口腔鳞状细胞癌的预后标记物及靶向药物研究

发布时间：2018-05-29 15:08

本文选题：口腔鳞状细胞癌 + 生存率　；参考：《郑州大学》2017年硕士论文

【摘要】：研究背景:口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)又称口腔鳞癌,是常见的发生于口腔颌面部的恶性肿瘤之一。尽管国内外目前对口腔鳞癌的流行病学调查和回顾性研究层出不穷。但对于河南省本地区的相关研究为数不多。加强对河南地区口腔鳞癌患者发病特点及生存率等因素的研究,有利于提高全省在口腔鳞癌治疗中的总体水平。micro RNA(mi RNA)是目前对于口腔鳞癌的研究热点方向。研究证实,mi RNA这类非编码小RNA,是通过靶向、特异性作用m RNA或启动因子,导致靶RNA降解或者抑制其翻译,降低相关靶基因蛋白质的表达,从而发挥重要的生理和病理调控作用。目前研究已发现,mi RNA的异常表达与疾病尤其是癌症有着密切的关系。癌症进展中的一个关键步骤是增殖失控。最近有证据显示mi RNA作为肿瘤癌基因或抑癌基因异常表达是影响肿瘤细胞生长的一个关键因素。在前期的基因芯片研究中我们已经发现口腔鳞癌患者中mi RNA-602的高表达趋势,并发现了EHMT1(常染色质组蛋白甲基转移酶1)基因可以通过甲基化组蛋白从而在转录水平抑制MAL基因的表达,而mi R-602可对已经发生转录的MAL m RNA进一步发挥抑制作用,EHMT1/mi R-602转录本通过在转录水平及转录后水平协同影响MAL基因的稳定性,从而对口腔鳞癌的转移发挥调控作用。本研究拟对EHMT1在口腔鳞癌组织中的表达水平进行探索,为口腔鳞癌的转移寻找可靠的生物标记物。目前对于口腔鳞状细胞癌的标准化治疗包括手术切除及随后的辅助放疗和化疗。最近国内外的研究在口腔鳞癌的综合治疗方面取得了一些进展,但总体的预后情况仍然不太乐观。新辅助化疗(NACT)在口腔鳞癌治疗中的作用,成为目前探索的重要领域。新辅助化疗是指在实施局部治疗方法(如手术或放疗)前所做的全身化疗,目的是使肿块缩小、及早杀灭看不见的转移细胞,以利于后续的手术、放疗等治疗[36-49]。其目的是减少手术切除的范围,改善局部区域功能,控制和减少远端转移,从而降低死亡率和复发率,改善治疗结果。然而,新辅助化疗在口腔癌中的适应症尚未明确定义,大多数研究未能显示新辅助化疗在可切除的口腔鳞癌中,对局部区域病变的控制和总体生存率的明显影响。在一些对局部病变晚期和不可切除口腔鳞癌的病人的研究中,新辅助化疗可以显著的控制肿瘤的生长,缩小病变的范围并给手术切除创造机会。尽管这些研究阐述了新辅助化疗对极晚期和不可切除的口腔鳞癌,远端转移和可切除性及预后功能的一些积极影响,但需要进一步验证。目前临床上主要通过氟尿嘧啶、顺铂、阿霉素等药物进行化疗治疗,但这些药物在发挥抗癌作用的同时易产生耐药性,而且毒副作用很大。因此,急需找到一个新型的有效的抗肿瘤药物以提高患者的生存率。砷剂(主要是亚砷酸钠,Na As O2)在血液系统肿瘤方法有较多的研究,在胃癌、肺癌等实体肿瘤中也有研究,但总体研究较少,且对口腔鳞状细胞癌的研究尚不明确。因此,本研究检测亚砷酸钠对体外培养的人口腔鳞状细胞癌HN12细胞增殖凋亡的影响,并探讨其机制,以期为亚砷酸钠的临床影响提供理论基础。第一部分:对河南地区461例口腔鳞癌患者的回顾性分析目的:通过对2007年至2012年在郑州大学第一附属医院口腔颌面外科就诊的口腔鳞癌患者的病例资料进行搜集和整理,总结河南地区口腔鳞癌发病的特点。分析与口腔鳞癌发病及预后生存率和复发率相关的因素。为河南地区口腔鳞癌患者的治疗方法选择和复发的预防以及预后的评估提供参考。方法:搜集和整理2007年至2012年于郑州大学第一附属医院口腔颌面外科住院手术治疗的口腔鳞癌患者的病历资料,用SPSS21.0,Graph Pad6.0等统计软件对纳入的患者病例资料进行统计学分析,分别统计患者的年龄、性别、地域、肿瘤分化程度、病理TNM分期等特点。研究对象为符合筛选条件的河南地区口腔鳞癌患者,对研究中涉及到的复发率分析采用卡方检验进行数据分析。选择α=0.05作为检验水准,当P0.05认为具有统计学意义。在对影响术后生存时间的因素的研究中,采用Kaplan-Meier生存分析法绘制生存曲线,并使用Log-rank法对所得出的数据进行检验。结果:1河南地区461例口腔鳞癌患者的基本情况分析在461名口腔鳞癌患者中男性280人,女性181人;发病年龄主要集中在50-79岁;郑州市就诊患者显著高于其他地区患者,另外豫西北地区三门峡市、济源市、鹤壁市、洛阳市就诊患者分布数量较少,相比而言豫东地区周口市、开封市、商丘市就诊患者分布相对较多;就诊患者中,中-高分化的口腔鳞癌患者占大多数;从发病部位上看,就诊患者中舌,颊粘膜,牙龈发病率位列前三。2.对95例河南地区口腔鳞癌患者的复发率及生存率分析在前期研究中的461例患者中随机选取95例符纳入标准的病例进行后续研究。观察到这些患者的复发率和生存率研究中,年龄、肿瘤的临床分期对生存率产生影响;研究中还观察到肿瘤的分化程度和肿瘤的临床分期对复发率可以产生影响;对于晚期(IV期)的患者而言辅助化疗对患者的生存率存在影响;对于分化程度较低的口腔鳞癌患者辅助化疗对患者的复发率存在影响。小结:1河南地区口腔鳞癌患者发病特点研究表明,口腔鳞癌在河南地区的分布存在地域差异。造成这一现象的原因可能主要有交通条件差异,各地市级医院或基层医院的医疗条件差异等。从本研究的结果中观察到,河南地区口腔鳞癌的发病特点与全国基本相符。但在发病年龄段上,位于70-79岁的患者数量也尤为突出,而造成这一结果的具体因素还有待进一步研究。在这些患者中分化程度较低的患者数量较少,这可能与Broders/WHO组织学分级系统本身存在的固有缺陷有关。2影响口腔鳞癌术后复发率的相关因素本研究结果显示,就研究所涉及的口腔鳞癌患者而言,年龄,性别对口腔鳞癌的复发率没有影响。而在研究中观察到,病理分级和临床分期对口腔鳞癌的术后复发率存在影响。对于低分化口腔鳞癌的患者而言,化疗对其术后复发率也存在着影响。3影响口腔鳞癌术后生存率的相关因素研究发现对于口腔鳞癌患者而言年龄和肿瘤的临床分期可能是影响患者术后生存率的因素。高年龄组的患者术后生存时间低于低年龄组,I期患者的术后生存率高于IV期患者。研究同时还发现对于晚期(IV期)口腔鳞癌患者而言,接受化疗有助于改善患者的术后生存率。提示我们对于晚期患者的辅助化疗在晚期口腔鳞癌的治疗领域仍然值得研究。第二部分:EHMT1在口腔鳞癌组织中的表达目的:了解EHMT1在口腔鳞癌组织中的表达情况,探究EHMT1的表达与口腔鳞癌进展的关系。方法:利用SP免疫组化染色法,对前期研究的随访患者中随机选取95名患者调取病理组织蜡块,切片染色。结果判断结合细胞计数法和光密度分析法。使用SPSS及Graph Pad等统计软件用t检验和Mann-Whitney秩和检验及Kaplan-Meier生存分析法对数据进行分析。结果:1.根据EHMT1的免疫组化染色结果,95例口腔鳞癌患者的组织切片染色平均光密度(Mean optical density,MOD)值在34.53~167.38之间。取中位数119.86。将患者分为两组:EHMT1低表达组,即MOD值小于119.86的患者;EHMT1高表达组,即MOD值大于等于119.86的患者。采用Kaplan-Meier生存分析法用GraphPad6.0绘制生存曲线,并使用Log-rank法对所得出的数据进行检验。