Objective To investigate the protein expression of Omi/HtrA2 in NRK-52E apoptosis models and to observe the change of apoptosis after Omi/HtrA2 was inhibited.Methods The Omi/HtrA2-shRNA expression plas-mids 251(Pgenesil-1/Omi/HtrA1 shRNA1),252(Pgenesil-1/Omi/HtrA2 shRNA2),and negative plasmid HK(Pgene-sil-1/HK) were transfected into NRK-52E by liposome-mediated method.Protein expression of Omi/HtrA2 and cas-pase3 were quantified by Western blotting.Apoptosis in every group was analyzed by DNA ladder.Results Success-fully transfected cells were presented with green fluorescent NRK-52E.Western blotting showed higher expression of Omi/HtrA2 in the control group than in normal group.There was a significant reduction in Omi/HtrA2 expression in the groups transfected with 251 and 252 than in the controls(P0.05),while statistical difference between the groups transfected 251 and 252 was not found.The protein expression of caspase3 of the control group increased comparing to the normal(P0.05).The protein expression of caspase3 in the groups with inhibited Omi/HtrA2 was lower than that in the controls(P0.05).The distinctive ladder pattern was visible in the control group but appeared weaker in the 251 and 252 groups.Conclusion RNA interference technique was successfully used to inhibit the expression of Omi/HtrA2 in the hypoxia-reoxygenation model,which resulted in mitigation of apoptosis.

Objective To explore a new way for establishing a model of hypoxia/reoxygenation injury in rat renal tubular cell line NRK-52E. Methods Rat renal tubular cell line NRK-52E was divided into control group and experimental group.The cells in experimental group were cultured under the condition of N294%+O21% + CO25% for 2,4 and 6 h and then 95% air+CO25% for 1,12 and 24 h with 10% fetal bovine serum DMEM/F12.The cells in control group were cultured with 10% fetal bovine serum DMEM/F12 under the condition of 95% air+CO25%.Trypan blue exclusion was used for detecting cell count and cell viability.Spectrophotometric method was used for determining the activity of lactate dehydrogenase (LDH) in the culture medium. Results Compared with control group,trypan blue exclusion rate was significantly higher after hypoxia followed by reoxygenation, cell count and cell viability increased significantly,and the activity of LDH significantly increased(P0.05). Conclusion This method for establishing hypoxia/reoxygenation injury model is more simple,easier and more reliable.

Objective To investigate the mechanisms and effects of Omi/HtrA2 short hairpin RNA (shRNA) on the apoptosis of rat renal tubular epithelial cells (NRK-52E) as a result from hypoxia-reoxygenation damage. Methods The cells were divided into 5 groups: normal group (cultured under routine condition), model group (subjected to hypoxia-reoxygenation), HK group (transfected with Pgenesil-1/HK), shRNA1 group (transfected with Pgenesil-1/Omi/HtrA2 shRNA1), and shRNA2 group (transfected with Pgenesil-1/Omi/HtrA2 shRNA2). The fluorescent microscopy was used to examine the expression of fluorescent protein. The expressions of Omi/HtrA2 and caspase-3/-9 were examined by Western blotting and the activity of caspase-3/-9 was analyzed by colorimetry. Results The expression of fluorescent protein was detected in transfected cells but not in nontransfected ones. Compared to model group, the expressions of Omi/HtrA2 and caspase-3/-9 were significantly decreased in shRNA1 and shRNA2 groups (P<0.01). Compared to normal groups, the expressions of Omi/HtrA2 and caspase-3/-9 were obviously increased in model group (P<0.01). The expressions of Omi/HtrA2 and caspase-3/-9 did not differ either between model group and HK group or between shRNA1 group and shRNA2 group. The activity of caspase-3/-9 well correlated with the expressions of caspase-3/-9 in all groups. Conclusion The Pgenesil-1/Omi/HtrA2 shRNA1 and Pgenesil-1/Omi/HtrA2 shRNA2 may markedly inhibit the expressions of Omi/HtrA2 in NRK-52E. Apoptosis of NRK-52E from hypoxia-reoxygenation injury was shown to be inhibited through blockage of caspase-9 activation and subsequently of caspase-3.

Objective To construct a eukaryotic expression plasmid pcDNA3.1-adrenomedullin(pcDNA3.1-ADM),and to explore its expression in rat renal epithelial cells(NRK-52E).Methods The total RNA was extracted from the adrenal glands in Wistar rats.The full-length cDNA of rat ADM was amplified by RT-PCR and cloned into pMT-18T vector using TA cloning,and then identified with double digestion and sequencing.The pMT-18TADM and plasmid pcDNA3.1 were double digested with BamHⅠ and EcoRⅠ respectively,and the target gene was cloned into pcDNA3.1 directionally.The recombinant plasmid pcDNA3.1-ADM was identified by double digestion,and then transected into NRK-52E by liposome.Expression of ADM in pcDNA3.1-ADM transferred cells were detected by RT-PCR.Results The destination gene,585 bp,was distinctively amplified by PCR,and inserted successfully into the plasmid pcDNA3.1.Expression of ADM in cells transfected with pcDNA3.1-ADM was much higher than in controls.Conclusion The eukaryotic expression plasmid pcDNA3.1-ADM is successfully constructed and expressed in NRK-52E.