肿瘤坏死因子-α对肾入球动脉平滑肌细胞胞内钙离子浓度的影响

目的观察肿瘤坏死因子-α(TNF-α)对肾入球动脉平滑肌细胞(RASMCs)内钙离子浓度(Ca2+]i)的影响。方法应用筛网及酶消化法进行原代雄性SD大鼠RASMCs的分离与培养,将原代培养的RASMCs间接免疫荧光α-肌动蛋白(α-actin)抗体、平滑肌肌球蛋白重链(SM)单克隆抗体染色,进行鉴定。实验分组:A.无钙缓冲液组;B.无钙缓冲液+TNF-α组;C.无钙缓冲液+内皮素组;D.无钙缓冲液+2-氨基乙基二苯硼酸盐(2-APB)+内皮素组;E.无钙缓冲液+TNF-α+内皮素组;F.无钙缓冲液+TNF-α+2-APB+内皮素组。采用confocal显微镜测定单个活细胞内Ca2+荧光强度,计算细胞内Ca2+]i。结果 A组和B组RASMCs内Ca2+]i分别为(109.51±52.60)和(128.07±78.56)nmol/L,两组间无显著差异(P>0.05);C组和E组分别为(240.36±56.78)和(375.45±60.39)nmol/L,两组间有显著差异(P<0.01),与前A组和B组比较也均有显著差异(P<0.01);D组和F组分别为(101.66±48.03)和(116.35±53.48)nmol/L,与A组比较无显著差异(P>0.05)。结论 TNF-α可增强内皮素刺激引起的RASMCs内Ca2+]i增加,且通过1,4,5-三磷酸肌醇受体(IP3R)发挥作用。

The effect of TNF-α on [ Ca2 + ] i in renal afferent arterial smooth muscle cells

Purpose To investigate the effect of tumor necrosis factor-α(TNF-α) on Ca2+]i in renal afferent arterial smooth muscle cells(RASMCs).Methods RASMCs were originally isolated and cultured from kidney cortex of male SD rats by sieves and enzyme digestion.Cells were characterized as RASMCs by positive indirect immunofluorescence staining for α-smooth muscle action and myosin heavy chain and visible myoneme by transmission electron microscope.Study was performed by the Ca2+-sensitive dye Fluo-3/AM with confocal imaging.Cells were cultured without TNF-α,with TNF-α for 24 h,stimulated by endothelin for 10 min or with TNF-α for 24 h,then stimulated by endothelin for 10 min in Ca2+-free buffer.Furthermore,2-aminoethoxydiphenylborate(2-APB) was added to cells 10 minutes before endothelin stimulation in order to observe the blockade effect of 2-APB on inositol 1,4,5-trisphosphate receptors(IP3Rs).Results Without endothelin stimulation,there is no difference on Ca2+]i of RASMCs in the two groups(109.51±52.60,128.07±78.56 nmol/L,P>0.05).Endothelin caused a rapid increase in Ca2+]i of RASMCs,that was obviously enhanced by TNF-α(240.36 ±56.78,375.45±60.39 nmol/L,P < 0.01).Pretreatment of RASMCs with the 2-APB for 10 min fully eliminated the difference in changes of Ca2+]i between control group and TNF-α treated group(101.66±48.03,116.35±53.48 nmol/L,P>0.05).Conclusion The interaction between TNF-α and IP3R led to the important modulatory influence on release of stored Ca2+of RASMCs induced by endothelin.