P=0.03850.05,结果表明EHMT1低表达组患者的生存率高于EHMT1高表达组,差异具有统计学意义。2.根据患者的肿瘤转移情况将患者分为转移组(metastasis)和非转移组(non-metastasis),对两组患者的EHMT1表达情况进行分析,用t检验验证两组的MOD值是否存在差异。结果为:P=0.0070.05,这说明两组患者的EHMT1表达存在显著差异。3.根据第一部分研究中的临床病理参数分组后得出不同参数的阳性率。运用Mann-Whitney秩和检验检测各参数的统计学差异。结果显示晚期口腔鳞癌患者的EHMT1表达与早期患者相比较存在统计学差异。P0.05小结:1.EHMT1在口腔鳞癌中的表达对患者术后生存率存在影响,患者EHMT1表达水平与其术后生存时间呈负相关。2.EHMT1在口腔鳞癌中的表达对患者肿瘤转移存在影响,患者EHMT1表达水平与其肿瘤转移率呈正相关。3.晚期患者的EHMT1阳性率与早期患者相比存在显著统计学差异,提示EHMT1的表达可能与肿瘤的进展有关。第三部分:亚砷酸钠抑制口腔鳞状细胞癌增殖、促凋亡及机制研究目的:利用CCK8法、Annexin V/PI双染法及Western blot等方法探讨亚砷酸钠调控Wnt信号通路对口腔鳞状细胞癌增殖及凋亡的影响。方法:1.25、2.5、5、10、20μmol/L的亚砷酸钠处理人口腔鳞状细胞癌细胞株HN12,24、48、72h后,CCK8实验检测细胞增殖;0、10μmol/L的亚砷酸钠处理HN12细胞48h后,流式细胞仪检测细胞凋亡,Western blot检测Cleaved caspase3、β-catenin、Cyclin D1蛋白表达;后续实验分为对照组、亚砷酸钠组、激活剂+亚砷酸钠组,各组细胞处理48h后,CCK8实验、流式细胞术及Western blot分别检测三组细胞的增殖、凋亡及Cleaved caspase3、β-catenin、Cyclin D1蛋白表达。结果:1.随着时间及浓度增加细胞抑制率均显著升高,具有时间和浓度依赖性(P0.05或P0.01),根据IC50选择10μmol/L的亚砷酸钠作为后续研究;2.10μmol/L的亚砷酸钠组细胞凋亡率及Cleaved caspase3蛋白表达显著高于0μmol/L组,β-catenin、Cyclin D1蛋白表达显著低于0μmol/L组(P0.01);3.亚砷酸钠组及激活剂+亚砷酸钠组细胞凋亡率、细胞抑制率及Cleaved caspase3蛋白表达均显著高于对照组,β-catenin、Cyclin D1蛋白表达显著低于对照组(P0.01);4.激活剂+亚砷酸钠组细胞凋亡率、细胞抑制率及Cleaved caspase3蛋白表达均显著低于亚砷酸钠组,β-catenin、Cyclin D1蛋白表达显著高于亚砷酸钠组(P0.01)。小结:1.研究证明亚砷酸钠能够抑制HN12细胞的增值同时促进其凋亡。2.经过亚砷酸钠处理的HN12细胞其凋亡相关蛋白Cleaved caspase3表达升高,而Wnt信号通路相关的β-catenin、Cyclin D1蛋白表达降低。3.激活Wnt信号通路后亚砷酸钠的促凋亡作用受到抑制。结论:1.河南地区口腔鳞癌的发病特点及患者术后的生存率和复发率与国内发达地区相关报道情况大致相同。表明我省的口腔鳞癌的治疗水平处于全国前列。2.EHMT1在口腔鳞癌中的异常表达,特别是在转移性患者癌组织中的高表达提示EHMT1可能成为口腔鳞癌转移的生物标志物。3.亚砷酸钠可抑制口腔鳞状细胞癌增殖及促进凋亡,其机制与Wnt信号通路的调控有关。
[Abstract]:Background: oral squamous cell carcinoma (OSCC), also known as oral squamous cell carcinoma, is one of the most common malignant tumors occurring in the oral and maxillofacial region. Although the epidemiological investigation and retrospective study of oral squamous cell carcinoma are emerging at home and abroad, there are few related studies in Henan province. The study of the characteristics and survival rate of oral squamous cell carcinoma in Henan is helpful to improve the overall level of.Micro RNA (MI RNA) in oral squamous cell carcinoma (oral squamous cell carcinoma), which is a hot research direction for oral squamous cell carcinoma. It has been proved that MI RNA, a non coded small RNA, is the result of the targeting and specific action of M RNA or starting factor. The target RNA degrades or inhibits its translation and reduces the expression of related target gene proteins and plays an important role in physiological and pathological regulation. The current study has found that the abnormal expression of MI RNA is closely related to the disease, especially cancer. A key step in cancer progression is to proliferate out of control. Recent evidence shows that MI RNA is used as a RNA. The abnormal expression of tumor oncogene or tumor suppressor gene is a key factor affecting the growth of tumor cells. In the early gene chip study, we have found the high expression of MI RNA-602 in oral squamous cell carcinoma, and found that the EHMT1 (normal chromatin methyltransferase 1) gene can be transcribed by methylation histone Level inhibits the expression of MAL gene, while mi R-602 can further inhibit the