Establish Mesangial Cell Activation Model And Its Biological Effects On Monocyte Macrophages

Objective:To investigate the optimal conditions for the activation of human kidney mesangial cells (Human mesangial cells, HMCs) and activated HMCs’ cell phenotype and molecular markers, and the biological effects between activated HMCs and human monocyte-macrophage cells (Human monocyte macrophages cells, HMMCs).Methods:1. The human cell phenotype and molecular markers of activation of research and research HMCs after activation:with 10ng/ml,20ng/ml,50ng/ml of recombinant human platelet-derived growth factor (Recombinant Human Platelet Derived Growth Factor-BB, PDGF -BB) were stimulated HMCs 2h,6h,12h,24h,48h, 72h, Real-time quantitative detection of nucleic acid amplification systems technology (Real-time Quantitative PCR Detecting System, qPCR) to find and identify the best stimulus concentration and time; Cell count (Cell Counting K-8, CCK-8) to detect the proliferation of HMCs after activation; Cell count scratch test cell chemokines and migratory ability HMCs after activation; Immunofluorescence staining detection activatedthe ability to detect the proliferation of activated HMCs the HMCs of Desmin, a-SMA, PDGFR-β, Transgelin, Tensin expression and using qPCR technique can detect the duration of HMCs in activating expression of state.2. Biological effects of activated human mesangial cells to human monocyte macrophages:detected by qPCR HMCs PDGF-BB stimulated secretion induced HMMCs whether activation of cytokine secretion and the presence of activated molecular markers HMMCs; the PDGF-BB induced HMCs and HMMCs placed in the Transwell co-culture system, select 2h,6h,12h,24h,48h,72h time node, study the biological effects of HMCs activated on HMMCs of; CCK-8 detected after co-culture of proliferation HMMCs cells after co-culture assay scratch HMMCs chemotaxis and migration capabilities.Results:1. The person HMCs activation and activated cellular phenotype and molecular markers:(1) Cytokines HMCs different points of time under different concentrations of stimulation, the qPCR detected after activation of IL-1, IL-6, CCL-2, PDGFR-β, Transgelin, Tensin mRNA expression results showed that:When using a concentration of 20ng/ml of PDGF-BB to stimulate HMCs, the effect is most obvious, and to stimulate the 48h time to observe the effect of the cytokine secretion of the best and most significant,72h observed but there is a downward trend, indicating HMCs activation in a time of 48h peak. (2) Different concentrations of PDGF-BB stimulation HMCs proliferation of cells:cell count and CCK-8 showed:Add PDGF-BB stimulation of HMCs compared to the control group, were 20ng/ml concentration of PDGF-BB by stimulating the proliferation of the most significant (p<0.05). (3) Changes in migratory ability of HMCs after activation:scratches were 20ng/ml of PDGF-BB stimulation of HMCs after 48h and the control group in the six-well plates, showed that the experimental group was significantly less cell last remaining scar area in the control group, the area is calculated statistically significant (p<0.05). (4) Proteomics change of the cell activation phenotypes:Immunofluorescence staining showed that activated phenotype Desmin, a-SMA, PDGFR-β, Transgelin, Tensin expression levels were enhanced when compared with the control group, NF-kB in the nucleus transport is also significantly stronger than the control group. (5) HMCs in duration can be activated under high expression status:HMCs at 48h at the peak of activation when over expressed in 48h anchored, qPCR detection techniques revealed that the activation status remain 48h after this gradually tends to none.2.The interactive biological effects of activated human mesangial cells and monocyte-macrophage cells:(1) PDGF-BB stimulation of HMCs cells can induce the secretion of HMMCs activation cytokines TNF and IL-4, but not secretory activation HMMCs molecular markers CCL-1 and CCL-15 and CCL-17. (2) Activation of HMCs and HMMCs co-culture:co-cultured HMMCs state does not secrete CCL-17(M2 type), but will secrete CCL-15 (M1 type) and CCL-1 (Regulatory) and 24h time highest. (3) The proliferation and migration of chemotaxis and activation of the HMCs and HMMCs after co-culture cells were significantly higher than the co-cultured HMMCs (P<0.05).Conclusion:1.PDGF-BB stimulation HMCs and that the optimum concentration and the best time of their activation were 20ng/ml and stimulation 48h, under this condition, HMCs proliferation and migration ability are the highest point (p<0.05), immunofluorescence also shows various molecular markers and cell phenotype are the highest expression at this time. In the case of such activation state became weaker still be maintained for about 48h, sufficient to stimulate and activate HMMCs.2.Activated HMCs and HMMCs activation can induce secretion of cytokines secreted HMMCs without activated cell phenotype and molecular markers, false-positive results when post-co-culture can be excluded. Cells co-cultured 24h HMMCs highest phenotypic expression, HMCs only after activation stimulus HMMCs produce Ml macrophages (pro-inflammatory) and regulatory Giant,but not stimulate a M2 macrophages (suppressing inflammation).