外源性锌离子培养/低氧环境下大鼠Müller细胞活力、锌离子浓度变化及促红细胞生成素的调节作用观察

Changes of activity and zinc concentration of rat Müller cells in the environment of exogenous zinc ion culture/hypoxia and regulation of erythropoietin

目的观察加入外源性锌离子培养/低氧环境下大鼠Müller细胞系rMC-1细胞活力、锌离子浓度变化及促红细胞生成素(EPO)的调节作用。方法将rMC-1细胞分为ZnCl2组、ZnCl2+EPO组、正常对照组,分别加入ZnCl2、ZnCl2+EPO、等量培养基,继续培养24 h后,采用MTT法检测各组细胞光密度值,计算细胞活力,采用TUNEL法检测各组细胞凋亡率。将rMC-1细胞分为CoCl2组、CoCl2+EPO组、正常对照组,分别加入CoCl2(诱导低氧环境)、CoCl2+EPO、等量培养基,采用锌离子探针FluoZin-3 AM检测各组细胞荧光强度(以荧光强度表示细胞内锌离子浓度),采用qRT-PCR法检测各组rMC-1细胞内ZnT8 mRNA,采用Western blotting法检测各组rMC-1细胞内ZnT8蛋白。结果ZnCl2组、ZnCl2+EPO组、正常对照组rMC-1细胞活力分别为0.252±0.034、0.342±0.031、0.513±0.040,组间相比,P均<0.05;ZnCl2组、ZnCl2+EPO组、正常对照组rMC-1细胞凋亡率分别为60.326%±5.230%、28.268%±3.756%、15.124%±3.521%,组间相比,P均<0.01。CoCl2组、CoCl2+EPO组、正常对照组rMC-1细胞内锌离子浓度分别为6.712%±1.251%、4.723%±0.892%、3.428%±0.522%,组间相比,P均<0.05;CoCl2组rMC-1细胞中ZnT8 mRNA和蛋白的相对表达量分别为0.224±0.038、2.436±0.187,CoCl2+EPO组分别为0.314±0.030、3.082±0.247,正常对照组分别为0.385±0.025、3.583±0.256,组间相比,P均<0.01。结论外源性锌离子可引起rMC-1细胞活力下降、细胞凋亡率增加,EPO可拮抗此作用。低氧状态下rMC-1细胞内锌离子浓度增加,加入EPO后降低,其机制可能与ZnT8表达升高有关。

Objective To observe the changes of activity/zinc concentration of rat Muller cell line rMC-1 and the reg ulation of erythropoietin under the environment of exogenous zinc ion culture/hypoxia.Methods The rMC-1 cells were divided into the ZnCl group,ZnCl+EPO group,and normal control group.ZnCl,ZnCl+EPO and equal amount of medium were added respectively.After the continuous culture of 24 h,the cell optical density value(cell viability)and apoptosis rate of each group were measured by MTT and TUNEL method,respectively.rMC-1 cells were divided into the CoCl group,CoCl+EPO group,and normal control group.CoCl(hypoxia induction environment),CoCl+EPO and e qual amount of culture medium were added respectively.Fluorescence intensity of cells in each group was detected by Fluo-Zin-3 AM(zinc concentration in cells was expressed by fluorescence intensity).ZnT8 mRNA was detected by q-RT PCR,and ZnT8 protein was detected by Western blotting.Results The cell viabilities of rMC-1 cells in the ZnCl group,ZnCl+EPO group and normal control group were 0.252±0.034,0.342±0.031,and 0.513±0.040,respectively,P<0.05.The apoptosis rates of rMC-1 cells in ZnCl group,ZnCl+EPO group and normal control group were 60.326%±5.230%,28.268%±3.756%,15.124%±3.521%,respectively,P<0.01.The concentrations of zinc in rMC-1 cells of the CoCl group,CoCl+EPO group,and normal control group were 6.712%±1.251%,4.723%±0.892%and 3.428%±0.522%,respectively,P<0.05.The relative expression levels of ZnT8 mRNA and protein in rMC-1 cells of the CoCl2 group were 0.224±0.038 and 2.436±0.187,respectively,those of the CoCl2+EPO group were 0.314±0.030 and 3.082±0.247,respectively,and those of the normal control group were 0.385±0.025 and 3.583±0.256,respectively(all P<0.01).Conclusions Exogenous zinc ions can decrease the activity of rMC-1 cells and increase the apoptosis rate,which can be antagonized by EPO.Under hypoxia condition,the concentration of zinc in rMC-1 cells increases,and then decreased after adding EPO,which may be related to the increase of ZnT8 expression.