| Objective:To explore the changes in morphology of peripheral blood mononuclear cells (PBMCs) in uremic patients on haemodialysis using atomic force microscopy (AFM), and to investigate its correlation with microinflammation.Methods:66uremic patients on haemodialysis (MHD group),30uremic patients neither treated by haemodialysis nor with a history of acute cardiovascular events (WHD group), and20healthy volunteers (CON group) were assessed. Of the66MHD patients,36patients had a history of acute cardiovascular events (MHD1group) and30patients did not (MHD2group). PMBCs were separated using ficoll density gradient centrifugation with lymphocyte separation medium, and were observed under inverted microscope for morphology and cell counts. AFM images were analyzed for volume, mean height, half-maximumm amplitude, average roughness and diameter of surface granules in PBMCs. Levels of IL-17and IL-10in PBMCs supernatant were detected by enzyme-linked immunosorbent assay (ELISA).Results:No significant differences were observed under inverted microscope in morphology of PBMCs among study subjects. Uremic patients had decreased PBMCs counts as compared to healthy volunteers (P<0.05); among uremic patients, PBMCs counts of WHD group were less than that of MHD group (P<0.05), which of MHD1group were less than MHD2group (P<0.05). AFM images showed PBMCs were larger in volume, mean height, half-maximumm amplitude and average roughness in uremic patients as compared to healthy volunteers, with granular processes or caveolae of uneven size distributed over cell surface. Moreover, they had higher IL-17levels and lower IL-10levels in cell supernatant than healthy volunteers (P<0.05). PBMCs volume, mean height, half-maximumm amplitude, average roughness, diameter of surface granules and IL-17levels were higher in WHD group as compared to those in MHD, while IL-10level was lower. PBMCs volume, mean height, half-maximumm amplitude, average roughness, diameter of surface granules and IL-17levels were higher in MHD1group than in MHD2group (P<0.05), while IL-10levels were lower (P<0.05). PBMCs volume, mean height, half-maximumm amplitude, average roughness were in positive and negative correlations with IL-17and IL-10levels, respectively, in uremic patients. PBMCs volume, mean height, half-maximumm amplitude, average roughness and IL-17and IL-10levels in PBMCs supernatant were close related factors in MHD patients with cardiovascular diseases, but not independent risk factors.Conclusions:Haemodialysis had better efficacy on microinflammatory state in uremic patients, whose PBMCs morphological change observed under AFM was related with the degree of microinflammatory state. PBMCs volume, mean height, half-maximumm amplitude, average roughness and levels of IL-17and IL-10were close related factors in MHD patients with cardiovascular disease. Observation and determination of morphological change of PBMCs in uremic patients on haemodialysis have potential to provide new clue for early recognition and diagnosis of cardiovascular complications in uremia. Objective:To explore the regulatory T cell (Treg)/T-helper17(Th17) cells functional disequilibrium accelerated cardiovascular events in uremic patients on haemodialysis by promoting vascular calcification.Methods:66uremic patients on haemodialysis (MHD group),30uremic patients neither treated by haemodialysis nor with a history of acute cardiovascular events (WHD group), and20healthy volunteers (CON group) were assessed. Of the66MHD patients,36patients had a history of acute cardiovascular events (MHD1group) and30patients did not (MHD2group). The extents of coronary artery calcification were assessed with coronary artery calcification score (CACS). The Treg and Th17cell frequencies were measured by flow cytometry (FCM). Expression of the retinoic acid receptor-related orphan receptor-yt (ROR-yt) and Foxp3were measured by real-time quantitative polymerase chain reaction (real-time qPCR). Levels of IL-17, IL-10, and BMP-2in cell supernatants were detected by ELISA, Protein expressions of BMP-2were measured by Western-blotting.Results:Uremic patients exhibited a functional disequilibrium of Treg/Th17when compared to healthy controls, displaying higher Treg and Th17cell frequencies, expression of Foxp3and ROR-yt, levels of IL-17and BMP-2, expressions of BMP-2and CACS. In contrast, IL-10was significantly lower in uremic patients (P<0.05). This disequilibrium was also significantly between WHD group and MHD group, MHD1group and MHD2group. IL-17and BMP-2levels were negatively correlated with Treg cell frequency, positively correlated with Th17cell frequency; IL-10levels were negatively correlated with Th17cell frequency, positively correlated with Treg cell frequency; BMP-2levels were positively correlated with IL-17, but negatively correlated with IL-10.Conclusions:Haemodialysis can efficiently reduce the microinflammatory state in uremic patients. Disequilibrium of Treg/Th17cell in uremic patients on MHD may induce secretion of inflammatory cytokines and aggravate vascular calcification, leading to cardiovascular events. Objective:To explore the mode of cardiovascular disease induced by Treg and Th17cells in uremic patients on haemodialysis.Methods:Treg and Th17cells were isolated from PBMCs of four groups by magnetic cell separation (MACS), and were co-cultured with CD14+monocytes. Cells were grouped as follows:A) CD14+monocytes were cultured without T cells; B) CD14+monocytes were cultured with Treg cells at a1:1ratio; C) CD14+monocytes were cultured with Th17cells at a1:1ratio; D) CD14+monocytes were cultured with Th17cells at a1:1ratio in the presence of100ng/ml soluble anti-IL-17mAb. Cell proliferations were detected by MTT assay. Levels of IL-6, IL-10and BMP-2in cell supernatant were detected by ELISA. The expressions of costimulatory molecules (CD80and CD86) were measured by FCM.Results:Cell proliferation activity was peak at48hours after cell co-cultured, and then gradually reduces. Compared to healthy volunteers, uremic patients had significantly higher levels of IL-6and BMP-2and expressions of CD80and CD86, while the levels of IL-10were significantly lower in uremic patients (P<0.05). Except WHD group and MHD1group, All pair-wise inter-group comparisons (WHD group and MHD1group, WHD1group and MHD2group) also revealed significantly differences (P<0.05).Among co-cultured cells groups, levels of IL-6, IL-10and BMP-2and expressions of CD80and CD86were not significantly different in health volunteers and MHD2patients (P>0.05). However, Compared to the other co-cultured cells groups, C group had significantly higher levels of IL-6and BMP-2and expressions of CD80and CD86, while the levels of IL-10were significantly lower in MHD1patients and WHD patients (P<0.05). These differences were also significantly displayed between B and A group, B and D group, A and D group in MHD1patients and WHD patients (P<0.05).Conclusions:Treg cells inhibit the activation of monocytes and the secretion of inflammatory cytokines and costimulatory molecules by cell-cell contact, while Th17cells promote the activation and secretion by increase IL-17, which aggravate vascular calcification, and then induce cardiovascular disease.
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