Disinfection Bolt Intervention Cervical Hr-hpv Infection Of The Local Immune Microenvironment Of The Experimental And Clinical Studies

Object:1. This research from the view of local lesion immune microenvironment, through researching the immunohistochemical expression of immune microenvironment related transcription factor T-bet, GATA3, Foxp3 in cervical intraepithelial neoplasia tissues, from the cellular level explored the relationship between cervical intraepithelial neoplasia progression and the number of Thl,Th2 and Treg in local lesion immune microenvironment.2. SiHa cervical cancer cells in the supernatant, CTL, Treg cell co-culture system as an object of study, observation of Treg, Qingdu tied to CTLA-4, PD-1 protein expression to explore the Treg cells in the tumor immune escape mechanism of traditional Chinese medicine suppository on disinfection effect local immune microenvironment.3. By detecting Qingdu Suppository, AnDafen and followed up for observation of the three groups observed after treatment viral load after 3 months and changes in clinical symptom score, judge drug suppository medicine clear clinical efficacy.Method:Empirical study:1. Select by HE staining, the pathological diagnosis of cervical inflammation and cervical intraepithelial neoplasia of the cervix 128 patients, divided into cervical inflammation, CINⅠgrade, CINⅡgrade and CINⅢgrade 4, in which cervical inflammation, CINⅡgrade lesions 32 Cases, CINⅠgrade of 34 cases and CINⅢgrade lesions of 30 cases.2. CCK-8 determination of the growth curve of PBMC.3. SiHa cells with different concentrations of supernatant role in CTL cells and Treg cells, flow cytometry CD8, Treg cell percentage changes.4. Isolation of human peripheral blood CD8+T cells, Treg cells, flow cytometry cell sorting the purity.5. CFSE labeling technique using cell value to understand the Treg cells to inhibit the function of CTL cells.6.4μg/ml,6μg/ml,8μg/ml ofβ-elemene could promote the proliferation of CTL, select a higher concentration of the drug as a follow-up experiments 8p.g/ml concentration of drug intervention. 1μl/ml,1.5μl/ml,2μl/ml the disinfection of water sink plug ethanol solution could promote the proliferation of CTL, select a higher drug concentration 2μl/ml follow-up experiments as the concentration of drug intervention7. Application of Western-Blot test and compare the CTL normal culture (control group), CTL 50% SiHa supernatants group (cancer supernatant group), CTL+Treg 50% SiHa supernatants group (training group), CTL+ Treg 50% SiHa supernatants+8μg/mlβ-elemene intervention group (β-elemene group), CTL+ Treg 50% SiHa supernatants+2μl/ml ethanol Qingdu suppository water precipitating solution group (Qingdu Suppository Group) five groups CTLA4, PD1 protein expression.Clinical research:1. The investigated group comprised 90 patients suffering from cervical HR-HPV infection, which all visited the DongFang Hospital of Beijing University of Chinese Medical, since Aug.2009 to Jan.2011. Randomly divided into treatment group Qingdu Suppository, AnDafen control group, followed up the observation group were treated three groups observed for 3 months. HCⅡdetected by cervical HPVDNA content (viral load) changes, to determine high-risk Qingdu suppository in the treatment of cervical HPV infection in clinical efficacy, but statistical analysis of the clinical symptom score.Results:1. With the progression of cervical intraepithelial neoplasia,the number of transcription factors T-bet positive Thl cells were significantly increased (P< 0.05).The number of transcription factors GATA3 positive Th2 cells were decreased, including Th2 celluar number change between cervical inflammation and CIN I was not significant P>0.05),the number changes among every grade of cervical intraepithelial neoplasia were significant (P<0.05).The number of transcription factors Foxp3 positive Treg cells were increased including Treg celluar number changes among cervical inflammation, CINⅠand CINⅡwere not significant (P>0.05),the number of Treg in CINIII was increased significantly (P<0.05)2. Application of CCK-8 determination of mononuclear cells (PBMC) found that the growth of cell growth and stagnation in the previous 48h, according to cell growth curve, the following experiment testing should be carried out in the 72h-120h.3. Supernatant of SiHa cells with different concentrations of the role of the CD8+CTL cells, compared with blank control group, serum,75% of the culture supernatant were significantly different (P<0.05); while the effect on Treg cells, 50% of the culture supernatant of group And 75% of the culture supernatant compared with the control group, there are significant differences (P<0.05),75% SiHa supernatant was chosen as the experimental intervention in the following concentrations.4. Isolation of human peripheral blood CD8+T and CD4+CD25+T cells by flow cytometry CD8+ purity was 87.37±0.93%, CD4+CD25+T cell purity was 89.12±2.17%.5. CFSE labeled by CTL,4 days after co-culture with Treg, CFSE showed a single peak, no significant proliferation, and CTL 4 days cultured alone, showing a bimodal CFSE, which began to appear in cell proliferation, suggesting that Treg proliferation of CTL inhibit.6.4μg/ml,6μg/ml,8μg/ml ofβ-elemene could promote the proliferation of CTL, select a higher concentration of the drug as a follow-up experiments 8μg/ml concentration of drug intervention.1μl/ml,1.5μl/ml,2μl/ml the disinfection of water sink plug ethanol solution could promote the proliferation of CTL, select a higher drug concentration 2μl/ml follow-up experiments as the concentration of drug intervention.7. Application of Western-Blot test and compare the CTL normal culture (control group), CTL 50% SiHa supernatants group (cancer supernatant group), CTL + Treg 50% SiHa supernatants group (training group), CTL + Treg 50% SiHa supernatants +8μg/mlβ-elemene intervention group (β-elemene group), CTL + Treg 50% SiHa supernatants+2μl/ml ethanol Qingdu suppository water precipitating solution group (clear Drug suppository group) five groups CTLA4, PD1 protein expression.8. Qingdu suppository and AnDafen can effectively reduce HC-ⅡHPVRlu co ratios that HPVDNA viral load, self-control results before and after treatment was statistically significant (P<0.05), showed no significant difference, and with the clinic observed group differ (P<0.05); also found that the two drugs on clinical symptoms and signs improved significantly effect (P<0.01), and followed up for observation group was significantly different (P<0.01).Conclusion:1. The local immune microenvironment in cervical lesions in the Th1, Th2 type cells and Treg cells are involved in cervical intraepithelial neoplasia of the immune response, Treg cells may progress through the immunosuppressive effects in disease2. Qingdu suppository can reduce the CTL in CTLA4, PD1 protein expression probably by inhibiting the function of Treg cells to reduce the CTLA4, PD1 protein expression, thus changing the local immune microenvironment to inhibit tumor effect.3. Qingdu suppository can reduce cervical high-risk human papillomavirus (HR-HPV) infection in viral load, also can effectively improve the clinical symptoms and signs.