The Study Of Different Death Patterns And Cytokines Of Lupus Nephritis Patients' Peripheral Blood CD4~+CD25~+T Regulatory Cells Induced By Polyphyllin â…¡

CD4+CD25+T lymphocyte is one of the subsets of regulatory T cells. The amounts of the CD4 CD25 Tregs are small and constitute approximately 5%-10% of CD4+T cells in the healthy people's peripheral blood. Studies suggests that Foxp3 is recognized as CD4+CD25fTregs' special signs and can reflect the level and functional activity of CD4+CD25+Treg in some extent. It is involved in the molecular mechanisms of immune tolerance and it's important to maintenance and regulation of the normal immune function.The abnormity of quantity or function of CD4+CD25+Tregs might lead to genesis of autoimmune diseases. There is evidence that, amount of the SLE patients'CD4+CD25+Tregs is significant decrease compared with human pepole'For its biological activity, CD4+CD25+Tregs secrete at least two types of inhibitory cytokines, namely TGF-βand IL-10. Study have found that TGF-βcan induce the expression of Foxp3 in native peripheral blood CD4+CD25+Tregs and can translate the initial CD4+CD25+Tregs to the activity Tregs. TGF-βcan inhibit immune effector cell proliferation, differentiation and inhibit cytokine production to exert immunosuppressive effects. IL-10 can regulate immune and can decrease antigen-specific T cell proliferation through direct or indirect mechanisms. It can also inhibit the production of IL-12, which is the key effector of Thl cell differentiation. In addition, IL-10 can prevent TCR-mediated activation of CD4+T cell.As important immune response regulator cells, CD4+CD25+Tregs also have the process of self-regulation. The own regulation of CD4+CD25+Tregs'programmed cell death (PCD) is one of the important mechanisms of immune cells'homeostasis regulation. According to morphology, PCD were divided into autophagy, apoptosis, oncosis and so on.Phizoma Paridis, also known as Paris polyphyll, its main active ingredient is steroidal saponins, include polyphyllin (PP)Ⅰ,Ⅱ,Ⅲ,Ⅳ,Ⅴ,Ⅵ,Ⅶ,and so on. Early results show that, Phizoma Paridis can signifycant reduce the FN mRNA level in renal tissue and help to reduce kidney disease. PP has the effect of enhancing the humoral and cellular immune, PP II is the potent immune regulator. But there are few research of PPII about that how to regulate immune, how to affect kidney diseases et al.In this experiment, the pathogenesis mechanism of immune regulation of CD4+CD25+Tregs in LN was investigated by observing the relationship between the PCD (autophagy, apoptosis, oncosis) and the Foxp3 expression, TGF-(3 and IL-10 secretion induced by PP II in healthy and LN people's peripheral blood CD4+CD25+Tregs. Objective:Separate the CD4+CD25+Tregs from healthy and LN people's peripheral blood and be cultured with PP11, then compare with DXM group. Through the research of the change of PCD (autophagy, apoptosis and oncosis)^ the secretion of TGF-βand IL-10 and the expression of Foxp3, to know the role of CD4+CD25+Tregs in the pathogenesis of LN and the possible mechanism of PP II in the treatment of LN.Methods:Peripheral blood CD4+CD25+Tregs and CD4+CD25- T lymphocytes of healthy people and LN patients were separated with percoll(1.073g/ml) and harvested by filtration through nylon column and were separated after magnetic separation. The purity of the isolated cells was analyzed by flow cytometry. The biggest non-toxic concentration of PP II was found and used MTT to test the cells viability. The CD4+CD25+Tregs and the CD4+CD25 lymphocytes were cultured while divided into PP II group (different concentration) and DXM group. Cell morphology was observed by electron microscope after cell culture. Autophagy, apoptosis,oncosis and Foxp3 expression were analyzed by flow cytometry. The levels of TGF-βIL-10 were analyzed by ELISA.Results:1. The purity of healthy people'peripheral blood T cells was 75.4%～98.3%. CD4'T lymphocyte's purity was 85.54～97.5%, CD4+CD25+Tregs'purity was 78.57～91.06%, CD4+CD25-T lymphocyte's purity was 75.05～85.02%。2. The purity of LN patients'peripheral blood T cells was 73.5%～94.1%, CD4+T lymphocyte's purity was 81.4～91.2%, CD4+CD25+Tregs' purity was 73.57～88.6%, CD4+CD25T lymphocyte's purity was 72.7～80.6%。3. lOμg/ml of PPⅡat 72h could increase the autophagy and apoptosis of healthy people and LN patients'peripheral blood CD4+CD25+Tregs, but oncosis was not observed significant changes. Correlation analysis showed that autophagy, apoptosis and oncosis changed independently induced by PP II.4.10μg/ml of PPII at 72h could not increase the autophagy and apoptosis of healthy people's peripheral blood CD4+CD25- lymphocytes. Correlation analysis showed that autophagy, apoptosis and oncosis of changed independently induced by PPⅡ.5. Healthy people and LN patients'peripheral blood CD4+CD25+Tregs secreted TGF-βand IL-10 and could secrete more induced by PPII. The ability of secretion of TGF-βand IL-10 of CD4+CD25T lymphocyte was much lower than CD4+CD25+Tregs, and couldn't secrete more induced by PP II.6. There was significant negative correlation among the apoptosis and the Foxp3 expression and the secretion of TGF-βIL-10. 7. PPⅡfailed to significantly change the Foxp3 expression between healthy people and LN patients'peripheral blood CD4+CD25+Tregs, and there was significant positive correlation between TGF-(3 and IL-10 secretion and Foxp3 expression.Conclusions:1. MACS can separate CD4+CD25+Tregs successfully and the purity is>70%.2. PPⅡcould increase the autophagy and apoptosis of healthy people and LN patients'peripheral blood CD4+CD25+Tregs, couldn't increase the oncosis.3. Correlation analysis showed that autophagy, apoptosis and oncosis of healthy people and LN patients'peripheral blood CD4+CD25+Tregs changed independently induced by PP II and had no significant correlation.4. Healthy and LN people'peripheral blood CD4+CD25+Tregs can secrete TGF-(3 and IL-10 and can secrete more induced by PP II;5. PP II failed to significantly change the Foxp3 expression between healthy people and LN patients'peripheral blood CD4+CD25Tregs, and there was significant positive correlation between TGF-βand IL-10 secretion and Foxp3 expression.6. There was significant negative correlation among the apoptosis and the Foxp3 expression and the secretion of TGF-β,IL-10.