抑制小鼠HL-1心肌细胞桥粒斑蛋白基因表达对缝隙连接蛋白43结构和功能的影响

Inhibition of desmoplakin expression induces connexin 43 remodeling in mouse HL-1 myocardial cells

目的:采用基因沉默技术抑制小鼠HL-1心肌细胞桥粒斑蛋白(DSP)基因表达以明确DSP与缝隙连接蛋白43(Cx43)的结构和功能关系。方法:用基因沉默技术抑制DSP基因的表达,Western blotting和流式细胞术检测HL-1细胞DSP和Cx43蛋白的表达,用双免疫荧光方法检测DSP与Cx43蛋白的表达与定位情况,并用划痕标记染料示踪技术检测细胞缝隙连接通讯状况。结果:与空白组和对照组相比,siRNA-DSP组的DSP和Cx43蛋白表达量降低(P<0.05)。免疫荧光检测发现空白组和对照组DSP与Cx43蛋白存在共定位情况,而siRNA-DSP组DSP和Cx43蛋白共定位遭到破坏,Cx43蛋白出现再分布,在细胞内检测到Cx43蛋白,并且划痕标记染料示踪技术检测发现siRNA-DSP组Lucifer yellow通过HL-1细胞缝隙连接的传输功能降低。结论:DSP表达抑制不仅使Cx43出现再分布,而且影响缝隙连接传导功能。

AIM： To evaluate the content, distribution, and function of connexin 43 （Cx43） gap junctions in mouse HL-1 myocardial cells under the condition of desmoplakin （DSP） silencing. METHODS： The technique of RNA interference was used to inhibit the protein expression of DSP in HL-1 cells. The protein expression of DSP and Cx43 was analyzed by Western blotting and flow cytometry. The immunofluorescence staining was used to detect the distribution and co-localization of DSP and Cx43. The techniques of scrape loading and dye transfer were also used to determine the function of Cx43 gap junctions. RESULTS： Compared with non-treatment group and negative control group, the protein expression of DSP and Cx43 was reduced （ P 〈 0.05 ）. Co-localization to the site of cell-cell contact was apparent in untreated and control conditions, but loss of DSP expression induced by siRNA-DSP correlated with a drastic redistribution of Cx43. In- stead, Cx43 was found mostly within the intracellular space. The results of dye transfer assay indicated a significant de- crease in the function of Cx43 gap junctions of the ceils treated with siRNA-DSP. CONCLUSION ： Inhibition of DSP ex- pression induces redistribution of Cx43 and decrease in dye coupling between ceils.