ABSTRACT
<p><b>OBJECTIVE</b>To study the role of serum from asphyxiated neonates in the inducement of human renal proximal tubular epithelial cells (HK-2) adhesion to neutrophils and possible mechanisms.</p><p><b>METHODS</b>HK-2 cells were cultured randomly with 20% serum from neonates (1, 3, and 7 days after asphyxia), pyrrolidine dithiocarbamate (PDTC) or placebo. The activity of myeloperoxidase (MPO), an indicator of adhesion ability of HK-2 cells to neutrophils in suspensions, was detected by the biochemistry assay. Intercellular adhesion molecule-1 (ICAM-1) and nuclear factor-kappaB (NF-kappaB) of HK-2 cells were examined with the immunohistochemical staining.</p><p><b>RESULTS</b>The expression of MPO in the post-asphyxial 1-day serum treatment group were significantly higher than that in the PDTC treatment and the control groups as well as the post-asphyxial 3 and 7-day serum treatment groups (P<0.01). The expression of ICAM-1 and NF-kappaB in the post-asphyxial 1-day serum treatment group was also significantly higher than that in the other groups (P<0.01).</p><p><b>CONCLUSIONS</b>Serum from asphyxiated neonates can induce HK-2 cell adhesion to neutrophils, possibly through activating NF-kappaB and increasing the synthesis and expression of ICAM-1 on the surface of renal tubular epithelial cells.</p>
Subject(s)
Humans , Infant, Newborn , Asphyxia Neonatorum , Blood , Cell Adhesion , Cells, Cultured , Intercellular Adhesion Molecule-1 , Kidney Tubules , Pathology , NF-kappa B , Metabolism , Neutrophils , PhysiologyABSTRACT
Objective To investigate the role of postasphyxial-serum of neonate in inducing injury of human renal proximal tubular cells(HK-2 cells).Methods HK-2 cells were used as target cell.The neonatal different concentration postasphyxial-serum of 1,3,7 days after asphyxia were used as attacking means.The experimental groups were divided into 15 groups:the 2.5%,5.0%,10.0%,(20.0%) attacking concertration groups of 1,3,7 day after asphyxia and control group of each concertration.The culture medium and concertration of the control group and the experimental groups were the same.The changes of morphology were observed under inverted microscope,the cell viability was measured by 3-(4,5-dimethyl-2-thiazoly1)-2,5-diphenyl-2H-tetrazolium bromide(MTT) method and the leakage rate of lactate dehydrogenase(LDH) was determined by biochemical methods.Results Compared with control group,the changes in morphology of HK-2 were most serious and obvious,the cell viability were obviously decreased(all P
ABSTRACT
Objective To investigate the role and intracellular signal transduction mechanism in the injury of renal tubular cells induced by postasphyxial-serum in neonate.Methods Human renal proximal tubular cell(HK-2 cell) was used as target cell. The experiment was designed as:control group, asphyxia group ,and pyrrolidine dithiocarbamate (PDTC)blocking group. The attacking concentration of serum was 20%, and the apoptosis rate of HK-2 cells was detected by flow cytometer.Results Compared with controls[(13.3?1.70)%],after being stimulated with postasphyxial-serum, the apoptosis rate of HK-2 cells of asphyxia group [(46.73?3.68)%] and PDTC blocking group [(31.19?2.79)%]were significantly increased(P
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Objective To explore the effect of postasphyxial-serum in neonate on expression of serine protease Omi/HtrA2 in renal tubular cells(HK-2).Methods Human renal proximal tubular cell line HK-2 cell was used as target cell.The cultural cells in orifice were divided into control group and asphyxia-serum attacking group.Blood was cowected from asphyxia newborns by means of femoral venous puncture,then the serum was garthered,anticoagulated by liquemie,3 000 r/min centrifuged 20 min,abstracted serum,thermostatic waterbathed the serum at 56 ℃,so that to inactivate addiment,filtered germ by micropore filte,the attacking concentrtion of serum was 200 mL/L,the cells of the asphyxia-serum attacking group were attacked by asphyxia-serum,and the cells of control group were cultivated with normal nutritive medium when the cells was needed.After 24 hours,the cells were tixed,then the expression of Omi/HtrA2 in cytoplast was detected by the use of immunohistochemical method.Results Omi/HtrA2 was inaurate or yellow brown and localized to the cytoplast.The rate of the cell expressed Omi/HtrA2 was(9.0?2.5)% in control group,after stimulated with postasphyxial-serum,in asphyxia group the rate of the cell expressed Omi/HtrA2 was(25.15?3.5)%,there was significant difference between 2 groups(t=-15.322 P
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Objective To investigate the role of erythropoietin(EPO)in relieving the injury of human renal tubular cells (HK-2) induced by postasphyxial-serum of neonates.Methods Human renal proximal tubular cell(HK-2) was used as the target cell.The experiment was designed as control group, asphyxia group,and group of pretreatment with EPO. The attacking concentration of serum was 200 mL/L,then the changes of morphology were observed under inverted microscope,and the cell viability was measured by 3-(4,5-dimethy lthiazcl-2-yl)-2,5-diphenyl-tetazolium bromide(MTT) methods,and the leakage rate of lactate dehydrogenase(LDH) was determined by biochemical methods.Results Compared with control group,the change in morphology of HK-2 was most serious and obvious,and the leakage rate of LDH increased significantly,and the cell viability decreased obviously in asphyxia group.But compared with asphyxia group,the change in morphology of HK-2 was obviously improved,and the leakage rate of LDH decreased and the cell viability increased in group of pretreatment with EPO in a dose-dependent manner except the group of 1 IU/mL.Conclusion EPO can play the role in relieving the injury of renal tubular cells induced by postasphyxial-serum in neonates.
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Objective To explore the effect of postasphyxial-serum in neonate on the expressions of Bcl-2-antagonist of cell death(BAD)and Bcl-2-associated X protein(BAX)in renal tubular cells(HK-2).Methods HK-2 cells were used as target cells.The experiment were divided into control group,asphyxia group and pyrrolodine dithiocarbamate(PDTC)blocking group.Control group:DMEM culture fluid was not contained asphyxia blood serum in every group;asphxia group:DMEM culture fluid contained 20 mL/L asphyxia blood serum in every group;PDTC blocking group:DMEM culture fluid contained 20 mL/L asphyxia blood serum and 40 ?mol/L PDTC in every group.The expressions of both BAD and BAX on cytoplast were detected by immunohistochemical method.Results Calculated Points according to HSCORE,compared with controls group[(1.97?0.26)and(1.77?0.11)],after stimulated with postasphyxial-serum,the expressions of both BAD and BAX of HK-2 cells of asphyxia group[(2.73?0.20)and(2.44?0.13)] and PDTC blocking group[(2.38?0.13)and(2.17?0.08)] significantly increased[F(BAD)=28.61,F(BAX)=15.51 Pa