Transforming Growth Factor ¦Â1 Ica Operon, Biofilm Formation, The Biological Materials Bacterial Lung Cancer Patients

The first part:TGF-β1in non-cellule lung cancer lung cancer patient circumference blood and lung The cancer organizes the expression and with organism immune state relational[Objective]:Discusses in the non-cellule lung cancer patient circumference blood to transform the growth factor (31(TGF-β1) the level, as well as nearby the lung cancer organization, the cancer organizes, in the normal lung organization TGF-P1level and organism immunologic function change and with the non-cellule lung cancer by stages, the pathology type relations.[method]:October,2010to October,2011,In the Third Affiliated Hospital of Kunming Medical University chest heart surgical department in hospital patient, the pathology confirmation for the non-cellule lung cancer (NSCLC). and the seventh edition of lung cancer international frame work method which the international anticancer treatment alliance (UICC) and the international lung cancer research board (IASLC) announces based on2009is Ⅰ-ⅣTime patient; Same time the hospital physical examination branch normal health comparison crowd42examples are the control groups. Unites the immunity adsorption law using the enzyme (ELISA) to examine in the empty stomach circumference venous blood TGF-β1content. The immunity group technology (IHC) two steps examine nearby the NSCLC patient lung cancer organization, the cancer to organize, in the normal lung organization TGF-β1level, uses the class type cell meter to examine the NSCLC patient and in same time the control group circumference blood CD3+, CD4+, CD8+, CD4+/CD8+and the CD4+CD25+cell proportion, and carries on the lamination statistical analysis to above result.[Results]:1.cases of Staphylococcus epidermidis isolated from68clinical isolates, extraction of DNA after PCR product gel electrophoresis test, icaA gene-positive isolates was31, the positive rate was45.6%(31/68), icaD gene-positive isolates was31. the positive rate was45.6%(31/68), icaA, icaD gene positive strains in25patients with PIA positive,80.6%(25/31); icaA, icaD gene negative strains of only PIA was positive in3cases, accounting for8.1%(3/37).Comparing the two, icaA, icaD gene expression of DNA positive clinical strains and the positive expression of PIA was positively related (x2=25.37, P<0.01).2PBMCs mediated, after24h,5,10,20ng/ml TGF beta1in group XWLC-05cell lines cultured in CD3+, CD4+, CD8+cell ratio is below Ong/ml TGF beta1group (P<0.01); a dose effect negative correlation.CD4+/CD8+and CD4+CD25+cell ratio higher than without TGF beta1group (P<0.01); a dose effect correlation.5,10,20ng/ml TGF beta1in group XWLC-05cell lines supernatant of IL-2, TNF, IFN gamma factor levels lower than without TGF beta1group (P<0.01); a dose effect negative correlation.IL-6levels higher than without TGF beta1group (P<0.01); a dose effect correlation.IL-4, IL-10factor levels are not adding TGF beta1groups showed no change (P>0.05).3with different concentration of TGF beta1supernatant of Staphylococcus epidermidis biofilm positive standard strains and clinical isolates of PIA+and medical polyurethane were cultured for24hours,10,20ng/ml group biofilm forming ability and thickness of more than0,5ng/ml TGF beta1group (P<0.01); Staphylococcus epidermidis film negative, PIA album album album called negative clinical isolates in four TGF beta1levels of bacterial biofilm formation ability and thickness no change (P>0.05).[Conclusion]:TGF-betal and of CD4+of CD25+level of the patient's immune function of NSCLC patients peripheral blood showed a negative correlation, the TNM staging more night, the lower the immune function of TGF-β1levels of CD4+CD25+cell ratio of the higher; In the lung cancer organization TGF-β1protein masculine gender expression rate is higher than nearby the cancer the organization and the normal lung organization, nearby the NSCLC patient cancer organization and the peripheral normal lung organization TGF-β1protein masculine gender expression rate non-significance difference. The second part:The relationship of Central venous catheter-related Staphylococcus epidermidis icaA, icaD expression and TGF-β1gene with biofilms in lung cancer patients[Objective] To analyze the impact of lung cancer patients with central venous catheter-related Staphylococcus epidermidis icaA, icaD icaR mRNA expression and PIA factor in the different levels of transforming growth factor β1role in the formation of bacterial biofilms.