人乳头瘤病毒抑制FAS介导的凋亡通路在食管鳞状细胞癌中的作用机制
发布时间：2018-08-01 17:00
【摘要】：目的:我国是食管癌(Esophageal Carcinoma,EC)的高发地区,每年因食管癌死亡者大约15万人,约占全部恶性肿瘤死亡数的近四分之一。我国食管癌人口死亡率最低的为云南省(105/10万),与死亡率最高的河南省相差31倍,可见食管癌的发病率存在明显的地域差异。近年来,环境致癌因素(如真菌毒素、病毒等)在各类肿瘤的发生发展中已成为研究热点之一。人类乳头瘤病毒(Human papillomavirus HPV)与食管癌发生的关系最早是1982年由Syrjanen提出,他的研究发现40%(24/60)的食管癌发生的组织学形态改变与生殖道湿疣改变极其相似,随后他与同事用免疫组织化学法在食管鳞状上皮细胞癌(Esophageal squamous Cell Carcinoma,ESCC)中发现了HPV的结构蛋白。HPV作为重要致癌因素在宫颈癌发生中的作用已得到证实。随着研究的深入,有学者发现口腔、食管鳞状细胞癌的发生还与HPV感染有关。2008年,WHO认定HPV感染与人口咽部鳞状细胞癌发生存在直接关系。然而HPV与食管鳞状细胞癌的发生关系及其作用尚未阐明。Fas属于肿瘤坏死因子和神经生长因子受体家族成员,当Fas与它相应配体Fas L(CD95L)结合后,Fas受体三聚体化而活化,激活的受体与FADD结合,再与caspase-8相互作用使后者活化,形成死亡诱导信号复合物,再激活一系列的Caspase-1,3,7等,其中Caspase-3的切割底物为PARP,调控凋亡最终步骤的执行。FADD作为Fas通路的关键蛋白,可以触发凋亡级联反应。研究发现在人结肠腺癌细胞系HCT116细胞中高表达HPV16 E6,可以通过影响TRAIL蛋白介导的凋亡信号途径,避免宿主细胞凋亡。研究还发现HPV E6蛋白可以诱导FADD和caspase-8的泛素化,从而抑制Fas信号通路的激活来抑制细胞凋亡的发生。我们在前期实验中已经了解到HPV感染可以降低食管鳞状细胞癌中Fas蛋白的表达,为进一步明确HPV在食管鳞状细胞癌发生发展中的作用,本研究首先明确我省食管鳞状细胞癌组织中FADD、caspase-8蛋白的表达情况,分析HPV感染与FADD、caspase-8蛋白表达的关系,其次对正常食管上皮细胞和高分化食管鳞状细胞癌细胞株转染携带HPV16 E6基因片段的质粒,在过表达E6蛋白的情况下观察Fas/Fas L凋亡通路相关蛋白(Fas/Fas L、FADD、caspase-8)的变化情况。在整体水平和细胞水平探讨HPV对Fas介导的细胞凋亡通路相关蛋白的影响,从细胞凋亡角度进一步阐明HPV与食管癌发生的关系及其作用机制,为食管癌发生的病毒学机制的研究提供新的思路和视角,为食管癌的临床治疗、预后判断和预防提供科学依据。方法:1标本收集收集河北医科大学第二医院2009年1月到2015年12月间符合条件的食管鳞状细胞癌(原发性食管鳞状细胞癌,手术前未经放疗和化疗)的石蜡标本,共159例。收集临床病理资料,包括年龄,性别,淋巴结转移情况和癌组织的分化程度。2检测病毒DNA将石蜡组织切成薄片,使用石蜡组织DNA提取盒提取组织DNA。以HPV L1基因MY09/MY11为引物,用PCR方法检测各实验组与对照组中HPV L1基因的存在。使用β-actin作为内参,使用三蒸水代替引物完成PCR反应,作为阴性对照,使用确诊HPV阳性的宫颈癌石蜡组织提取的DNA作为阳性对照。3检测食管鳞状细胞癌组织的FADD、caspase-8蛋白表达情况应用免疫组化SP法检测食管鳞状细胞癌组织中FADD、caspase-8蛋白表达情况,FADD、caspase-8蛋白阳性反应均定位于细胞质。4 EX-GS3066-M90(HPV16 E6)、EX-NEG-M90(空载)质粒提纯及浓度检测。5 HPV16 E6质粒转染对体外培养的Het-1A(人正常食管上皮细胞)和Eca-109(人食管高分化鳞状细胞癌株)的Fas介导凋亡通路相关蛋白(Fas/Fas L、FADD、caspase-8)的影响体外培养Het-1A和Eca-109,转染HPV16 E6质粒和空载质粒(阴性对照),应用半定量RT-PCR、Western blot方法分别检测两组Het-1A和Eca-109细胞FADD、caspase-8的m RNA和蛋白表达变化情况,分析HPV16 E6蛋白对Fas凋亡通路的可能影响和机制。为了进一步明确HPV16 E6对人食管正常上皮Fas介导的凋亡通路的影响,转染Het-1A后于12h、24h、48h收集细胞,观察细胞内Fas/Fas L、FADD和caspase-8蛋白表达在转染后的变化趋势。6统计分析所有数据采用SPSS19.0软件,计数资料采用χ2检验及Pearson相关分析法,P0.05认为差异有统计学意义。计量资料以均数±标准差((?)±s)表示,单因素方差分析P0.05时认为差异有统计学意义。结果:1.1食管鳞状细胞癌(ESCC)病理形态学观察结果与临床病理分析本组ESCC病例共159例,年龄跨度为27-87岁,中位数为62岁;男性患者124例,女性患者35例;经病理形态学观察:淋巴结转移患者47例;ESCC高分化患者39例,中分化72例,低分化48例。低分化组与高中分化组的淋巴结转移率无明显差异(P0.05)。1.2 ESCC组织中HPV的感染情况与临床病理特征的关系159例ESCC的HPV感染率为32.1%。HPV阳性组高中分化鳞癌占56.9%,明显低于HPV阴性组的75.9%;HPV阳性组低分化鳞癌占43.1%,明显高于HPV阴性组的24.1%(P0.05);HPV感染与年龄,性别,淋巴结转移之间无明显差异(P0.05)。2免疫组化结果2.1 ESCC组织中FADD、caspase-8蛋白表达情况159例ESCC组织中FADD蛋白表达阳性81例,阴性78例;caspase-8蛋白表达阳性93例,阴性66例。癌旁组织中FADD蛋白表达阳性150例,阴性9例;caspase-8蛋白表达阳性131例,阴性28例。FADD和caspase-8在ESCC组织中蛋白表达的阳性率分别为50.9%和58.5%,明显低于癌旁组织的94.3%和82.4%(P0.05)。2.2 ESCC组织中FADD、caspase-8蛋白表达与HPV的关系HPV阳性组FADD蛋白表达阳性19例,阴性32例,caspase-8蛋白表达阳性24例,阴性27例;HPV阴性组FADD蛋白表达阳性62例,阴性46例;caspase-8蛋白表达阳性69例,阴性39例,FADD和caspase-8在HPV阳性组蛋白表达的阳性率分别为37.3%和47.1%,明显低于阴性组的57.4%和63.9%(P0.05)。2.3 ESCC组织中FADD和caspase-8蛋白表达与临床病理特征的关系男性FADD蛋白表达阳性率为59.7%,明显高于女性的20.0%(P0.05);FADD蛋白表达与年龄,淋巴结转移及肿瘤分化程度之间无明显差异(P0.05)。男性caspase-8蛋白表达阳性率63.7%,明显高于女性的40.0%(P0.05);caspase-8蛋白表达与年龄,淋巴结转移及肿瘤分化程度之间无明显差异(P0.05)。2.4 HPV与FADD和caspase-8蛋白表达与临床病理特征的关系HPV感染阳性的ESCC组织:62岁以上(包括62岁)患者FADD蛋白阳性表达率24.1%,明显低于62岁以下患者的54.5%(P0.05);低分化组FADD蛋白阳性表达率为13.6%,明显低于高中分化的55.2%;FADD蛋白表达与性别和淋巴结转移之间无明显差异(P0.05)。HPV感染阳性的ESCC组织:低分化组caspase-8蛋白阳性率为27.3%,明显低于高中分化组的62.1%(P0.05),caspase-8蛋白表达与年龄,性别和淋巴结转移之间无明显差异(P0.05)。3细胞实验转染后HPV16 E6、Fas、Fas L、FADD、caspase-8蛋白表达情况3.1 RT-PCR结果显示:Het-1A细胞转染E6基因后与空载质粒组相比,E6基因相对表达升高(P0.05);FADD、caspase-8基因相对表达量降低(P0.05);Fas基因相对表达量升高(P0.05);Fas L无明显变化(P0.05)。Eca-109细胞转染E6基因后与空载质粒组相比,E6基因相对表达量升高(P0.05);FADD、caspase-8、Fas基因相对表达量升高(P0.05);Fas L无明显变化(P0.05)。