MAL m RNA that has been transcribed. The EHMT1/mi R-602 transcript affects the stability of the MAL gene by synergistic effect at the transcriptional and post transcriptional levels, thus regulating the metastasis of oral squamous cell carcinoma. This study is intended to be used in EHMT1 in oral squamous cell carcinoma. The level of expression is explored to find reliable biomarkers for the metastasis of oral squamous cell carcinoma. The current standardized treatment for oral squamous cell carcinoma includes surgical resection and subsequent adjuvant radiotherapy and chemotherapy. Recent domestic and foreign studies have made some progress in the comprehensive treatment of oral squamous cell carcinoma, but the overall prognosis is still not. Tai Le Guan. The role of neoadjuvant chemotherapy (NACT) in the treatment of oral squamous cell carcinoma has become an important field of exploration. The new adjuvant chemotherapy refers to the systemic chemotherapy before the implementation of local treatment methods (such as surgery or radiotherapy). The purpose is to reduce the mass and to kill the invisible transfer cells as early as possible for the follow-up operation and radiotherapy for the treatment of [36-4. 9]. aims to reduce the range of surgical excision, improve local regional function, control and reduce distal metastasis, thereby reduce mortality and recurrence, and improve treatment results. However, the adaptation of neoadjuvant chemotherapy in oral cancer has not been clearly defined. Most studies have failed to show neoadjuvant chemotherapy in resectable oral squamous cell carcinoma. The control of regional lesions and the significant impact of the overall survival rate. In the study of some patients with advanced local lesions and non resectable oral squamous cell carcinoma, neoadjuvant chemotherapy can significantly control the growth of the tumor, narrow the range of the lesion and create an opportunity for surgical excision. Resectable oral squamous cell carcinoma, distal metastasis and resectability and prognostic function have some positive effects, but need to be further verified. Currently, chemotherapy is performed mainly through fluorouracil, cisplatin, adriamycin and other drugs. However, these drugs are susceptible to drug resistance while exerting anti-cancer effects. It is necessary to find a new effective antitumor drug to improve the patient's survival rate. Arsenic (mainly arsenite, Na As O2) has been studied more in the blood system tumor methods, in gastric cancer, lung cancer and other solid tumors, but the overall study is less, and the study of oral squamous cell carcinoma is not clear. Therefore, this study is examined. The effect of sodium arsenite on the proliferation and apoptosis of human oral squamous cell carcinoma HN12 cells in vitro and its mechanism are discussed in order to provide a theoretical basis for the clinical effect of sodium arsenite. Part 1: retrospective analysis of 461 cases of oral squamous cell carcinoma in Henan: through the First Affiliated Hospital of Zhengzhou University from 2007 to 2012 The case data of oral squamous cell carcinoma patients in oral and