【作者】 乌恩奇；

【导师】 吴永革； 孔维；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 宫颈癌(Cervical Cancer, CC)是最常见的女性恶性肿瘤之一。高危型人乳头瘤病毒(Human Papillomavirus, HPV)已被证实为是引发宫颈癌的主要病原体。明确某一地区HPV主要流行型别对指导HPV疫苗的应用推广和新一代疫苗的设计优化有着重要意义。同时,了解宫颈癌中HPV的物理状态和基因突变情况对深入研究HPV生活史和致癌机理也有着重要意义。本实验从我国四川省,内蒙古通辽市和吉林省长春市收集宫颈癌及其相关组织,考察HPV整体感染和各型别分布情况,根据HPV型别调查结果,对在各地区流行最广泛的HPV16的E6基因的突变情况进行了分析。此外,本实验还对在东亚地区有着广泛流行的HPV58在宫颈癌组织内的物理状态和基因突变进行了考察。对来自四川,通辽和长春的样品进行的HPV检测结果表明,在我国一些地区,一些人群中,除了HPV16,18之外,HPV45、52、58、59等型别也有着广泛的流行,在针对我国人群的下一代宫颈癌疫苗的研发工作中应当充分考虑这些型别的流行特点。对109例HPV16的E6基因序列分析中,共检测出20处点突变;系统进化分析结果表明分离自我国的所有HPV16突变体均属于欧洲原型突变组。对宫颈癌组织中HPV58的基因突变及物理状态的分析表明,78.4%的样品中的HPV58以完全整合形态存在,其中近一半的HPV58有多点断裂现象。在HPV58基因组中有118个位点发生了突变;系统进化分析结果表明,分离自我国的所有HPV58突变体均与HPV58原型处于同一分支,而另一分支主要由分离自非洲的突变株组成。这一结果提示,与HPV16一样,HPV58不同的突变株在世界各地的分布也有着明显的地域性特征。更多还原

【Abstract】 Cervical cancer (CC) is the second most common cancer affecting women worldwide, and it is generally accepted that HPV is the primary causal agent of cervical carcinoma. The distribution of HPV types, subtypes and the variants varies greatly worldwide, which is closely related to the corresponding risk of cervical cancer. The determination of the prevalence of HPV types is important to the evaluation of the potential impact of current HPV vaccines and can assist in the identification of those additional HPV types, which should be considered for the development of future generation HPV prophylactic vaccines. Besides, the detailed delineation of the HPV gene variants and the pattern of viral integration could provide useful insights into understanding of mechanism of HPV-linked carcinogenesis and the natural history of HPV infections.At present study, we have evaluated the prevalence and distribution of HPV types in cervical specimens collected from Sichuan province, Tongliao city of Inner Mongolia and Changchun city of Jilin province by combination of nested PCR and sequencing method. And also, we sequenced the E6 gene of HPV16, which is predominant HPV type in all of those three areas, and determined the subtype of the HPV16 based on the sequence of the E6 gene. In addition, we investigated the physical status and the gene variants of HPV58, which was reported to have a relatively high frequency in CC patients from East Asia.Of the 190 CC specimens isolated from Sichuan province, HPV16 was found to be the most prevalent HPV genotypes. HPV 58, 52 and 59, detected in more than 10% of specimens, respectively, were quite prevalent in cervical cancer specimens from Sichuan province. HPV18, the second most prevalent HPV type worldwide, was found in 8.4% of specimens, which is lower than the rate reported in worldwide (12.8%). It is interesting that many HPV16, 52, 58 and 59 infections detected in this study existed with lower viral load, and was only detected by nested PCR methods. The role of such high prevalence of these types with low viral load in development of cervical cancer needs further investigation.In the result of HPV detection in 175 archival cervical lesions collected from Tongliao, HPV16 was found to be the most predominant HPV type in all cervical lesions, and HPV 45 was found as the second most predominant HPV type in CC and HSIL (High-grade Squamous Intraepithelial Lesion). The prevalence of HPV 45 in Tongliao area is significantly higher than those previous studies on other region of China, and the infection rate of HPV 45 was found to increase as the severity of cervical lesions increased, which indicate that HPV 45 may play a more significant role in the etiology of cervical cancer in Tongliao area than other region of China. Comparing the HPV detection rates in Han Chinese and Mongolian, the prevalence of HPV 18 in Mongolian CC patient was found to be significantly higher than that in Han Chinese patients.In Changchun area, HPV16 was found to be the most predominant HPV type in both CC and normal cervical swab specimens. The infection rates of other high risk HPV types, such as HPV 18, 45, 52, 58 and 59, which were reported to have high frequency in CC from different region of China, were quite low in CC specimens from Changchun. However, the prevalence of HPV58, which is prevalent in Southern China, and HPV 62, which is prevalent in Korea, were found to be quite prevalent in normal cervical samples from this area. The uncommon higher prevalence of HPV58 and 62 in normal cervical samples than that of cervical cancer in Changchun area may have some association with the change of population flow during recent decades since the open-door policy; and because the development of cervical cancer is usually slow, and occurs over