Het-1A细胞转染E6基因后与空载质粒组相比,E6基因在12h、24h和48h相对表达量呈上升趋势(P0.05);FADD和caspase-8基因在12h、24h和48h相对表达量呈下降趋势(P0.05);Fas基因在12h、24h和48h相对表达量呈上升趋势(P0.05);Fas L无明显变化(P0.05)。3.2 Western blot结果显示:Het-1A细胞转染E6基因后与空载质粒组相比,E6蛋白相对表达升高(P0.05);FADD、caspase-8、Fas蛋白相对表达量降低(P0.05);Fas L无明显变化(P0.05)。Eca-109细胞转染E6基因后与空载质粒组相比,E6蛋白相对表达量升高(P0.05);FADD、caspase-8、Fas蛋白相对表达量降低(P0.05);Fas L无明显变化(P0.05)。Het-1A细胞转染E6基因后与空载质粒组相比,E6蛋白在12h、24h和48h相对表达量呈上升趋势(P0.05);FADD和caspase-8蛋白在12h、24h和48h相对表达量呈下降趋势(P0.05);Fas蛋白在12h和24h相对表达量升高(P0.05),在48h相对表达量降低(P0.05);Fas L无明显变化(P0.05)。结论:1本组食管鳞状细胞癌组织HPV的检出率为32.1%,HPV阳性的癌组织中低分化鳞状细胞癌所占比例高于HPV阴性组,提示HPV与肿瘤分化存在相关关系。2食管鳞状细胞癌中FADD和caspase-8蛋白表达降低,而且HPV可进一步降低两者的蛋白表达水平。3 HPV16 E6基因可以降低Het-1A和Eca-109细胞FADD、caspase-8、和Fas蛋白表达水平。4组织学和细胞学水平研究结果提示,HPV可能通过抑制FAS介导的凋亡通路,在食管鳞状细胞癌发生发展中发挥一定作用。
[Abstract]:Objective: China is a high incidence area of Esophageal Carcinoma (EC), about 150 thousand people died of esophageal cancer every year, accounting for nearly 1/4 of the deaths of all malignant tumors. The lowest mortality rate of esophageal cancer in China is in Yunnan province (105/10 million), which is 31 times the difference between the highest death rate in Henan Province, and the incidence of esophageal cancer is found to exist. In recent years, environmental carcinogens (such as mycotoxins, viruses, etc.) have become one of the hotspots in the development of various tumors. The relationship between human papillomavirus (Human papillomavirus HPV) and the occurrence of esophageal cancer was first proposed by Syrjanen in 1982. His study found a group of 40% (24/60) of the cancer of the esophagus. The morphological changes were very similar to the changes in genital warts, and then he and his colleagues found the structural protein.HPV of HPV in Esophageal squamous Cell Carcinoma, ESCC as an important carcinogenic factor in the carcinogenesis of the cervix. Researchers found that the occurrence of oral and esophageal squamous cell carcinoma is also associated with HPV infection for.2008 years. WHO affirms that HPV infection has a direct relationship with the occurrence of squamous cell carcinoma of the pharynx. However, the relationship between HPV and the occurrence of squamous cell carcinoma of the esophagus and its role do not clarify that.Fas is a member of the tumor's bad cause of death and a member of the nerve growth factor receptor family, when Fas After binding with its corresponding ligand Fas L (CD95L), the Fas receptor is trimeric and activated, the activated receptor is combined with FADD, and then the latter activates the latter to form a death induced signal complex, and then activates a series of Caspase-1,3,7, in which the Caspase-3's cutting substrate is PARP, and the execution of.FADD as a Fa.FADD is used as Fa. The key protein in the s pathway can trigger the apoptosis cascade reaction. The study found that the high expression of HPV16 E6 in human colon adenocarcinoma cell line HCT116 cells can prevent the apoptosis of host cells by affecting the apoptosis signaling pathway mediated by TRAIL protein. The study also found that HPV E6 protein can induce the ubiquitination of FADD and caspase-8, thus inhibiting the Fas signal. The activation of the pathway inhibits the occurrence of apoptosis. In our previous experiments, we have learned that HPV infection can reduce the expression of Fas protein in the squamous cell carcinoma of the esophagus, and to further clarify the role of HPV in the development of squamous cell carcinoma of the esophagus. This study first identified the FADD, caspase-8 protein in the