| Part 1 Study on the correlation between multiple HPV infection and cervical cancerObjectives:To analyze the characteristic of clinical distribution of HPV genotypes infection between control group and cervical cancer group, and investigate the role of multiple HPV infection in the occurrence and development of cervical cancer.Methods:According to the principle of informed consent,224 cervical cancer tissue samples,from female patients treated in the Third Affiliated Hospital of Kunming Medical University from September 2013 to December 2015,were collected,and,1011 cervical vaginal secretion samples,from females who took physical examination in the same period, were selected as control group. We tested HPV genotype by gene chip and knew about HPV infection,then analyzed the HPV genotype infection in normal population(the control group) and the cervical cancer group respectively.Results:(1) The result of HPV typing test in 1011 cases of the control group shows that there are 135 HPV positive patients,and the total infection rate is 13.35%.Genotypes of HPV infection mainly are HPV52 (3.17%),16 (2.67%),58 (1.68%),18 (0.99%) and 39 (0.89%).The single infection rate of HPV52 (2.37%),16 (1.98%) and 58 (1.19%) are on the top three.The multiple infection rate of HPV52 (2.37%),16 (1.98%) and 58 (1.19%) are on the top three (multiple infection is counted repeatedly according to infection genotype). However,there are 216 HPV positive cases among 224 cervical cancer cases, and the total infection rate reaches 96.43%.In these cases,genotypes of HPV infection are mainly HPV 16 (86.61%),18 (19.64%),31 (4.46%),51 (3.13%) and 58 (3.13%).The single infection rate of HPV16 (59.82%),18 (3.57%) and 58 (1.34%) are on the top three.The multiple infection rate of HPV16 (26.79%),18 (16.07%) and 31 (3.57%) are on the top three.(2)The data of the control and cervical cancer group,which both were divided into 6 age stages(<21,21-30,31-40,41-50,51-60,and>61),was conducted the statistic analysis.The HPV positive rate of different age in the control group respectively is 0.40%,3.76%,4.95%,3.36%,0.69% and 0.18%,while in the cervical cancer group is 0.00%,6.25%,24.11%,36.16%,23.66% and 6.25%.The infection rate in the control and cervical cancer group both distribute as a single-peak model.HPV infection in the control group reach to peak at an age stage from 31 to 40,while the cervical cancer group is from 41 to 50,which is 10 years older than the former.(3)The study shows that,the mixed infection rate in the control group,including two or more than two genotypes, is 2.47%(25/1011),while multiple infection rate in the cervical cancer group is 27.68%(62/224) and all with high-risk mixed infection.Differences of multiple infection rate in 4 age stages(21-30,31-40,41-50 and 51-60) of the control group and the cervical cancer group respectively,is statistically significant (P<0.001). Among multiple infection,the co-infection is the main part, occupying 22.77%(51/224).The most common type of co-infection is HPV16+18,accounting for 52.94%(27/51),and next is HPV16+31(17.65%,9/51).Conclusions:(1) Showed as the research,HPV geboype distribution change with the increase of cervical lesion severity.The main type of HPV in patients with cervical cancer is HPV 16 and 18,which suggests that HPV 16 and 18 positive patients with cervical lesion,found in the cervical cancer screening,should be closely observed and with timely intervention and treatment, to prevent its progression to cervical cancer.Besides,bivalent HPV 16,18 vaccine, or quadrivalent HPV 16,18,6 and 11 exist potential strong prevention ability in Yunnan region.(2) HPV infection shows a single-peak distribution with the change of age,and it reaches to peak at an age stage from 41 to 50 in the cervical cancer group,which is 10 years older than the control group.Thus, perimenopause and menopause women with high-risk HPV infection should arouse attention and perform a cytology testing.In addition,the phenomenon——no cervical cancer patients younger than 21 years old was found in the process of the study,matches up with the description in the NCCN that persons younger than 21 have no need of cervical cancer screening.(3) The study indicates that multiple HPV infection in cervical cancer all are high-risk HPV mixed infection,whose infection rate in the cervical cancer group apparently higher than the control group.The results indicate that multiple HPV infection is closely rated to cervical lesion——the chances of multiple HPV infection increases with the exacerbation of cervical lesion.Thus,multiple HPV infection will increase the risk of cervical cancer.Part 2 Study on the correlation between cervical cancer and the mutation of HPV 16 E6 and E7 geneObjectives:To analyze the mutation characteristic of E6 and E7 gene,the main oncogenes of HPV 16, and explore the mutation site of HPV 16 E6 and E7 gene associated with the occurrence and development of cervical cancer.Methods:194 cervical cancer tissue samples,from HPV16 positive female patients with cervical cancer treated in the Third Affiliated Hospital of Kunming Medical University from September 2013 to December 2015,were enrolled,and,197 cervical vaginal secretion samples,from HPV 16 positive females without cervical lesion who took physical examination in the same period, were selected as the control group.The process of sample collection,diagnosis and treatment were conducted under patients informed consent,and pathological diagnosis is considered as the gold standard of cervical lesion.After samples were collected,we extracted the DNA of specimens for HPV types, sequenced integrated gene section of E6 and E7,obtained after PCR amplified E6 and E7 gene fragments of HPV 16 positive samples, then looked for variation characteristic of HPV 16 E6 and E7, and conducted a stratified analysis of the common mutation site combined with clinical data.Results:(1) Without African type 1 and type 2, Asian -American type,and North American variant, only Asian variant and European variant,were found in the Phylogenetic analysis result of HPV 16 E6、E7 amplified products.There are no significant difference of variant in the infection group and the cervical cancer group(P>0.05).(2)In all HPV 16 positive samples,E6 mutation rate in the group HPV infection but no cervical lesion and the cervical cancer group respectively is 70.00%(126/180) and 73.18%(131/179).The most common mutation site of HPV16 E6 gene in the infection group and the cervical cancer group is T178G/A (D25E),A276G (N58S) and T350G (L83V),but the results of statistical analysis show no statistical significance of the mutation distribution in the infection group and the cervical cancer group.(3)C627T,G666A,T843C,T846C,A647G (N29S) and C749T (S63F) are the common mutation sites of E7 gene.Among them,the difference of C749T(S63F) between cancer patients and infected persons is significant (P<0.01)Conclusions:(1) Asian variant and European variant were found in the Phylogenetic analysis result. Shows no relationship with the occurrence and development of cervical cancer,(2)The most common mutation site of HPV16 E6 gene in the infection group and the cervical cancer group is T178G/A (D25E),A276G (N58S) and T350G (L83V),and the mutation distribution between infection group and cervical cancer group has no statistical significance difference.(3) A significant difference in the mutation of HPV 16 E7 gene C749T(S63F)site was seen between the infection group and the cervical cancer group.It suggests that C749T (S63F) mutation may exist relationship with the occurrence of cervical cancer.But the mechanism of the mutation related to cervical lesion need to be further investigated.In the result of the study, the correlation between development of cervical lesion did not been found,and do not match the description previously reported.It may because that the occurrence of cervical cancer is a result of the concerted action of manifold factors and multi-gene.Beside HPV infection,gene mutation of host cells also plays an important role.Therefore,we simultaneously researched p53 and pRb gene polymorphism,and synthesized the results of E6 and E7 mutation and p53 and pRb gene polymorphism to further analyze. |
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