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目的探讨瞬时受体电位M7(transient receptor potential melastatin 7,TRPM7)对dHL-60细胞极性化的影响及可能的分子机制。方法通过siRNA技术下调dHL-60细胞TRPM7蛋白表达,分为转染无义干扰片段的对照组和TRPM7干扰组,用Zigmond小室观察细胞极性化的变化,采用Western blot检测TRPM7和磷酸化AKT的表达,并用细胞免疫荧光方法观察磷酸化AKT的定位情况。结果与转染无义干扰片段的对照组相比,转染TRPM7靶向siRNA的dHL-60细胞中,TRPM7蛋白表达水平明显降低(P<0.05);干扰TRPM7后dHL-60细胞的极性化率与对照组相比显著增加(P<0.05);磷酸化AKT表达明显上调,并从细胞质转向了细胞膜,呈极性分布在细胞质膜上,而总AKT的表达则无明显变化。AKT的特异性抑制剂Triciribine可使dHL-60细胞在fMLP刺激下的磷酸化AKT的极性分布消失。结论 TRPM7通过AKT信号通路调控细胞极性相关分子,进而影响dHL-60细胞的极性化能力。
Objective To investigate the effect of transient receptor potential melastatin 7 (TRPM7) on the dHL-60 cell polarity and its possible molecular mechanism. Methods The expression of TRPM7 protein in dHL-60 cells was down-regulated by siRNA. The transfected cells were divided into control group and TRPM7 interference group. Zigmond cells were used to observe the change of cell polarity. Western blot was used to detect TRPM7 and phosphorylated AKT The phosphorylation of AKT was observed by immunofluorescence staining. Results The expression of TRPM7 protein in dHL-60 cells transfected with TRPM7 siRNA was significantly lower than that in control cells transfected with nonsense interfering fragments (P <0.05) (P <0.05). The phosphorylated AKT expression was significantly up-regulated and turned from the cytoplasm to the cell membrane, with a polarity distribution on the plasma membrane, while the total AKT expression did not change significantly. Triciribine, a specific inhibitor of AKT, abolished the polarity distribution of phosphorylated AKT in dHL-60 cells stimulated by fMLP. Conclusion TRPM7 regulates cell polarity-related molecules through the AKT signaling pathway, thereby affecting the polarity of dHL-60 cells.