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目的本研究旨在应用聚合酶链反应技术(PCR)联合DNA探针技术对河北省中南部地区食管鳞癌患者人乳头瘤病毒(HPV)感染情况进行研究,以明确二者之间的关系;同时应用单链构象多态性(SSCP)技术对标本p16基因第二外显子突变进行初筛,以明确其在食管癌发病过程中的变化以及HPV感染与p16基因突变的关系。方法对47名河北省中南部地区食管鳞癌切除术患者的癌组织(实验组)以及37名相同地域健康志愿者食管黏膜组织(对照组)进行取材,提取DNA进行HPV分型检测,同时以HPV E6基因引物对标本DNA HPV感染情况进行复检;除试剂盒阳性、阴性对照外,同时采用HPV阳性宫颈癌组织做阳性对照,DEPC水做阴性对照。HPV感染检测完成之后,以p16基因第二外显子引物对组织DNA进行SSCP分析,判断HPV感染时是否伴有p16基因突变。统计学分析采用SPSS13.0软件完成,其中以病理分期为标记分组,采用K个独立样本的非参数检验方法检验不同病理分期的p16基因是否呈现差异;采用pearson相关分析检验p16基因第二外显子在食管鳞癌中与性别、年龄、吸烟史、饮酒史、家族史及淋巴结转移的关系。结果食管鳞癌患者中HPV感染率0%,正常对照组HPV感染率为0%,试剂盒阳性对照呈现阳性,宫颈癌标本HPV感染呈现阳性;复检食管癌患者中HPV E6基因为0%,正常对照组HPV E6基因为0%,宫颈癌标本HPV E6基因为阳性,此结果和试剂盒检测结果一致。食管鳞癌患者中p16第二外显子基因缺失率为0%,基因突变率为44.7%;正常对照组p16第二外显子基因缺失率为0%,基因突变率为0%。p16基因突变与不同病理分期的食管鳞癌的关系中,渐近显著性P值为0.035,卡方值为6.691,认为病理分期越晚,p16第二外显子基因突变率越高;Pearson相关分析中,p16基因第二外显子在食管鳞癌中与性别、年龄、吸烟史、饮酒史、家族史及淋巴结转移的渐进显著性P值分别为0.496、0.143、0.000、0.547、0.113、0.005;以HPV感染为标记分组,检验HPV感染与p16基因变异(Fisher确切概率法)P>0.05,无统计学意义。结论 (1)河北省中南部地区HPV感染与食管鳞癌无关,HPV感染可能不是食管鳞癌的病因。(2)p16基因第二外显子基因变异与食管鳞癌有关。食管鳞癌病理分期越晚,p16基因第二外显子突变越明显。(3)p16基因第二外显子突变与患者吸烟史及淋巴结转移情况有关。(4)HPV感染可能并非食管鳞癌中p16基因第二外显子突变的原因。
Objective To investigate the relationship between human papillomavirus (HPV) infection and esophageal squamous cell carcinoma (HPV) in central and southern Hebei Province by polymerase chain reaction (PCR) and DNA probe technique. Single-strand conformation polymorphism (SSCP) technique was also used to screen the second exon mutation of p16 gene in order to clarify its relationship with the pathogenesis of esophageal cancer and the relationship between HPV infection and p16 gene mutation. Methods Forty-seven patients with esophageal squamous cell carcinoma of esophageal squamous cell carcinoma (experimental group) and 37 esophageal mucosa (control group) from healthy volunteers in the same area of Hebei province were collected and DNA was extracted for genotyping. In addition to the positive and negative control of the kit, the positive control of HPV-positive cervical cancer tissue was also used as the negative control of DEPC water. After HPV infection was detected, SSCP analysis of the tissue DNA using the second exon primer of p16 gene was performed to determine whether there was a p16 gene mutation in HPV infection. Statistical analysis was done by using SPSS 13.0 software. The pathological staging was used as a marker group, and K independent samples were used to test whether there was difference in p16 gene in different pathological stages. Pearson correlation analysis was used to test the second appearance of p16 gene The relationship between son and esophageal squamous cell carcinoma in terms of sex, age, smoking history, drinking history, family history and lymph node metastasis. Results The HPV infection rate was 0% in esophageal squamous cell carcinoma patients and 0% in the normal control group. Positive samples of the positive samples showed positive HPV infection and HPV positive specimens of cervical cancer specimens. The HPV E6 gene was 0% The normal control group of HPV E6 gene was 0%, HPV E6 cervical cancer specimens were positive, the test results and the kit results. Esophageal squamous cell carcinoma in p16 exon 2 gene deletion rate was 0%, gene mutation rate was 44.7%; normal control group p16 second exon gene deletion rate was 0%, the gene mutation rate was 0%. In the relationship between the mutation of p16 gene and esophageal squamous cell carcinoma of different pathological stages, the asymptotic significance P value was 0.035 and the chi-square value was 6.691. The later the pathological stage was, the higher the mutation rate of p16 exon 2 gene was. Pearson correlation Analysis, p16 gene exon 2 in esophageal squamous cell carcinoma and gender, age, smoking history, drinking history, family history and lymph node metastasis of the progressive significant P values were 0.496,0.143,0.000,0.547,0.113,0.005 ; HPV infection as a marker group, test HPV infection and p16 gene mutation (Fisher exact probability method) P> 0.05, no statistical significance. Conclusion (1) HPV infection in central and southern Hebei Province has nothing to do with esophageal squamous cell carcinoma, HPV infection may not be the cause of esophageal squamous cell carcinoma. (2) The second exon gene mutation of p16 gene is associated with esophageal squamous cell carcinoma. The later the pathological stage of esophageal squamous cell carcinoma, the more obvious the mutation of the second exon of p16 gene. (3) The mutation of the second exon of p16 gene was related to smoking history and lymph node metastasis in patients. (4) HPV infection may not be the cause of the second exon mutation of p16 gene in esophageal squamous cell carcinoma.