tissue of the squamous cell carcinoma of our province. The relationship between HPV infection and the expression of FADD and caspase-8 protein was analyzed. Secondly, the plasmids carrying HPV16 E6 gene fragment were transfected into normal esophageal epithelial cells and highly differentiated squamous cell carcinoma cell lines, and the changes of the Fas/Fas L apoptotic pathway Guan Danbai (Fas/Fas L, FADD, Caspase-8) were observed under the overexpression of E6 protein. To explore the effect of HPV on Fas mediated apoptosis pathway related proteins at the overall level and cell level, further elucidate the relationship between HPV and the pathogenesis of esophageal cancer from the angle of cell apoptosis, and provide new ideas and perspectives for the study of the virological mechanism of the occurrence of esophageal cancer, for the clinical treatment of esophageal cancer, the prognosis and the prediction of the prognosis. Methods: 1 specimens were collected and collected from second hospitals of Hebei Medical University from January 2009 to December 2015. The paraffin specimens of esophageal squamous cell carcinoma (primary squamous cell carcinoma of the esophagus, without radiotherapy and chemotherapy) were collected from January 2009 to December 2015. A total of 159 cases were collected, including age, sex and lymph node metastasis. The differentiation degree of the cancer tissue.2 detected the paraffin tissue into thin slices, the paraffin tissue DNA extraction box was used to extract the tissue DNA. and the HPV L1 gene MY09/MY11 was used as primer. The HPV L1 gene in the experimental group and the control group was detected by PCR method. The beta -actin was used as the internal parameter to make the PCR reaction with three water instead of primers. In sex control, the FADD of esophageal squamous cell carcinoma tissue was detected by the DNA extracted from the paraffin tissue of cervix cancer with positive HPV. The expression of caspase-8 protein was detected by immunohistochemical SP method and the expression of FADD, Caspase-8 protein in the tissues of squamous cell carcinoma of the esophagus was detected by immunohistochemistry, and the positive reaction of caspase-8 protein was located in the cells. Qualitative.4 EX-GS3066-M90 (HPV16 E6), EX-NEG-M90 (unloaded) plasmid purification and concentration detection of.5 HPV16 E6 plasmids transfection on Het-1A (human normal esophageal epithelial cells) and Eca-109 (human esophageal highly differentiated squamous cell carcinoma strain) HPV16 E6 plasmids and negative plasmids (negative controls) were transfected into two groups of Het-1A and Eca-109 cells FADD, Caspase-8 m RNA and protein expression changes were detected by semi quantitative RT-PCR and Western blot methods. Effect of mediated apoptosis pathway, after transfection of Het-1A, cells were collected at 12h, 24h, 48h, and the expression of Fas/Fas L, FADD and caspase-8 protein in the cells was observed after transfection. All data were analyzed by SPSS19.0 software. The count data were analyzed by chi 2 test and Pearson correlation analysis. P0.05 believed the difference was statistically significant. Measurement was statistically significant. The results showed that the difference was statistically significant when the single factor variance analysis ((?) + s) showed that the difference was statistically significant when the single factor variance analysis was P0.05. Results: 1.1 the pathological morphological observation of esophageal squamous cell carcinoma (ESCC) and clinicopathological analysis were 159 cases in