| Objective To study HPV infection status and HPV genotype feature in different grade of cervical lesions and to investigate the effects of cidofovir on Hela cell and Siha cell lines derived from cervical carcinoma infected by human papillomavirus, and to evaluate antivirus and antitumor activity of cidofovir.Methods 1. Clinical study:①having received colposcopic examination and multiple biopsies, 690 patients with high-risk HPV and TCT were analyzed retrospectively.②153 patients with paraffin-embedded examples of different grade of cervical lesions were analyzed retrospectively. DNA was extracted and HPV genotypes were checked by HPV gene chips. 2. In vitro:①The growth rate of Hela cell and Siha cell treated with cidofovir was determined with MTT assay and colony forming assay.②By real-time quantitative PCR, the effects of cidofovir on HPV16 E6/E7and HPV18 E6/E7 mRNA were studied.③By Western-blot, the effects of cidofovir on p53, Rb in Hela cell and Siha cell were investigated.④Cell cycle and apoptosis of Hela cell and Siha cell treated with cidofovir were observed with flow cytometer.⑤By TRAP-ELISA, the effects of cidofovir on telomerase activation in Hela cell and Siha cell were evalued. 3. In vivo: Athymic mice were injected 2×106 Hela cells and Siha cells respectively. Once tumors were established, the mice were divided into 3 groups randomly and intratumorally injected with PBS, cidofovir at a dose of 2.5mg/ml (0.25mg/ injection) and 5.0 mg/ml (0.5mg/injection) respectively three per week.①During 3 weeks, volumn and weight of transplantation tumors were measured and compared.②By Western-blot, the expression of p53 in tumors was observed.③By immunohistochemical method, the expression of PCNA was observed in transplantation tumors of cidofovir-treated and control group. Moreover, the effects of cidofovir on cell apoptosis in transplantation tumors were noted by TUNEL technology. Results 1. Clinical study:①The positive rate of high-risk HPV of CIN II/III and invasive cancer were over 90.00%. The higher the cytology and pathology grade of cervical lesions, the more the infection rate of HPV.②The more HPV DNA load, the severer cervical lesions.③Different HPV genotype resulted in different grade of cervical lesions. HPV16 is the most genotype.④Mixed HPV infection rate was associated with the severity of cervical lesions. 2. In vitro:①It was confirmed by MTT assay and colony forming assay that cidofovir can significantly inhibit the proliferation of Hela cell and Siha cell ( P<0.05) .②By real-time quantitative PCR, cidofovir can result in inhibition of HPV16 and HPV18 E6/E7 mRNA.③By Western-blot, cidofovir can increase the expression of p53 in Hela cell and Siha cell dose-dependently. In addition, cidofovir can increase the expression of p-Rb and decrease the expression of p-pRb.④Cidofovir made Hela cell and Siha cell cycle arrest in S stage and G1 stage. Cell apoptosis were observed with flow cytometer.⑤By TRAP-ELISA, cidofovir can significantly inhibit telomerase activation in Hela cell and Siha cell ( P<0.01) . 3. In vivo:①During 3 weeks, transplantation tumors administered cidofovir showed a statistically significant reduction in volumn and weight, compared with control tumors( P<0.01).②By Western-blot, cidofovir can increase the expression of p53 in transplantation tumors.③By immunohistochemical method, fewer proliferating cells were noted in the cidofovir-treated transplantation tumors, whereas control tumors were characterized by higher expression of PCNA. Moreover, cidofovir can induce apoptosis in transplantation tumors, as evidenced by TUNEL technology.Conclusion High-risk HPV infection is a basic etiological factor of cervical carcinoma. The clearance of HPV holds the key to prophylaxis of cervical carcinoma. Cidofovir can inhibit the growth of human cervical carcinoma Hela cell and Siha cell lines infected by human papillomavirus and induce cell apoptosis. |
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