多还原

【Abstract】 Objective:Oral squamous cell carcinoma is a common malignant tumor in oral and maxillofacial region. Generally, it is treated by sequence therapy consisting of surgery, radiotherapy, chemotherapy, biological therapy. However, in the recent20years, the five-year survival rate of patients with oral malignancy has been remaining at50%, which is even lower with advanced tumor. In order to regulate gene expression, Histone deacetylase (HDAC) and Histone acetyltransferases (HAT) dynamically adjust the affinity of histone and DNA moderate chromatin. Previously, researchers found that many tumors developed with an abnormal regulation of HDAC. Valproic acid, a classic anti-epileptic drug, may become a new type of anticancer drugs because recent study found that it could inhibit HDAC and consequently affected the biological behavior of the tumor, such as proliferation, apoptosis, cell cycle and differentiation. This study aimed to investigate how valproic acid affected oral squamous cell carcinoma cell lines CAL-27as well as the potential mechanism for oral tumor treatment.Methods:1. To observe cell morphology change of CAL-27induced by valprioc acid via inverted phase contrast microscope.2. To detect the toxicity and inhibitory effects of valproic acid on the growth of CAL-27cells via methylthiazolyltetrazolium assay (MTT assay)3. To detect the apoptosis and cell cycle of CAL-27induced by valproic acid via flow-cytometry.4. To detect the mRNA change of SUMO related genes during CAL-27differentiation and apoptosis induced by valproic acid via real time fluorescence quantitative PCR. Results:1. With valproic acid concentration increased, inhibitory effect on growth of CAL-27gradually strengthened as well as the apoptosis rate, but at a concentration of2mM perform significantly. CAL-27cells were treated with valproic acid for24hours, when cell cycle arrest with G1phase increased, S phase decreased, and the G2phase changed unobviously, statistically significant difference.2. An indicator of CAL-27cell differentiation, involucrin, mRNA level increased significantly when cells were treated with valproic acid at2mM up to24hours.3. During the process of CAL-27cell differentiation and apoptosis, SENP1mRNA level downregulated at12hours, while SENP3, SUM01and SUM02increased.Conclusions:1. Valproic acid could inhibit the growth of oral squamous cell carcinoma cell line CAL-27cells. During the process, cell cycle arrested, cell apoptosis increased and cell began to differentiate.2. Some of small ubiquitin-like modifier (SUMO) gene profile changed during CAL-27cell apoptosis and differentiation induced by valproic acid, the SUMO pathway possibly participate the process.更多还原