this group of ESCC cases, the age span was 27-87 years and the median was 62 years, 124 cases in male and 35 in female patients. Morphological observation: 47 cases of lymph node metastasis, 39 cases of ESCC high differentiation, 72 cases of middle differentiation, 48 cases of low differentiation. There was no significant difference in lymph node metastasis rate between the low differentiation group and the high school differentiation group (P0.05) the relationship between the HPV infection and the clinicopathological features in the.1.2 ESCC tissue 159 cases of HPV infection rate of ESCC were the grade of differentiated high school in the 32.1%.HPV positive group. Cancer accounted for 56.9%, obviously lower than 75.9% in HPV negative group, and 43.1% in HPV positive group, significantly higher than 24.1% in HPV negative group (P0.05). There was no significant difference between HPV infection and age, sex, lymph node metastasis (P0.05).2 immunization results 2.1 ESCC tissues FADD, Caspase-8 protein expression in 159 cases ESCC tissue of FADD protein expression Yang There were 81 cases, 78 cases negative, 93 cases of caspase-8 protein expression positive, 66 cases negative, 150 cases of FADD protein expression in the paracancerous tissues, 9 cases negative, 131 cases of caspase-8 protein expression, 50.9% and 58.5% in ESCC tissues of negative.FADD and caspase-8 negative, obviously lower than 94.3% and 82.4% (P0.05).2.2 E (P0.05).2.2 E adjacent to the paracancerous tissue. The relationship between the expression of FADD, Caspase-8 protein and HPV in SCC tissue was 19 cases of FADD positive expression in HPV positive group, 32 cases negative, 24 cases of caspase-8 protein expression, 27 cases negative, 62 cases of FADD protein expression in HPV negative group and 46 cases negative, 69 cases of caspase-8 protein expression positive, 39 cases of negative sex, FADD and caspase-8 in the positive group protein expression positive. The rates were 37.3% and 47.1% respectively, which were significantly lower than those of 57.4% and 63.9% (P0.05).2.3 ESCC of the negative group. The expression of FADD and caspase-8 protein was associated with the clinicopathological features, the positive rate of the male FADD protein was 59.7%, significantly higher than that of the female (P0.05), and there was no significant difference between the expression of FADD protein and age, lymph node metastasis and the degree of tumor differentiation. Difference (P0.05). The positive rate of male caspase-8 protein expression was 63.7%, significantly higher than that of female 40% (P0.05); there was no significant difference between the expression of caspase-8 protein and age, lymph node metastasis and tumor differentiation (P0.05) the relationship between.2.4 HPV and FADD and caspase-8 protein expression and clinical pathophysiology: ESCC tissue of HPV infection positive: over 62 years of age (packet The positive expression rate of FADD protein was 24.1%, which was significantly lower than 54.5% (P0.05) in patients under 62 years of age, and the positive rate of FADD protein in low differentiation group was 13.6%, significantly lower than that in high school differentiation, and there was no significant difference between FADD protein expression and sex and lymph node metastasis (P0.05).HPV positive ESCC tissue: low differentiation group caspase-8 protein The positive rate was 27.3%, obviously lower than that of the high school group (62.1% (P0.05)). There was no significant difference between the expression of caspase-8 protein and age, sex and lymph node metastasis (P0.05) HPV16 E6 after transfection of.3 cells, Fas, Fas L, FADD, Caspase-8 protein expression 3.1 RT-PCR nodes showed that the transfected cells were compared with the empty plasmid group after transfection. The relative expression of gene increased (P0.05), the relative expression of FADD, Caspase-8 gene decreased (P0.05), the relative expression of Fas gene increased (P0.05), and Fas L had no obvious change (P0.05).Eca-109 cells transfected to the E6 gene, and the relative expression of the E6 gene was higher than that of the empty plasmid group. The relative expression of E6 gene in 12h, 24h and 48h showed an upward trend (P0.05) in 12h, 24h and 48h after transfection of E6 gene without significant change (P0.05), and the relative expression of FADD and caspase-8 genes decreased. P0.05).3.2 Western blot results showed that the relative expression of E6 protein increased (P0.05) and the relative expression of FADD, Caspase-8, Fas protein decreased (P0.05) in Het-1A cells transfected with E6 gene, and the relative expression of FADD, Caspase-8, Fas protein decreased (P0.05). The relative expression of DD, Caspase-8 and Fas decreased (P0.05), and there was no obvious change in Fas L (P0.05).Het-1A cells transfected to the E6 gene and E6 protein in 12h. The expression amount increased (P0.05), the relative expression of 48h decreased (P0.05) and Fas L had no obvious change (P0.05). Conclusion: 1 the detection rate of HPV in esophageal squamous cell carcinoma was 32.1%, and the proportion of low differentiated squamous cell carcinoma in HPV positive carcinoma tissues was higher than that in the negative group. It was suggested that HPV was associated with tumor differentiation in.2 esophageal squamous cell carcinoma. The expression of ADD and caspase-8 protein decreased, and HPV could further reduce the protein expression level of both.3 HPV16 E6 gene, which could reduce FADD, Caspase-8, and Fas protein expression level.4 histology and cytology, suggesting that the apoptosis pathway may be inhibited in the squamous cell carcinoma of the esophagus. Play a role in the development of life.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.1
[Abstract]:Objective: China is a high incidence area of Esophageal Carcinoma (EC), about 150 thousand people died of esophageal cancer every year, accounting for nearly 1/4 of the deaths of all malignant tumors. The lowest mortality rate of esophageal cancer in China is in Yunnan province (105/10 million), which is 31 times the difference between the highest death rate in Henan Province, and the incidence of esophageal cancer is found to exist. In recent years, environmental carcinogens (such as mycotoxins, viruses, etc.) have become one of the hotspots in the development of various tumors. The relationship between human papillomavirus (Human papillomavirus HPV) and the occurrence of esophageal cancer was first proposed by Syrjanen in 1982. His study found a group of 40% (24/60) of the cancer of the esophagus. The morphological changes were very similar to the changes in genital warts, and then he and his colleagues found the structural protein.HPV of HPV in Esophageal squamous Cell Carcinoma, ESCC as an important carcinogenic factor in the carcinogenesis of the cervix. Researchers found that the occurrence of oral and esophageal squamous cell carcinoma is also associated with HPV infection for.2008 years. WHO affirms that HPV infection has a direct relationship with the occurrence of squamous cell carcinoma of the pharynx. However, the relationship between HPV and the occurrence of squamous cell