| Objective 1 The patients with early cervical lesions will be screened through detecting Human papillomavirus(HPV)infection in cervical exfoliated cells.2 The plasmid carrying HPV18E6 and E7 fusion gene were transfected into Hela cells,and the effect of HPV18E6 and E7 fusion gene on Hela cells was analyzed,which will be providing scientific basis for the prevention and treatment of HPV18 infection-related diseases.Methods 1 The cervical exfoliated cell specimens and their clinicopathological data were collected.The 15 High-risk HPV(HR-HPV)types in the samples were detected by fluorescence quantitative PCR,and the relationships between the distribution of HR-HPV and clinicopathological features were analyzed.2 HPV infection was detected using MY09/11 primers and HPV58 was detected using HPV58 E6 specific primers;The copy number of HPV58 E6,E2 and human housekeeper gene β-actin was detected by quantitative fluorescence PCR,and the integration status of HPV58 was determined by the ratio of E2 to E6;At the same time,the position of HPV58 integrated into the host chromosome was analyzed through oncogene transcription amplification.3 The primers of Gal K,HPV18-w E6E7 and HPV18-m E6E7 with 50 bp ORF55 homologous arms were designed,and PCR amplifications were conducted for these corresponding gene fragments,the PCR products were purified.The Gal K was electroporated to SW102-TBAC competent cells and screened in medium containing galactose and chloramphenicol.SW102-T-ORF55-Galk-BAC clones were obtained,and the competent cells were prepared.SW102-T-ORF55-HPV18-w E6E7-BAC and SW102-T-ORf55-HPV18-m E6E7-BAC were obtained through homologous recombination and resistance screening in medium containing 2-deoxygalactose and chloramphenicol.The plasmid DNA was extracted and verified by PCR.The correct plasmid DNA was transfected into Hela cells.The expressions of HPV18-w E6E7 and HPV18-m E6E7 in transfected cells were verified by reverse transcription PCR and sequencing.The morphological changes of Hela cells in transfected groups and the untransfected group were observed under microscope.The effect of HPV18-m E6E7 fusion gene on tumor cells was detected by soft AGAR clone assay and CCK8 assay.Results 1 725 specimens were 15 HR-HPV positive in 2605 cervical exfoliated cell samples,including 620 cases with pathological changes,and the positive rate of high-risk HPV increased with the severity of the disease.2 295 HPV positive were detected in1269 exfoliated cervical cell samples by HPVL1 primers,among them 34 HPV58 positive were detected by HPV58 specific primers.The results showed that 4 cases were pure episome,8 cases were pure integration,22 cases were mixture(including integration and episome)by analyzed the integration status of HPV58 positive samples.Statistical analysis showed that there were no significant correlation among the degree of cervical lesions with the viral load and integration status of HPV58.Eight HPV58 integration samples were integrated into chromosomes 1,2,5,8,and 17,respectively.3 1400 bp Gal K,803 bp HPV18-w E6E7 and 803 bp HPV18-m E6 E were obtained by PCR amplification.The clones of SW102-T-ORF55-HPV18-w E6E7-BAC and SW102-TORF55-HPV18-m E6E7-BAC were obtained after electrotransformation,recombination and cloning screening.The expressions of HPV18-w E6E7 and HPV18-m E6E7 in transfected Hela cells were verified by sequencing and reverse transcription PCR.There were contact inhibition and no overlapping growth for transfected Hela cells with SW102-T-ORF55-HPV18-m E6E7-BAC through morphological observation under inverted microscope.The results of soft AGAR cloning showed that no clones were formed in Hela cells with SW102-T-ORF55-HPVm E6E7-BAC.CCK8 results showed that the growth rate of Hela cells with SW102-T-ORF55-HPV18-m E6E7-BAC was slower than that of other groups.These results showed that HPV18-m E6E7 inhibited Hela cells.Conclusions 1 The infection rate of HR-HPV were increased with the aggravation of cervical lesions,which suggested that cervical lesions will be screened through HR-HPV detection.2 HR-HPV HPVE6 and E7 genes integration is random.3 The constructed mutant fusion gene of HPV18-m E6E7 can inhibit the growth of HPV18 positive tumor cells.Figure 20;Table 10;Reference 149... |
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