| Objective1 By focusing on vaginal immune microenvironment, the objective of this study was to explore the association between the possible shift of cytokine network and the disease progression and probe the potential indicator to predict further outcome and evaluate effect by measuring the levels of IFN-y as well as IL-10 in vaginal secretions in different stages of cervical disease.2 The second aim of this project was to study the effects of Qingdu Suppository (QDS) and interferonα-2b suppository on high risk HPV infection and the regulation of these two agents in vaginal immune microenvironment and explored the potential immune regulation mechanism of Qingdu Suppository.3 By focusing on MHC-I antigen presentation pathway, the aim of this research was also to investigate the molecular mechanism of Qingdu Suppository andβ-elemene in anti-virus and anti-tumor function based on cervical cancer cell lines HeLa (HPV 18 positive).MethodsClinical research1 A case-control study was carried out in this research. We enrolled women with normal cytology for the control,women with CIN 1 or CIN2-3 as the study groups, and sampled all the patients'vaginal washing fluid in their follicular phases. By using enzyme-linked immunosorbent assay(ELISA),the levels of IFN-y,IL-10 in vaginal washing fluid were tested. Differences in the cytokine concentrations between the groups were analyzed by the nonparametric Kruskal-Wallis.The correlation between the cytokine and stages of cervical lesion as well as that between cytokine and age were determined by Spearman analysis.2 In this study,the cutoff point for each cytokine was assigned by using the lower limit of 95% confidence interval for the mean of the control group. The study groups were classified into two groups,level-up group and level-down group. We used these two as independent variables and CIN as dependent variable. The association between level s of IFN-y and IL-10 and stages of cervical leslon was evaluated by single factor logistic regression. Related factors were analysed by multivariate logistic regression and a regression model was established. Finally,the predictive function of the regression model was evaluated. 3 According to the options of patients,we classified them into three groups: the QDS group, interferonα-2b group and follow-up group as the control group. The treatment lasted for 3 months. After that, all patients received a follow-up interview in 1 or 2 months later and the vaginal washing fluid was sampled. By using ELISA, the levels of IFN-γand IL-10 in the vaginal washing fluid were measured. The effects of these two drugs were analysed and the relationship between treatments,effects and the shifts of vaginal immune microenvironment were explored.Experimental research1 The appropriate inhibitory concentrations and time points of QDS,β-elemene and IFN-α-2b were detected by methyl thiazolyl tetrazolium (MTT)assay for next experiment.2 To assess the regulation of these drugs on HLA-I,flow cytometry(FCM) and Western blot were performed to test the protein expression level of HLA-I on the surface and inside the cells respectively,and real-time PCR for the mRNA expression level of HLA-A,B,C.3 To analyse the regulation of these drugs on MHC-I antigen presentation pathway,Western blot and real time-PCR were performed to test the protein and mRNA expression level of MHC-I antigen presentation pathway associated molecules TAP1,TAP2,LMP2 and LMP7 respectively.Results1 In the levels of IFN-γof the vaginal washing fluid, there were marked differences between these three groups,which were associated with disease progression. There were also significant cross-group differences in the levels of IL-10,but not associated with disease progression.2 The down-regulation of IFN-y in the vaginal washing fluid (OR:6.003,95%CI 2.759～13.062) and up-regulat ion of IL-10 (OR:2.367,95%CI 1.201～4.667) could be considered as risk factors of CIN. The positive predictive value of the regression model was 84.9% while the negative predictive value was 51.7% and accuracy value was 71.2%.According to these results,the regression model still cannot be used in clinic.3 The HPV clearance rates in the QDS group, IFNα-2b group and control group were respectively 38.46%,30.00% and 20.00%, with no significant difference.4 After the treatments with QDS and IFNα-2b, the levels of IFN-y in the vaginal washing fluid increased markedly(p<0.05),whereas no difference was observed for IL-10.5 Water-extraction and alcohol-precipitation of Qingdu Suppository (QDS),β-elemene and IFNα-2b up-regulated the protein expression of HLA-I at the cell surface significantly (p<0.05). In detail, IFNα-2b up-regulated constitutive molecule HLA-A mRNA expression, whileβ-elemene and Qingdu Suppository both up-regulated constitutive molecules HLA-A, B mRNA expression.6 These three agents increased the protein expression of antigen presentation pathway associated molecules TAP1, TAP2, LMP2 and LMP7 in cell line HeLa. Qingdu Suppository up-regulated the mRNA expression of these four molecules, whereas no significant difference was observed after treatments withβ-elemene and IFNα-2b.7 These three agents increased the protein expression of endoplasmic reticulum chaperones CNX and CRT, while IFNα-2b andβ-elemene both increased the protein expression of BIP. These three agents all up-regulated the mRNA expression of molecules above, but there was no significant difference.Conclusions1 We conclude by clinical research that the IFN-y in the vaginal washing fluid may be a useful indicator of local immune responses for the stage of the cervical lesions. The down-regulat ion of IFN-y and up-regulat ion of IL-10 in the vaginal washing fluid could be considered as the risk factors of cervical intraepi thel ial neoplasia. Qingdu Suppository can up-regulate the level of IFN-γin vaginal immune microenvironment,which is a potential anti-viral mechanism for it.2 On the basis of the experimental research, the following conclusion can be drawn that Qingdu Suppository,β-elemene and IFNα-2b can promote antigen presentation through up-regulating the gene and protein expression of antigen presentation associated molecules,which is a potential mechanism of anti-virus and anti-tumor function for these agents. |
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