maxillofacial surgery were collected and arranged to summarize the characteristics of oral squamous cell carcinoma in Henan area. The factors related to the incidence of oral squamous cell carcinoma and the survival rate and recurrence rate were analyzed. The selection and recurrence prevention of oral squamous cell carcinoma in Henan area and the evaluation of the prognosis were evaluated. Methods: to collect and collate the medical records of oral squamous cell carcinoma patients in oral and maxillofacial surgery in the First Affiliated Hospital of Zhengzhou University from 2007 to 2012. The data of patients' cases were statistically analyzed with SPSS21.0, Graph Pad6.0 and other statistical software, and the age, sex, region, and tumor score of the patients were statistically analyzed. Degree, pathological TNM staging and other characteristics. The object of the study was the oral squamous cell carcinoma in Henan area, which was in accordance with the screening conditions. The recurrence rate analysis involved in the study was analyzed with chi square test. The selection of alpha =0.05 as a test level, when P0.05 considered a statistical significance. In the study of factors affecting the survival time after the operation, The survival curve was plotted by Kaplan-Meier survival analysis and the data obtained by Log-rank were tested. Results: the basic analysis of 461 cases of oral squamous cell carcinoma in 1 Henan areas was analyzed in 461 cases of oral squamous cell carcinoma in 280 men and 181 women; the age of onset was mainly at the age of 50-79; the patient was significantly higher than that of the 461 patients. The patients in his region, in addition to Sanmenxia, Jiyuan, Hebi and Luoyang in Northwest Henan were less distributed, compared with Zhoukou City, Kaifeng City and Shangqiu city in Eastern Henan Province, and the majority of patients with middle and high differentiation of oral squamous cell carcinoma were found in the patients. The recurrence rate and survival rate of the buccal mucosa and gingival incidence were three.2. in 95 cases of oral squamous cell carcinoma in Henan area. A follow-up study was conducted in 461 patients in the early study. The recurrence rate and survival rate of these patients were investigated, age, and the survival rate of the tumor. It was also observed that the degree of tumor differentiation and the clinical stage of the tumor had an impact on the recurrence rate; for patients in the late stage (IV phase), adjuvant chemotherapy had an effect on the survival rate of the patients; for patients with low differentiation, the adjuvant therapy of oral squamous cell carcinoma had an impact on the recurrence rate of the patients. Summary: 1 Henan The distribution of oral squamous cell carcinoma in Henan region shows that there are regional differences in the distribution of oral squamous cell carcinoma in the region. The reasons for this phenomenon may be mainly due to the difference of traffic conditions, the difference of medical conditions in local hospitals or primary hospitals. From the results of this study, the incidence of oral squamous cell carcinoma in Henan area is