carcinoma of the esophagus and its role do not clarify that.Fas is a member of the tumor's bad cause of death and a member of the nerve growth factor receptor family, when Fas After binding with its corresponding ligand Fas L (CD95L), the Fas receptor is trimeric and activated, the activated receptor is combined with FADD, and then the latter activates the latter to form a death induced signal complex, and then activates a series of Caspase-1,3,7, in which the Caspase-3's cutting substrate is PARP, and the execution of.FADD as a Fa.FADD is used as Fa. The key protein in the s pathway can trigger the apoptosis cascade reaction. The study found that the high expression of HPV16 E6 in human colon adenocarcinoma cell line HCT116 cells can prevent the apoptosis of host cells by affecting the apoptosis signaling pathway mediated by TRAIL protein. The study also found that HPV E6 protein can induce the ubiquitination of FADD and caspase-8, thus inhibiting the Fas signal. The activation of the pathway inhibits the occurrence of apoptosis. In our previous experiments, we have learned that HPV infection can reduce the expression of Fas protein in the squamous cell carcinoma of the esophagus, and to further clarify the role of HPV in the development of squamous cell carcinoma of the esophagus. This study first identified the FADD, caspase-8 protein in the tissue of the squamous cell carcinoma of our province. The relationship between HPV infection and the expression of FADD and caspase-8 protein was analyzed. Secondly, the plasmids carrying HPV16 E6 gene fragment were transfected into normal esophageal epithelial cells and highly differentiated squamous cell carcinoma cell lines, and the changes of the Fas/Fas L apoptotic pathway Guan Danbai (Fas/Fas L, FADD, Caspase-8) were observed under the overexpression of E6 protein. To explore the effect of HPV on Fas mediated apoptosis pathway related proteins at the overall level and cell level, further elucidate the relationship between HPV and the pathogenesis of esophageal cancer from the angle of cell apoptosis, and provide new ideas and perspectives for the study of the virological mechanism of the occurrence of esophageal cancer, for the clinical treatment of esophageal cancer, the prognosis and the prediction of the prognosis. Methods: 1 specimens were collected and collected from second hospitals of Hebei Medical University from January 2009 to December 2015. The paraffin specimens of esophageal squamous cell carcinoma (primary squamous cell carcinoma of the esophagus, without radiotherapy and chemotherapy) were collected from January 2009 to December 2015. A total of 159 cases were collected, including age, sex and lymph node metastasis. The differentiation degree of the cancer tissue.2 detected the paraffin tissue into thin slices, the paraffin tissue DNA extraction box was used to extract the tissue DNA. and the HPV L1 gene MY09/MY11 was used as primer. The HPV L1 gene in the experimental group and the control group was detected by PCR method. The beta -actin was used as the internal parameter to make the PCR reaction with three water instead of primers. In sex control, the FADD of esophageal squamous cell carcinoma tissue was detected by the DNA extracted from the paraffin tissue of cervix cancer with positive HPV. The expression of caspase-8 protein was detected by immunohistochemical SP method and the expression of FADD, Caspase-8 protein in the tissues of squamous cell carcinoma of the esophagus was detected by immunohistochemistry, and the positive reaction of caspase-8 protein was