缺血预适应对HK-2细胞缺氧/复氧损伤的保护作用及相关基因表达的调节

Protective effects and possible mechanism of ischemic preconditioning on HK-2 cell with anoxia-reoxygenation injury

目的探讨缺血预适应对HK-2(人类肾小管上皮细胞)细胞缺氧/复氧损伤的保护作用及机制。方法用300μmol.L-1CoCl2预处理HK-2细胞2h,然后更换正常的培养基培养24h后用无血清的培养基培养,建立预适应的细胞模型。MTT法测定细胞增殖,流式细胞技术测定细胞的凋亡,RT-PCR技术检测诱导型一氧化氮合酶(inducible ni-tric oxide synthase,iNOS)和凋亡相关基因的表达情况。结果 300μmol.L-1CoCl2预处理可以明显增加HK-2细胞在无血清刺激下的增殖能力,减少凋亡(P<0.05或0.01)。预适应可以上调iNOS和凋亡抑制基因Bcl-2的表达,下调凋亡促进基因Caspase-9的表达,而这些调节作用可被iNOS的抑制剂氨基胍(AG)抑制(P<0.05)。结论 300μmol.L-1CoCl2预处理HK-2细胞2h可以模拟预适应的保护作用;iNOS和凋亡相关基因在预适应中起到重要的作用。

Aim To establish a cell preconditioning model and clarify the regulation of inducible nitric oxide synthases and apoptosis related gene in ischemic preconditioning.Methods The HK-2 cell was pretreated with 300 μmol·L^-1CoCl2 for 2 hours,then stimulated in serum-free media after 24 hours cultivation in normal media.MTT method and FACS were used to detect the proliferation and apoptosis of cell.Then RT-PCR method was used to show the regulation of inducible nitric oxide synthases and apoptosis related gene in ischemic preconditioning.Results After pretreated with 300 μmol·L^-1 CoCl2 for 2 hours,cell proliferation increased and apoptosis decreased when cultured in serum-free media.Inducible nitric oxide synthases was upregulated after preconditioning,meanwhile Bcl-2 gene was upregualted and Caspase-9 gene was down-regulated.These regulation can be reversed by AG,the inhibitor of inducible nitric oxide synthases.Conclusion HK-2 cell pretreated with 300 μmol·L^-1 CoCl2 2 hours may be adopted as a cell model of ischemic preconditioning;and inducible nitric oxide synthases can mediate the regulation of Bcl-2 and Caspase-9 gene expression after preconditioning.