characterized and complete. The country is basically consistent. But at the age of onset, the number of patients at the age of 70-79 is also particularly prominent, and the specific factors that cause this result remain to be further studied. In these patients, the number of patients with lower degree of differentiation is less, which may be related to the inherent defects of the Broders/WHO histology classification system itself related to the impact of.2 on oral scales. The results of the related factors of recurrence rate after cancer show that age and sex have no effect on the recurrence rate of oral squamous cell carcinoma in the oral squamous cell cancer patients involved in the study. In the study, the pathological and clinical stages have an impact on the postoperative recurrence rate of oral squamous cell carcinoma. The postoperative recurrence rate also affects the postoperative survival rate of.3 in oral squamous cell carcinoma. It is found that the age and the clinical stage of the tumor may be the factors affecting the postoperative survival rate for the patients with oral squamous cell carcinoma. The survival time of the patients in the high age group is lower than the low age group, and the postoperative survival rate of the I patients is high. The study also found that chemotherapy is helpful to improve postoperative survival in patients with advanced (IV) oral squamous cell carcinoma, suggesting that adjuvant chemotherapy for advanced oral squamous cell carcinoma is still worth studying for advanced patients. Second: the second part: the expression of EHMT1 in oral squamous cell carcinoma: to understand EHM The expression of T1 in oral squamous cell carcinoma was used to explore the relationship between the expression of EHMT1 and the progress of oral squamous cell carcinoma. Methods: using SP immunohistochemical staining, 95 patients were randomly selected for pathological tissue wax block and stained. The results were combined with cell count and light density analysis. SPSS and Graph P were used. The data were analyzed by t test, Mann-Whitney rank test and Kaplan-Meier survival analysis. Results: 1. according to the immunohistochemical staining results of EHMT1, the mean optical density (Mean optical density, MOD) of 95 cases of oral squamous cell carcinoma was between 34.53~167.38. The median 119.86. was divided into the patients. The two group: EHMT1 low expression group, MOD value less than 119.86 patients; EHMT1 high expression group, that is, the MOD value is greater than 119.86. Using the Kaplan-Meier survival analysis method to use GraphPad6.0 to draw the survival curve, and use the Log-rank method to test the obtained data. The results show that the survival rate of the patients with low expression of EHMT1 is higher than EH. MT1 high expression group, the difference was statistically significant.2. was divided into the metastasis group (metastasis) and the non transfer group (non-metastasis) according to the tumor metastasis of the patients. The EHMT1 expression in the two groups was analyzed, and the t test was used to verify the difference in the MOD value of the two groups. The result