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目的研究不同浓度细胞外钙对HepG2细胞内钙的影响并初步阐明细胞内钙变化的机制。方法给予不同浓度钙培养液作用24h后,用Fluo-3/AM染色剂对HepG2细胞进行染色,应用流式细胞仪检测其细胞内钙浓度的变化;利用阻断剂对钙通道进行阻断,寻找细胞内钙变化的原因;应用siRNA干扰对找寻到的钙通道进行沉默,进一步确认引起钙变化的钙通道。结果细胞内钙浓度随细胞外钙浓度的升高而升高,当细胞外钙浓度为2.75mmol/L时,细胞内钙钙荧光强度值达到最大为(123.25±12.95);钙池操纵的钙通道(SOC)阻断剂—La3+可显著抑制外钙引起的内钙升高(P﹤0.01);应用siRNA沉默TRPC1通道后可显著抑制外钙引起的内钙变化。结论随细胞外钙浓度升高,肝细胞内钙浓度显著增高;TRPC1参与的钙池操纵的钙通道可能是引起细胞内钙离子升高的主要通道。
Objective To study the effects of different concentrations of extracellular calcium on intracellular calcium in HepG2 cells and to clarify the mechanism of intracellular calcium changes. Methods HepG2 cells were stained with Fluo-3 / AM for 24 hours after being treated with different concentrations of calcium. The changes of intracellular calcium concentration were detected by flow cytometry. The calcium channels were blocked by blocking agents, Looking for reasons for changes in intracellular calcium; using siRNA interference to find the calcium channel to be silenced, to further confirm the calcium changes caused by calcium channels. Results The intracellular calcium concentration increased with the increase of extracellular calcium concentration. When the extracellular calcium concentration was 2.75mmol / L, the intracellular calcium and calcium fluorescence intensity reached the maximum (123.25 ± 12.95) (SOC) blocker-La3 + could significantly inhibit the increase of intracellular calcium induced by extracellular calcium (P <0.01). The silencing of TRPC1 channel by siRNA could significantly inhibit the intracellular calcium induced by extracellular calcium. Conclusions With the increase of extracellular calcium concentration, the intracellular calcium concentration is significantly increased. The calcium channel operated by TRPC1 may be the main channel leading to the increase of intracellular calcium.