[Methods]1.separation of patients with lung cancer in vivo central venous catheter surface adhesion of Staphylococcus epidermidis clinical isolates, species identification of central venous catheter related bloodstream infection by Staphylococcus epidermidis type. line of bacterial genome DNA extraction, use PCR in biofilm formation of related genes icaA icaD icaRmRNA expression, Congo red agar culture method to detect PIA factor. PIA factor analysis of icaA. icaD, icaR operon carrying the expression of the relationship between; and to establish the stability of the PIA phenotype negative, positive GPB clinical isolates.2. in vitro culture of Xuanwei lung adenocarcinoma XWLC-05cell lines were stable after passage, adding different concentrations (0.5,10.20ng/ml) of TGF-β1and peripheral venous blood of patients with NSCLC isolated mononuclear cells (PBMCs) of co-culture supernatant of culture, after24hours by flow cytometry (FCM). groups of immune-related factors of CD3+and CD4+, CD8+, CD4+/CD8+and CD4+CD25+cell count and supernatant IL-2, IL-4, IL-6, IL-10, TNF and IFN-gamma factor levels.3.Group of four different concentrations of TGF-β1. Above and cultured for72hours XWLC-05cell line supernatant join Staphylococcus epidermidis biofilm negative and positive standard strains and clinical isolates of the PIA, the PIA positive epidermal aureus co-cultured and joined the medical polyurethane biomaterials, and the establishment of the Staphylococcus epidermidis biofilm (BF) model ARemoved at cultured for72hours, biofilm formation by semi-quantitative assay concentration gradient of TGF-β1factor GPB biofilm formation; with a laser scanning confocal microscope continuous observation of the situation and the thickness of the biofilm growth; scanning electron microscopy biofilm surface state.[Results]1. isolated from68cases of Staphylococcus epidermidis clinical isolates by RT-PCR products by gel electrophoresis, the icaA gene-positive strains of31the positive rate of45.6%(31/68), of which25PIA positive accounted for80.6%; icaD gene-positive strains24, the positive rate was35.2%(24/68), of which15PIA-positive, accounting for62.5%(15/68). Between icaA, icaDmRNA express positive clinical strains with PIA-positive expression was positively correlated (x2=25.37, P<0.01) on the40PIA-positive strains and28of the PIA-negative strains of icaA, icaD, icaR gene mRNA expression were detected. PIA-positive strains of icaR gene expression at the mRNA level is no longer, and icaA, icaD gene mRNA expression; the same time, the PIA-negative strains of icaA, icaD the mRNA expression of lack of icaR gene expression of normal.2. Mediated by PBMCs of24hours,5,10,20ng/ml of TGF-β1group XWLC-05cell lines in culture medium Ong/ml of TGF-β1group (P<0.01); a dose-dependent negative correlation. Of CD4+/CD8+and CD4+CD25+cell percentage higher than those without added TGF-β1group (P<0.01); positive correlation between the dose-dependent manner.5,10,20ng/ml TGF-β1group XWLC-05cell line supernatant dose effect of IL-2, TNF and IFN-γ-factor levels lower than those without added TGF-β1group (P<0.01); was negatively correlated relationship. IL-6levels than those without added TGF-β1group (P<0.01); positive correlation between the dose-dependent manner. IL-4, IL-10factor levels did not increase TGF-β1group did not change (P>0.05).(3) by adding5,10,20ng/ml of TGF-β1group supernatant of Staphylococcus epidermidis biofilm-positive standard strains and PIA+clinical isolates of medical polyurethane surface for72hours biofilm formation capacity and thickness greater than Ong/ml of TGF-β1group (P<0.01); Staphylococcus epidermidis biofilm negative, the PIA-negative clinical isolates in four of TGF-β1levels of bacterial biofilm formation capacity and thickness did not change (P>0.05).[Conclusion] Staphylococcus epidermidis related bloodstream infection in patients with lung cancer Staphylococcus epidermidis operon icaA icaD gene expression is closely related to the synthesis of PIA, icaR gene may play a role in the inhibition of PIA synthesis. PBMCs of dielectric conductivity of TGF-β1XWLC-05cell strain levels of CD3+and of CD4+of CD8+cytokines have inhibitory effects, and dose-dependent negative-related; XWLC-05cell strains of CD4+/of CD8+and of CD4+of CD25+cell factors have role in promoting and positively related to dose-dependent manner. TGF-β1can reduce XWLC-05cytokine release inhibition of cellular immunity.3. PBMCs of mediated and TGF-β1XWLC-05cell line Thl factors:IL-2, TNF and IFN-y inhibition and dose-dependent negative; Th2factor:IL-6levels role in a dose-dependent positive correlation, but no significant effect on IL-4, IL-10cytokine level. Of TGF-β1caused XWLC-05cell line Thl/Th2cytokine imbalance, causing Thl/Th2drift could easily cause a Thl cell dominant cellular immune function decline. High concentrations of TGF-β1factors have some role in promoting Staphylococcus epidermidis biofilm-positive standard strains and the PIA+clinical isolates in the polyurethane surface biofilm formation. The third part:TGF-β1in the tree shrew with biological materials for the center of Staphylococcus epidermidis infections (BCI) model of bacterial biofilm formation in vivo[Objective] To observe TGF-beta1factor in tree shrews and helper T cell subsets: Th1/Th2cytokine level.To explore the different levels of TGF-β1factor in tree shrews with central venous catheters for the center of Staphylococcus epidermidis biofilm formation impact.