| Objective: Inflammatory cell infiltration are important factors which directly lead to the development of kidney inflammation, fractalkine was the first cognition of the cell surface adhesion chemokines, can effectively mediate NK cells, monocytes, macrophages and T lymphocyte adhesion and chemotaxis. Fractalkine interaction with its specific receptor, CX3CR1, mediate the adhesion of chemokines and inflammatory cell,are involved in a variety of pathophysiological processes in the body. For example, in human acute crescentic glomerulonephritis and kidney transplantation of acute rejection of kidney, fractalkine significantly increased was an important factor in mediating renal mononuclear cell infiltration. TNF-? is a cytokine with a variety of biological activity, which has an important role in resistance to inflammation and promote repair under normal dose, but beyond its physiological levels will cause the body to produce immune balance destruction damage. Calcitriol(1,25-(OH) 2D3) is the active form of vitamin D3, which plays an important role in the body's calcium and phosphorus metabolism, but also has a wider role in immune regulation. Therefore, under inflammatory conditions the study of mechanisms and prevention and treatment in kidney injury with fractalkine have attracted an increasing attention in recent years.In this study, the intervention by TNF-? in renal tubular epithelial cells(Human kidneyproxiamal tubular cells-2, HK-2) simulated kidney inflammation injury model to study the expression of chemotactic factor fractalkine in renal tubular epithelial cells(HK-2) and its influence on THP-1 monocyte chemotactic effect. On this basis, after TNF-? stimulation in renal tubular epithelial cells(HK-2), it is aim to investigate the protective effect of calcitriol by adding calcitriol to intervene.Methods:1 Cell culture: human proximal tubular epithelial cell line HK-2 and THP-1 were saved in department of pathogenic biology and Immunology of our hospital.(1) HK-2 were cultured in D-MEM/F-12 medium supplemented with 10% fetal bovine serum to 80%~90% confluence, instead of D-MEM/F-12 medium supplemented with 0.05% fetal bovine serum for 24 h, to make them in the Go phase, and then tested according to the following experimental design: ?Normal control group. ?TNF-? treatment group: cells were treated with 40ng/ml TNF-? at different time nodes(0, 1, 6, 12, 24, 48 h) to detect the expression of fractalkine in HK-2 cells; cells were treated with different concentrations of TNF-?(0, 10, 20, 40ng/m L) for 24 h to detect the expression of fractalkine in HK-2 cells. ?Calcitriol action group: pretreated with 40ng/ml TNF-? for 24 h, cells were then treated with different concentrations(10-9mol/L, 10-10mol/L, 10-11mol/L) of calcitriol for 12 h to detect the expression of fractalkine in HK-2 cells.(2) THP-1 cells were cultured RMPI-1640 medium. ?Normal control group: adding only serum-free DMEM/F12 medium in the lower chamber to observe random movement of THP-1 cells. ? Single TNF-? group: To observe the effect of TNF-? chemotaxis prognosis of HK-2 cells to THP-1 cells.?Unstimulated group: HK-2 cells without stimulation by TNF-? for THP-1 cells chemotaxis. ?TNF-? experimental group: To observe the effect of TNF-? chemotaxis prognosis of HK-2 cells to THP-1 cells. ?Fractalkine neutralizing antibody group: adding(1ng/m L, 5ng/m L) neutralizing antibody to observe the impact of HK-2 cells to THP-1 cells chemotaxis. ?Calcitriol experimental group: To observe the effect of chemotaxis of HK-2 cells to THP-1 cells with 10-9mol/L calcitriol. These tests of each different concentrations and time points were equipped with multiple wells and repeated three times.2 The cell viability of HK-2 cells were evaluated by MTT method.3 Using Annexin V-FITC/PI staining to detect the cell apoptosis of each human renal tubular epithelial experimental group induced by TNF-?.4 The m RNA expression levels of fractalkine in each group with different intervention were determined by reverse transcription polymerase chain reaction(RT-PCR).5 Fractalkine in cell supernatant of each group were detected by enzyme-linked immunosorbent assay.6 The protein levels of fractalkine in groups with different intervention were determined by Western- blot.7 To observe the mainly expression location of fractalkine in HK-2 cells after TNF-? intervention by immunohistochemical.8 Using Transwell equipment of microporous membrane enzyme-containing polycarbonate 24-well plates, membrane diameter 6.5 mm, membrane pore size 8.0?m, to analyze the impact of HK-2 cells to THP-1 cells chemotaxis in each experimental group.Results:1 TNF-? and calcitriol intervention have significant effects on the survival rate of HK-2 cells.TNF-? affected the HK-2 cell viability in time and dose manner. HK-2 cell viability decreased with