was P=0.0070.05, which indicated the EHMT1 table of the two groups of patients. The positive rates of different parameters were obtained after grouping the clinicopathological parameters in the first part of the study. The statistical difference between the parameters were detected by the Mann-Whitney rank sum test. The results showed that the EHMT1 expression in the patients with advanced oral squamous cell carcinoma was compared with the early patients in a statistically significant difference of.P0.05: 1.EHMT1 at the mouth of the oral squamous cell carcinoma (1.EHMT1). The expression of EHMT1 in the squamous cell carcinoma has an effect on the postoperative survival rate. The expression level of the patient is negatively correlated with the survival time of the patients with the postoperative survival time. The expression of.2.EHMT1 in oral squamous cell carcinoma has an influence on the metastasis of the patients. The expression level of the patient's EHMT1 is positively related to the metastasis rate of the tumor, and the positive rate of EHMT1 in the late.3. patients is compared with the early patients. In the significant statistical difference, the expression of EHMT1 may be related to the progression of tumor. Third part: sodium arsenite inhibits the proliferation, apoptosis and mechanism of oral squamous cell carcinoma. CCK8, Annexin V/PI double staining and Western blot are used to investigate the proliferation and proliferation of Wnt signaling pathway by sodium arsenite to regulate the proliferation of oral squamous cell carcinoma. The effect of apoptosis. Methods: 1.25,2.5,5,10,20 mu mol/L sodium arsenite treated human oral squamous cell carcinoma cell line HN12,24,48,72h, and the cell proliferation was detected by CCK8 experiment. After HN12 cell 48h was treated with sodium arsenite of 0,10 mol/L, the apoptosis was detected by flow cytometry. Western blot was used to detect Cleaved Caspase3. The follow-up experiments were divided into control group, sodium arsenite group, activator and sodium arsenite group. After 48h, CCK8 experiment, flow cytometry and Western blot were used to detect the proliferation, apoptosis and the expression of Cleaved Caspase3, Cleaved Caspase3, -catenin and Cyclin D1 protein respectively. Results: 1. with time and concentration, the cell inhibition rate increased significantly, with the increase of cell inhibition rate. With time and concentration dependence (P0.05 or P0.01), sodium arsenite was selected according to IC50 as a follow-up study. The apoptosis rate and the expression of Cleaved Caspase3 protein in the 2.10 mol/L sodium arsenite group were significantly higher than that of the 0 mu mol/L group, and the expression of beta -catenin, Cyclin D1 protein was significantly lower than that of the 0 micron mol/L group; 3. sodium arsenite group and activator + subgroup + subgroup The apoptotic rate, cell inhibition rate and Cleaved Caspase3 protein expression in sodium arsenate group were significantly higher than those in control group, beta -catenin, Cyclin D1 eggs.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.8

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