Dissertation
Dissertation > Medicine, health > Oncology > Genitourinary tumors > Female genital tumors > Uterine tumors

The Relationship between Nitric Oxide and Different HPV Types in Cervical Microenvironment and the Effect on Hela/Caski Cells

Author WeiXueMin
Tutor SuiLong
School Fudan University
Course Obstetrics and Gynaecology
Keywords cervical cancer nitric oxide proliferation apoptosis HPV Infection
CLC R737.33
Type Master's thesis
Year 2011
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HPV (Human papillomavirus) infection is the most common sexually transmitted infections (STI). It is supposed that there are above 80% sexually active women who have a opportunity to infect HPV till 50 years old. Epidemiological studies from Beijing areas have shown that during 5552 sexually active women aged from 25 to 54, the general infection rate was 6.7%, however, that of whom without cervical lesions was 4.8%, and the most common subtypes were HPV 16,58 and 33.Although HPV DNA is detected in the majority of uterine cervical carcinomas, the persistence or progression of cervical lesions suggest that viral antigens are not adequately presented to the immune system. This infection heals spontaneously through immunological defense in the great majority of cases, but in some women HPV persists and this may lead to cervical cancer, which rarely arises without a preceding HPV infection. To understand the factors that affect the balance between viral persistence and viral clearance and the role of the immune system in these processes is important. A shift in this balance towards immunity enables most women to clear the infection. A shift towards immune tolerance facilitates persistence and may permit the development of cervical cancer.However, it is unknown why hr-HPV infection is cancerous in some women whereas in others it is eradicated. Individual differences in systemic or local immunological defense may be one explanation. Many putative cofactors for HPV-induced cervical cancer have been identified through epidemiologic case-control studies, including tobacco smoke, multi-parity, inflammation, and coinfection with Chlamydia or HSV-2. One possible factor involved could be nitric oxide (NO), a free radical gas produced through three nitric oxide synthases (NOSs) (neuronal, endothelial and inducible), which are all found in the human uterine cervix. Nitric oxide (NO) is both a cytotoxic agent and a tumor growth promoter. Cytotoxic effects of NO may occur by direct damage to DNA subsequent to inhibition of enzymes involved in nucleic acid synthesis. In contrast, NO may favor tumor dissemination by promoting angiogenesis. This study aimed to explore the relationship between nitric oxide within cervical microenvironment and different HPV types as well as the effect of SNP, a nitric oxide donor,on the proliferation and apoptosis of cervical cancer cell lines.To explore the relationship between nitric oxide in cervical microen-vironment and different HPV types. Normal cervical tissues without HPV infection were used as control and cervical intraepithelial neoplasia (CIN) with HPV infection as experimental group. At the same time,the nitric oxide (NOx) level of the endocervical canal was detected by Griss method. Furthermore, to study the effect of SNP,a nitric oxide donor,on the prolif eration and apoptosis of cervical cancer cell lines(Hela/Caski).P rolifer-ation of Caski and Hela cells was determined by methyl thiazolyl tetrazolium(MTT) assay,cell apoptosis was detected by flow cytometry after 24 hours; the expressions of HPV E6/E7 gene mRNA and P53 protein were detected by SYBR GreenⅠquantitative real-time PCR and Western blot.Section 1 The relationship between nitric oxide within cervical microenvironment and different HPV typesObjective To study the relationship between nitric oxide within cervical microenvironment and different HPV types.Method From December 1,2009 to August 5,2010, a total of 115 women, aged from 21 to 77 years, mean age was 35, referred to Cervical diseases department for colposcopy of Obstetrics and Gynecology Hospital of Fudan University were recruited for this study. The women participated in a cervical smear screening program in connection with a history of abnormal cervical cytology. Women with any visible vaginal bleeding had been excluded. Cervical mucus samples were collected from each woman as described before. Briefly, a Dacron swab kept in the cervical canal for precisely 20 s was flushed in 1.5mL of physiological saline for 2 s. The samples were assayed for nitrate/nitrite metabolites (NOx) spectrophotometrically using the Griess reaction. The detection limit of the assay for NOx was 0.8μmol/L. In order to reduce the impact of interassay variation, the samples were assayed in only seven batches. The subtypes of HPV in the Cervical mucus was studied by Hybribio-HPV 21 typing test. This study was approved by the Research Ethics Committee, Obstetrics and Gynecology Hospital, Fudan University,and with written informed consent from the study subjectsResult The cervical NOX release of women with hrHPV[(47.6±1.4)μmol/L] was higher compared to hr HPV negative women [(22.8±0.3)μmol/L)] (p<0.05);but there was no statistical difference between lr-HPV group[(24.1±1.2)μmol/L] and control group [(22.8±0.3)μmol/L)]. (P>0.05).Conclusion The presence of hr HPV is associated with an increased release of NO in uterine cervix; The increasing release of NO may play a role in regulating the elimination of HPV in cervical microenvironment, which is a part of mucosal immunity of cervix. Objective To study the effect of SNP,a nitric oxide donor,on the proliferation and apoptosis of cervical cancer cell lines and mechanism involvedMethod Proliferation of Caski and Hela cervical cells was determined by methyl thiazolyl tetrazolium(MTT) assay,cell apoptosis was detected by flow cytometry after 24 hours; the expressions of HPV E6/E7 gene mRNA and P53 protein were detected by SYBR GreenⅠquantitative real-time PCR and Western blot.Result After 24 hours,SNP inhibited the proliferation of Caski/Hela cells and increased apoptosis rate significantly, its optimal concentration was 1mmol/L. SNP reduced the expressions of HPV18E6/E7 mRNA and increased the protein expression of P53,there were statistically significant differences between adding SNP group and the control group (P<0.05),but there were no statistically significant differences in HPV 16 E6/E7 mRNA and that of P53 (P>0.05)Conclusion NO inhibited the growth and proliferation and enhance the apoptosis of cervical cancer cells, inhibit the expression of HPV18E6/E7mRNA in Hela cells and activate the expression of P53 protein,the mechanism may be due to higher sensitivity of Hela cell to SNP.

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