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用四甲基偶氮唑盐(MTT)比色法检测不同浓度EGCG处理HaCat细胞24、48和72 h的生长变化,确定EGCG的最适作用浓度;将细胞分为对照组、EGCG单独处理组、X射线单独照射组、EGCG联合X射线照射组,克隆形成实验检测EGCG对HaCat细胞的辐射保护作用;DCFH-DA检测细胞内ROS变化;AnnexinV-FITC检测细胞凋亡的变化;DNA片段分析出现凋亡的片段;流式细胞术检测细胞周期变化。结果表明,EGCG在0-50μmol/L浓度范围内可刺激HaCat细胞增殖,高浓度则抑制生长,其效应呈时间依赖性;50μmol/L的EGCG能提高放射后HaCat细胞的克隆形成率;EGCG能抑制X射线诱导的细胞凋亡,与单纯照射组相比,药物+照射组凋亡率由9.25%降至7.82%,能抑制X射线诱导的凋亡片段;相比于单纯照射组,EGCG降低了X射线诱导的HaCat细胞G2/M期阻滞。提示EGCG对人皮肤HaCat细胞具有辐射保护作用;EGCG可能通过抑制X射线引起的ROS,从而抑制细胞凋亡及降低辐射诱导的细胞G2/M期阻滞发挥辐射保护作用。
The growth of HaCat cells treated with different concentrations of EGCG for 24, 48 and 72 h were detected by MTT colorimetric assay, and the optimal concentration of EGCG was determined. The cells were divided into control group, EGCG group , X-ray single irradiation group, EGCG combined with X-ray irradiation group, clone formation experiment to detect the radiation protective effect of EGCG on HaCat cells; DCFH-DA detection of intracellular ROS; Annexin V-FITC detection of apoptosis; DNA fragment analysis Apoptotic fragments; flow cytometry to detect cell cycle changes. The results showed that EGCG could stimulate the proliferation of HaCat cells in the concentration range of 0-50μmol / L and the high concentration inhibited the growth of EGCG in a time-dependent manner. EGCG at 50μmol / L increased the colony formation rate of HaCat cells after irradiation; Compared with the irradiation alone group, the apoptotic rate of drug + irradiation group was reduced from 9.25% to 7.82%, which inhibited the X-ray-induced apoptosis. Compared with the irradiation alone group, EGCG decreased X-ray induced G2 / M phase arrest in HaCat cells. These results suggest that EGCG has a protective effect on human skin HaCat cells. EGCG may play an important role in radiation protection by inhibiting X-ray-induced ROS, inhibiting apoptosis and decreasing G2 / M arrest induced by radiation.