a period of years, the changes of prevalence of HPV types may has not been reflected in the HPV infection rates in cervical cancer.Comparing the HPV detection rates in cervical cancer from Sichuan, Tongliao and Changchun, no significant differences were observed in the overall prevalence of HPV infection, while the distribution of some certain HPV types varies greatly across populations. Combining with the results of previous studies in China, our result further confirmed that the distribution of HPV types in China is different from other region of the world, and there is also some difference in prevalence of HPV types among different region and different ethic group in China. Besides HPV16 and 18, which were most prevalent high-risk types worldwide, HPV 45, 52, 58 and 59 is prevalent in different region of China, and for optimal population coverage, these HPV carcinogenic types should be considered for second-generation HPV prophylactic vaccines.By sequencing 109 cases of HPV16 E6 gene, 20 nucleotide positions in E6 gene found to be variable, and among them, the nucleotide changes A121G、C158G、T377G、t429c、C434G have not been reported previously. The nucleotide change T96G was found to be the most common variant in present study, 27 samples contained this nucleotide change, and the nucleotide change T268G was found to be the second most common variant, 22 samples contained this nucleotide change. Of the 20 nucleotide changes, 17 lead to amino acid changes. Among them amino acid change R10G、T22S、H24Y、D25K、D25E、E29Q、K34E、A46G、R55K、N58S、L64E、S71C、H78Y、L83V、L119R、K121E、S138C were located in the HPV E6 epitope regions, suggests that these changes may alter their epitope specificity. The amino acid change D113E was located in the HPV E6 P53 binding and degeneration regions; indicate that this change may affect the binding affinity of E6 to p53.Phylogenetic analysis based on sequences of 33 variants isolated from in present study and 5 main HPV 16 subtypes show that 27 of 33 variants were classified as As (East-Asia) subtype, and 6 of variants were classified as E (European, prototype) subtype; all variants isolated in present studies were included in the Prototype-prototype like group. Both As subtype and E subtype were found in both Han Chinese and Mongolian from Sichuan, Inner Mongolia and Changchun, and. the As subtype seems to be more prevalent in Sichuan, which located at southern china, while the E subtype seems to be more prevalent in Changchun and Tongliao, where located at northern China.By investigating the physical status and the gene variants of 37 cases of HPV58, a pure integrated genome was found in 78.4 % ( 29/37) specimens, which is much higher than those in previous studies. In the 29 completely integrated HPV 58 isolates, 15 cases were found to have multiple disruptions in the HPV 58. There is, to our knowledge, no previous report on phenomena of the multiple disruptions found in the integrated HPV genome. The mechanism of action and biological activity of the multiple disruptions of HPV genome remain unclear and require further investigation. The sequence between nucleotides 7557 and 970 is conserved in all completely integrated HPV 58 genome, indicates that detection of this sequence might be an ideal marker for PCR-based determination of HPV 58 infections. Besides, all completely integrated HPV 58 genome isolated in this study have disrupted or lost at least part of nucleotide sequence between nucleotides 1883 and 3184, indicating that detection of the loss of this sequence might be an ideal marker for PCR-based determination of HPV 58 integration.In the entire HPV 58 genome, 119 (1.52 %) nucleotide changes were found; of these, 45 lead to amino acid changes when compared with the reference sequence. Among each region or ORF of the HPV58 genomes, the unncoding region between nt 4123-4244 had the highest proportion of variable nucleotide positions 3.31% (4/121); among each ORF of the HPV 58, the amino acid changes frequency in E7 was the highest 7.14% (7/98), followed by E4, E1, E6, E5, L2, L1 and E2. Phylogenetic analyses based on partial L1 sequences of 14 variants isolated in previous studies and this study shows that two main groups were observed in HPV 58 variants, the prototype or prototype-like group and non-prototype-like group. All variants identified in this study were included in the prototype/prototype-like group. No African specimens were included in the prototype/prototype-like group, and no Asian specimens in the non-prototype-like group, while European and American specimens were included in both groups.更多还原