located in the cells. Qualitative.4 EX-GS3066-M90 (HPV16 E6), EX-NEG-M90 (unloaded) plasmid purification and concentration detection of.5 HPV16 E6 plasmids transfection on Het-1A (human normal esophageal epithelial cells) and Eca-109 (human esophageal highly differentiated squamous cell carcinoma strain) HPV16 E6 plasmids and negative plasmids (negative controls) were transfected into two groups of Het-1A and Eca-109 cells FADD, Caspase-8 m RNA and protein expression changes were detected by semi quantitative RT-PCR and Western blot methods. Effect of mediated apoptosis pathway, after transfection of Het-1A, cells were collected at 12h, 24h, 48h, and the expression of Fas/Fas L, FADD and caspase-8 protein in the cells was observed after transfection. All data were analyzed by SPSS19.0 software. The count data were analyzed by chi 2 test and Pearson correlation analysis. P0.05 believed the difference was statistically significant. Measurement was statistically significant. The results showed that the difference was statistically significant when the single factor variance analysis ((?) + s) showed that the difference was statistically significant when the single factor variance analysis was P0.05. Results: 1.1 the pathological morphological observation of esophageal squamous cell carcinoma (ESCC) and clinicopathological analysis were 159 cases in this group of ESCC cases, the age span was 27-87 years and the median was 62 years, 124 cases in male and 35 in female patients. Morphological observation: 47 cases of lymph node metastasis, 39 cases of ESCC high differentiation, 72 cases of middle differentiation, 48 cases of low differentiation. There was no significant difference in lymph node metastasis rate between the low differentiation group and the high school differentiation group (P0.05) the relationship between the HPV infection and the clinicopathological features in the.1.2 ESCC tissue 159 cases of HPV infection rate of ESCC were the grade of differentiated high school in the 32.1%.HPV positive group. Cancer accounted for 56.9%, obviously lower than 75.9% in HPV negative group, and 43.1% in HPV positive group, significantly higher than 24.1% in HPV negative group (P0.05). There was no significant difference between HPV infection and age, sex, lymph node metastasis (P0.05).2 immunization results 2.1 ESCC tissues FADD, Caspase-8 protein expression in 159 cases ESCC tissue of FADD protein expression Yang There were 81 cases, 78 cases negative, 93 cases of caspase-8 protein expression positive, 66 cases negative, 150 cases of FADD protein expression in the paracancerous tissues, 9 cases negative, 131 cases of caspase-8 protein expression, 50.9% and 58.5% in ESCC tissues of negative.FADD and caspase-8 negative, obviously lower than 94.3% and 82.4% (P0.05).2.2 E (P0.05).2.2 E adjacent to the paracancerous tissue. The relationship between the expression of FADD, Caspase-8 protein and HPV in SCC tissue was 19 cases of FADD positive expression in HPV positive group, 32 cases negative, 24 cases of caspase-8 protein expression, 27 cases negative, 62 cases of FADD protein expression in HPV negative group and 46 cases negative, 69 cases of caspase-8 protein expression positive, 39 cases of negative sex, FADD and caspase-8 in the positive group protein expression positive. The rates were 37.3% and 47.1% respectively, which were significantly lower than those of 57.4% and 63.9% (P0.05).2.3 ESCC of the negative group. The expression of FADD and caspase-8 protein was associated with the clinicopathological features, the positive rate of the male