increasing TNF-? concentration and time compared with control group, the difference was statistically significant(P < 0.05). After TNF-? intervention adding with different concentrations of calcitriol we found that cell viability gradually increased and the HK-2 cell proliferation was most obvious at the dose of 10-9 mol/L compared with TNF-? stimulation group, the difference was statistically significant(P < 0.05).2 The effect of calcitriol on apoptosis of HK-2 cells induced by TNF-?.HK-2 cell apoptosis was significantly increased with TNF-? stimulation compared with the control group, the difference was statistically significant(P < 0.05). After treating with different doses of calcitriol, the HK-2 cell apoptosis of each group was significantly decreased compared with TNF-? stimulation group, the difference was statistically significant(P < 0.05). At the dose of 10-9 mol/L, HK-2 cell apoptosis rate decreased most significantly.3 The effect of calcitriol on fractalkine in HK-2 cells supernatants induced by TNF-?.The content of the fractalkine in HK-2 cells supernatants by TNF-? intervention increased compared with the control group(P < 0.05). After treating with different concentration gradients of calcitriol the content of the fractalkine in HK-2 cells supernatants all decreased compared with the TNF-? stimulation group, the difference was statistically significant(P < 0.05). Fractalkine in the HK-2 cells supernatant expressed at basal rate levels in the control group.4 HK-2 cells treatment with TNF-? up-regulated both the m RNA and protein expression of fractalkine in a time- and dose-dependent manner.HK-2cells were induced by 40ng/ml TNF-?, the expression of fractalkine was found to have a certain time-dependent manner,its expression gradually increased with increasing time, the normal group HK-2 cells expressed only a small amount of fractalkine. the difference was statistically significant compared with the normal group(P < 0.05). Similarly, HK-2 cells were treated with different concentrations of TNF-? for 24 h, the expression of fractalkine gradually increased with increasing concentrations of TNF-?.5 calcitriol reversed the TNF-? stimulation for HK-2 cells which decreased the expression of fractalkine in HK-2 cells and increased the expression of fractalkine in HK-2 cells VDR at both m RNA and protein level.HK-2 cells were pretreated with 40ng/ml TNF-? for 24 h and then were treated with different concentrations of calcitriol for 12 h, to detect the m RNA and protein expression fractalkine we found fractalkine expression significantly decreased compared with TNF-? single stimulation group(P < 0.05). Meanwhile, to detect the calcitriol receptors VDR in HK-2 cells, we found that TNF-? reduced the m RNA and protein expression of VDR(P < 0.05) while calcitriol could reverse TNF-? stimulation for HK-2 cells, up-regulated the m RNA and protein expressing of VDR(P < 0.05).6 Fractalkine subcellular mainly located in the cell membrane of HK-2 cells.With TNF-? intervene on HK-2 cells, TNF-? non-intervene were used as a control, we observed that green fluorescence was found mainly in cell site HK-2 cells under an inverted fluorescence microscope, which indicted fractalkine subcellular mainly located above the cell membrane.7 The chemotaxis of HK-2 cells to THP-1 cells was enhanced by TNF-? intervention, and its chemotaxis attenuated after adding the antibody and calcitriol.Normal control group and TNF-? alone group did not show the migration of THP-1 cells. Unstimulated HK-2 cells to THP-1 cells have a weak chemotaxis, which was statistically significant compared with the normal control group and single TNF-? difference between groups(P < 0.05). The chemotaxis of HK-2 cells to THP-1 cells has been enhanced between TNF-? stimulation 24 h group and the three groups, and the difference with statistical significance(P < 0.05). HK-2 cells of fractalkine neutralizing antibody group produced chemotaxis inhibition to THP-1 cells, the higher the concentration of neutralizing antibodies inhibited chemotaxis more obvious, the difference was statistical significance compared with TNF-? group(P < 0.05). Calcitriol intervention group attenuated HK-2 cells chemotaxis to THP-1 cells, the difference was statistical significance with TNF-? group(P < 0.05)Conclusions:1 TNF-? stimulated HK-2 tubular epithelial cells which up-regulated the expression of fractalkine in a time- and dose-dependent manner, fractalkine expression site was mainly in the cell membrane.2 Calcitriol could reverse the up-regulation effect of fractalkine by TNF-? which up-regulated chemotaxis of HK-2 cells to THP-1 cells while the antibody and calcitriol attenuated its chemotaxis. These mean calcitriol has a protective effect on tubular inflammatory injury. |
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