STIMI调控TRPC1参与HEK293细胞铅内流
首发时间:2019-08-23
摘要:目的:研究Pb2+进入HEK293细胞途径,探讨是否TRPC1参与Pb2+进入细胞。方法:MTT法检测细胞存活率;Hoechst 33342和Annexin V+PI染色检测Pb2+诱导人HEK293细胞的凋亡作用;Western Blot检测PARP蛋白表达的变化;过表达和干扰TRPC1,检测进入HEK293细胞Pb2+浓度;分别过表达STIM1 DD、TRPC1KK,共转染STIM1DD和TRPC1KK,检测进入HEK293细胞Pb2+浓度。结果:Pb2+导致HEK293细胞存活率下降且具有剂量-反应关系、Pb2+能导致HEK293细胞凋亡;过表达和TRPC1分别可促进和减少Pb2+进入HEK293细胞;转染TRPC1KK铅的进入量与对照组相比明显降低;共转STIM1DD+TRPC1KK铅的进入量比TRPC1KK组、STIM1DD组明显增多,差异具有统计学意义(P﹤0.001)。结论:Pb2+诱导的HEK293细胞凋亡。Pb2+铅可以通过SOCE通道进入细胞内,SOCE的重要的分子组成STIM1参与铅进入细胞,STIM1可以通过TRPC1参与铅进入细胞内。
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STIM1 regulate TRPC1 involved in Pb2+ influx in HEK293 cells
Abstract:Objective: invistigate the molecular mechanism of Pb2+ influx in HEK293 cells, whether TRPC1 invovle in Pb2+ influx. Method: The cell viability was measured with MTT colorimetric assay; The apoptosis of SH-SY5Y cells induced by MPP+ was confirmed by hoechst 33342 staining and Annexin V+PI assay ;Western-blotting was used to identify the protein expression changes of PARP; Transfect with wild-type TRPC1 or TRPC1 siRNA, followed 100μM Pb2+ treated for 24 hours, then mesure the amount of Pb2+ into the cells by ICP-STIM1 regulate TRPC1 involved in Pb2+ influx in HEK293 cellsMS. We transfect with STIM1DD、TRC1KK and STIM1DD+TRPC1KK, followed 100μM Pb2+ treated for 24 hours. The amount of Pb2+ into the cells is measured by ICP-MS. Results: overexpression of TRPC1– WT increase Pb2+ influx , on the contrary, knockdown the TRPC1 decrease the Pb2+ influx. The results suggested that TRPC1 take part in Pb2+ into cells. Pb2+ influx decreased obviously because TRPCKK lost the normal function of TRPC1. Pb2+ inflSTIM1 regulate TRPC1 involved in Pb2+ influx in HEK293 cellsux of cotransfection STIM1DD and TRPC1KK more than that of STIM1DD or TRPC1KK. Conclusion: Pb2+ influx the HEK293 cell through SOCE channel. STIM1 regulate TRPC1 through molecular mechanisms involved in Pb2+ into the HEK293 cells, meanwhile involved in the cell toxicity of Pb2+.
Keywords: Labor hygiene STIM1 TRPC1 SOC
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