FADD protein was 59.7%, significantly higher than that of the female (P0.05), and there was no significant difference between the expression of FADD protein and age, lymph node metastasis and the degree of tumor differentiation. Difference (P0.05). The positive rate of male caspase-8 protein expression was 63.7%, significantly higher than that of female 40% (P0.05); there was no significant difference between the expression of caspase-8 protein and age, lymph node metastasis and tumor differentiation (P0.05) the relationship between.2.4 HPV and FADD and caspase-8 protein expression and clinical pathophysiology: ESCC tissue of HPV infection positive: over 62 years of age (packet The positive expression rate of FADD protein was 24.1%, which was significantly lower than 54.5% (P0.05) in patients under 62 years of age, and the positive rate of FADD protein in low differentiation group was 13.6%, significantly lower than that in high school differentiation, and there was no significant difference between FADD protein expression and sex and lymph node metastasis (P0.05).HPV positive ESCC tissue: low differentiation group caspase-8 protein The positive rate was 27.3%, obviously lower than that of the high school group (62.1% (P0.05)). There was no significant difference between the expression of caspase-8 protein and age, sex and lymph node metastasis (P0.05) HPV16 E6 after transfection of.3 cells, Fas, Fas L, FADD, Caspase-8 protein expression 3.1 RT-PCR nodes showed that the transfected cells were compared with the empty plasmid group after transfection. The relative expression of gene increased (P0.05), the relative expression of FADD, Caspase-8 gene decreased (P0.05), the relative expression of Fas gene increased (P0.05), and Fas L had no obvious change (P0.05).Eca-109 cells transfected to the E6 gene, and the relative expression of the E6 gene was higher than that of the empty plasmid group. The relative expression of E6 gene in 12h, 24h and 48h showed an upward trend (P0.05) in 12h, 24h and 48h after transfection of E6 gene without significant change (P0.05), and the relative expression of FADD and caspase-8 genes decreased. P0.05).3.2 Western blot results showed that the relative expression of E6 protein increased (P0.05) and the relative expression of FADD, Caspase-8, Fas protein decreased (P0.05) in Het-1A cells transfected with E6 gene, and the relative expression of FADD, Caspase-8, Fas protein decreased (P0.05). The relative expression of DD, Caspase-8 and Fas decreased (P0.05), and there was no obvious change in Fas L (P0.05).Het-1A cells transfected to the E6 gene and E6 protein in 12h. The expression amount increased (P0.05), the relative expression of 48h decreased (P0.05) and Fas L had no obvious change (P0.05). Conclusion: 1 the detection rate of HPV in esophageal squamous cell carcinoma was 32.1%, and the proportion of low differentiated squamous cell carcinoma in HPV positive carcinoma tissues was higher than that in the negative group. It was suggested that HPV was associated with tumor differentiation in.2 esophageal squamous cell carcinoma. The expression of ADD and caspase-8 protein decreased, and HPV could further reduce the protein expression level of both.3 HPV16 E6 gene, which could reduce FADD, Caspase-8, and Fas protein expression level.4 histology and cytology, suggesting that the apoptosis pathway may be inhibited in the squamous cell carcinoma of the esophagus. Play a role in the development of life.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.1
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