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青岛地区宫颈病变组织中人乳头瘤病毒16型L1基因多态性分析
Analysis of HPV16L1Gene Variants of Different Cervical Lesions in Qingdao Abstract
【作者】 孙欣；
【导师】 王言奎；
【作者基本信息】 青岛大学 ， 妇产科学， 2012， 硕士
【摘要】 目的HPV病毒基因存在变异,且具有种族和地域特异性。本研究通过对不同宫颈病变组织中HPV16L1基因的检测和序列对比,分析了青岛地区宫颈病变患者HPV16L1基因结构特点和变异规律,探讨基因变异与宫颈癌的相关性,为高特异性疫苗的研制提供实验基础。材料与方法(1)收集山东青岛地区宫颈癌新鲜组织61例,经病理诊断为宫颈鳞状上皮细胞癌；LCT(液基细胞学检查)标本300例,其中正常细胞47例,LSIL(低度鳞状上皮内病变)100例,HSIL(高度鳞状上皮内病变)153例。300例LCT标本中有明确病理诊断者181例,其中慢性宫颈炎40例,宫颈上皮内瘤变(cervical intraepithelial neoplasia, CIN)Ⅰ～Ⅲ级141例。(2)用蛋白酶K法,分别提取新鲜组织和细胞DNA。(3)以提取的DNA为模板,采用PCR技术,以通用引物MY09/11筛选出感染HPV的标本,然后用HPV16型特异性引物筛选出HPV16阳性标本。(4)以HPV16阳性标本DNA为模板,扩增L1基因全序列。(5)所获PCR产物行基因全序列测序,应用DNASTAR软件,与德国HPV标准株进行比对分析,寻找HPV16L1的突变位点,分析核苷酸和氨基酸的变异,比较热点突变位点突变频率在各组的差异,并与其他国家其他地区突变位点进行比较。结果(1)慢性宫颈炎组、CIN组、宫颈癌组HPV感染率分别为75.0%(30/40)、89.4%(126/141)和93.4%(57/61),3组相比较,差异有统计学意义(X2=8.370,P=0.015)。正常细胞、LSIL、HSIL、宫颈癌细胞组HPV感染率分别为55.3%(26/47)、61.0%(61/100)、81.7%(125/153)和93.4%(57/61)。4组相比较,差异有统计学意义(X2=34.405,P=0.000)。(2)慢性宫颈炎组、CIN组、宫颈癌组HPV16感染率分别为30.0%(12/40)、41.1%(58/141)和65.6%(40/61),3组相比较,差异有统计学意义(X2=14.874,,)=0.001)。正常细胞、LSIL、HSIL、宫颈癌细胞组HPV16感染率分别为10.6%(5/47)、32.0％(32/100)、53.6%(82/153)和65.6%(40/61)。4组相比比较,差异有统计学意义(X2=44.303,P=0.000)。(3)所有经过病理诊断的标本中,共有96成功扩增出L1基因,其中慢性宫颈炎10例、CINⅠ～Ⅲ53例、宫颈癌33例,其扩增率分别为25.0%、37.6%和54.1%。所有LCT标本中,共有136例成功扩增出Ll基因，其中HSIL70例,LSIL28例,正常细胞抹片5例。(4)L1基因共发现13处变异,所有标本均发生C6240G、A6432G、6902insATC.6954de1GAT及G7061A(无义)5处突变。另一突变热点为A6178C,其在慢性宫颈炎、CIN、宫颈癌3组间频率分布没有显著性差异(X2=1.265,P=0.531),在正常细胞、LSIL、HSIL、宫颈癌细胞4组间频率分布亦没有显著性差异(X2=6.705,P=0.082)。结论(1)青岛地区HPV16L1基因存在多态性,最常见的突变位点为C6240G、A6432G、6902insATC、6954delGAT、G7061A无义)及A6178C。HPV16L1基因的多态性具有明显的地域性特征。(2)青岛地区HPV16L1基因的多态性可能导致病毒地方株的免疫原性和抗原性改变,但与宫颈癌的发生发展没有明显关系。
【Abstract】 ObjeetiveHuman papillomavirus (HPV) has a number of intratypic variants, with racial specificity and different geographical distribution.This study was performed to detect sequence variants of the HPV16late gene L1derived from Qingdao women with different cervical lesions,analyze the structural features and variation rule, discuss the association between the sequence variant and the cervical lesions, and to provide experiment foundation of the development of thigh specific vaccine.Materials and Methods(1)61cervical cancer biopsies.with were diagnosed squamous cervical cancer by pathology, and300cervical-scraping cell lysates (LCT) were collected in the Qingdao area. The LCT tissues include47normal tissues,100Lowgrade Squamous Intraepithelial Lesion (LSIL) tissues,and153Highgrade Squamous Intraepithelial Lesion (HSIL) tissues. In all LCT tissues.181tissues were pathologically diagnosed.including40chronic cervical inflammation and141cervical intraepithelial neoplasia (CIN) I～III.(2) DNA samples were extracted from the cervical scrapes and tissues.(3) DNA samples were amplified by polymerase chain reaction(PCR)、using specific primers MY09/11for HPV infection, specific HPV16primers to identify the positive tissues.(4) DNA samples extracted from the HPV16positive tissues were amplified by the primers for L1.(5) the PCR fragments of HPV16L1gene were sequenced and compared with the type stain by DNASTAR.Results (1) The positive rate of HPV was75.0%(30/40、89.4%(126/141) and93.4%(57/61) separately in inflammation. CIN and cervical cancer. The distribution showed statistical differences (X2=8.370, P=0.015). The positive rate of HPV was55.3%(26/47),61.0%(61/100)、81.7%(125/153)and93.4%(57/61)separately in normal cells, LSIL,HSIL and cervical cancer cells. The distribution showed statistical differences (X2=34.405, P=0.000)(2) The positive rate of HPV16was30.0%(12/40)、41.1%(58/141) and65.6%(40/61) separately in inflammation, CIN and cervical cancer. The distribution showed statistical differences (X2=14.874, P=0.001). The positive rate of HPV16was10.6%(5/47)、32.0%(32/100)、53.6%(82/153) and65.6%(40/61) separately in normal cells. LSIL,HSIL and cervical cancer cells. The distribution showed statistical differences (X2=44.303, P=0.000)(3) A total of96samples were amplified successfully in all samples diagnosed by pathology. The rate were25.0%(10/40)、37.6%(53/141) and54.1%(33/61) separately in inflammation. CIN and cervical cancer. In all LCT samples.136were amplified successfully, with70HSIL.28LSIL and5normal cells.(4) A total of13variants were found in the full length of L1gene. Five mutations, including C6240G、A6432G、6902insATC、6954delGAT and G7061A(nonsense). were found in all of the positive samples. Mutation of A6178C was another hot spot, whose distribution showed no significant difference among inflammation、CIN and cervical cancer (X2=1.265, P=0.531). and also no significant difference among normal cells. l.SIL,HSIL and cervical cancer cells(X2=6.705, P=0.082).Conclusions(1) The C6240G、A6432G、6902insATC、6954delGAT、G7061A(nonsense) and A6178C were the prevalent variants in Qingdao cervical lesions. There are significant geographical features in the HPV161.1gene variation. (2) The HPV16L1gene variation may cause certain changes concerning immunogenicity and antigenicity of the virus, nevertheless has no apparent connection to the development of cervical lesions.