[Methods]1.Under the weight of the tree shrew. via the tail vein injection of various concentrations of TGF-β1factor (100ng/kg,200ng/kg400ng/kg),48hours after the extraction1ml venous blood measured by flow cytometry of Th1cytokines: IL-2, TNF and IFN-γ and Th2cytokines:IL-6, IL-4, IL-10leve1.2In the tree shrew tree shrew biological material model in the femoral vein at the line central venous catheter (medical polyurethane) underwent catheter placement, injection of different concentrations of TGF-β1factor (100ng/kg200ng/kg,400ng/kg) and the control group were injected through the femoral vein catheter PIA phenotype negative and positive the GPB standard strains and clinical isolates, the formation of tree shrews biological materials, bacterial infection (BCI) model.3.72hours after intubation, remove the central venous catheter in the femoral vein, divided into three parts, observing different levels of TGF-β1factor in tree shrews BCI model and the experimental group and control group animals of central venous catheter BCI model:(1) API bacterial identification system identification of bacteria (2) semi-quantitative biofilm formation assay in each group of bacterial biofilm formation;(3) scanning electron microscopic observation of biofilm formation.[Results](1) intravenous injection of three concentrations of TGF-β1(100ng/kg,200ng/kg400ng/kg) group tree shrews Thl cytokines:IL-2and TNF and IFN-γ is lower than the normal group (F=18.124, P<0.05); further between any two groups q test Tip:100ng,200ng between the two groups no significant difference (P>0.05),400ng group was lower than100ng,200ng group (P<0.01). Th2cytokines:IL-6, IL-4, IL-10were higher than normal group (F=15.421, P <0.05). Further group of two-two q test:100ng,200ng between the two groups no significant difference (P>0.05), the400ng groups is higher than100ng,200ng group(p<0.01).(2) the establishment of tree shrews GPB PIA positive BCI model: table the the GPB PIA-positive standard strain (ATCC35984), after72hours, remove the femoral vein central venous catheter, using semi-quantitative biofilm formation experiment suggested:three concentration of TGF-β1(100ng/kg200ng/kg,400ng/kg) group in tree shrews biofilm formation of the positive rate of TGF-β1group (He=59.68, P<0.01) higher than those not injected; further pairwise comparisons prompt:100ng200ng,400ng biofilm-positive rate of TGF β1group without significant difference (t=3.26, P>0.05). Tree shrew central venous catheter biofilm Scanning Electron Microscope Tip:200ng,400ng of TGF- β1group GPB biofilm formation capacity is higher than100ng of TGF-β1group and control group.(3) tree shrews GPB PIA negative BCI model:the GPB PIA negative standard strain (ATCC12228),72hours later, removal of femoral vein in central venous catheters, semi quantitative biofilm formation experiment indicates that:three the concentration of TGF beta1(100ng/kg,200ng/kg,400ng/kg) group of tree shrew biofilm formation on positive rate and not injected TGF beta1group showed no significant difference (Hc=2.741, P>0.05); a further two two tips:100ng,200ng,400ng TGF beta1groups between biofilm positive rate had no significant difference (t=3.26, P>0.05).Tree shrew central venous catheter within biofilms were observed by scanning electron microscope tip:100ng,200ng,400ng TGF beta1group table aureus among biofilm morphology with no significant difference between the control group.[Conclusion](1).The tree shrews in vivo implantation of TGF beta1factor, on T helper cell subsets of factor Th1inhibition, and the TGF beta1concentration is negative correlation; for factor Th2in IL-6levels have stimulative effect, and with the TGF beta1concentrations are positively related to TGF beta; in1caused the tree shrews in vivo Th1/Th2cytokine imbalance, resulting in Th1/Th2drift, easy to cause the Thl cells leading to decreased cellular immunity.(2) TGF-β1factor to tree shrew Staphylococcus epidermidis the PIA+strains BCI model biofilm have beneficial effects; and TGF-β1factor had no significant effect on the tree shrew S. epidermidis PIA strains formed biofilm in vivo central venous catheter surface.