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目的研究电磁辐射后,瞬时受体电位通道蛋白3(transient receptor potential channel 3,TRPC3)是否参与诱导海马神经元凋亡。方法以65 mW/cm2电磁波辐照原代培养的新生24 h以内Wistar大鼠海马神经元20 min,以未接受电磁波辐照的神经元为对照组,将TRPC3-siRNA和对照寡核苷酸转染神经元后分别设立为辐照+TRPC3-siRNA干扰组和辐照+TRPC3假干扰组。采用实时定量RT-PCR法及蛋白质免疫印迹法检测TRPC3 mRNA和蛋白水平的表达,电磁辐射后用CCK8试剂盒比色法检测各组细胞存活率,Annexin V-FITC试剂盒染色后经流式细胞仪检测细胞凋亡,激光共聚焦显微镜观察胞内游离钙的变化。结果电磁辐射可明显增加神经元中TRPC3 mRNA和蛋白表达,细胞存活率为(58.8±3.6)%,凋亡率为(30.2±2.6)%,与对照组有显著差异(P<0.05);辐照+TRPC3干扰组细胞存活率为(86.3±4.1)%,凋亡率为(9.2±0.6)%,与辐照组有显著差异(P<0.05);辐射+TRPC3假干扰组与辐射组无明显差异(P>0.05)。辐照后胞内游离钙浓度显著升高,而TRPC3干扰后可以部分抑制其升高。结论 TRPC3表达增加所介导的细胞内游离钙升高可能是电磁辐射致海马神经元凋亡的原因之一。
Objective To investigate whether transient receptor potential channel 3 (TRPC3) is involved in the apoptosis of hippocampal neurons induced by electromagnetic radiation. Methods The hippocampal neurons of primary cultured neonatal Wistar rats were irradiated with 65 mW / cm2 electromagnetic wave for 20 min. The neurons irradiated with no electromagnetic wave were used as the control group. TRPC3-siRNA and control oligonucleotide The neurons were established as irradiation + TRPC3-siRNA interference group and irradiation + TRPC3 false interference group. Real-time quantitative RT-PCR and Western blotting were used to detect the expression of TRPC3 mRNA and protein. Cell viability was detected by CCK8 kit assay after staining with electroporation. Annexin V-FITC kit was stained with flow cytometry The apoptosis of cells was detected by laser confocal microscopy and the change of intracellular free calcium was observed. Results Electromagnetic radiation significantly increased the expression of TRPC3 mRNA and protein in neurons. The cell viability was (58.8 ± 3.6)% and the apoptosis rate was (30.2 ± 2.6)%, which was significantly different from the control group (P <0.05) The cell survival rate was (86.3 ± 4.1)% and the apoptosis rate was (9.2 ± 0.6)%, which was significantly different from that of irradiation group (P <0.05). The radiation + TRPC3 interference group was Significant difference (P> 0.05). After intracellular free calcium concentration was significantly increased, and TRPC3 interference can be partially inhibited after its increase. Conclusions Elevated intracellular free calcium mediated by increased expression of TRPC3 may be one of the reasons for the apoptosis of hippocampal neurons induced by electromagnetic radiation.