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目的研究中波紫外线(UVB)联合纳米碳酸钙处理对HaCaT细胞增殖、凋亡的影响及其机制。方法取对数生长期HaCaT细胞分为对照组、UVB照射组、纳米碳酸钙组和联合处理组4组,UVB照射组和联合处理组接受累计照射剂量为2.97×10-2J/cm2的UVB照射,对照组和纳米碳酸钙组给予同等条件下假照射。照射完毕后,对照组和UVB照射组分别用含体积分数为10.00%磷酸盐缓冲液的高糖达尔伯克氏改良伊格尔培养基(DMEM)继续培养,纳米碳酸钙组和联合处理组分别用含质量浓度为250 mg/L纳米碳酸钙的高糖DMEM继续培养。于培养后0、6、12、18、24 h收获细胞,采用噻唑蓝法检测细胞活性,流式细胞术检测细胞凋亡率,免疫印迹法检测凋亡相关蛋白P53和Caspase-3表达情况。结果与同时间点对照组比较,除UVB照射组在6 h时间点差异无统计学意义外(P>0.05),其余各处理组在6~24 h各个时间点HaCaT细胞活力均下降(P<0.05);联合处理组HaCaT细胞活力在6~24 h各个时间点均低于同时间点UVB照射组(P<0.05),在18和24 h均低于同时间点纳米碳酸钙组(P<0.05)。UVB照射组HaCaT细胞凋亡率高于对照组(P<0.05),联合处理组HaCaT细胞凋亡率高于其余3组(P<0.05)。P53、Caspase-3蛋白表达水平在UVB照射组和纳米碳酸钙组HaCaT细胞均较对照组表达上调;联合处理组HaCaT细胞P53和Caspase-3蛋白的表达水平均高于其余3组。结论 UVB联合纳米碳酸钙处理对HaCaT细胞的损伤效应较单一损伤因素更严重,表现为抑制细胞增殖,诱导细胞凋亡,促进P53及Caspase-3蛋白表达。
Objective To investigate the effects of UVB combined with nano-calcium carbonate on the proliferation and apoptosis of HaCaT cells and its mechanism. Methods The logarithmic growth phase HaCaT cells were divided into control group, UVB irradiation group, nano-calcium carbonate group and combined treatment group, UVB irradiation group and combined treatment group received cumulative radiation dose of 2.97 × 10-2 J / cm2 UVB irradiation The control group and the nano-CaCO3 group were given the same conditions of false illumination. After the irradiation, the control group and the UVB irradiation group were respectively cultured with high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10.00% phosphate buffer solution for a further time, respectively. The nano-calcium carbonate group and the combined treatment group Culture was continued with high glucose DMEM containing 250 mg / L nano-CaCO3. The cells were harvested at 0, 6, 12, 18 and 24 h after culture. Cell viability was detected by thiazolyl blue staining. Flow cytometry was used to detect the apoptotic rate. Western blotting was used to detect the expression of apoptosis-related proteins P53 and Caspase-3. Results Compared with the control group at the same time point, the activity of HaCaT cells in all the other treatment groups was decreased at all time points except 6 hours after UVB irradiation (P < 0.05). The activity of HaCaT cells in the combined treatment group was lower than that in the UVB irradiation group (P <0.05) at all time points of 6-24 h, but lower than that in the nano-calcium carbonate group at 18 and 24 h (P < 0.05). The apoptosis rate of HaCaT cells in UVB irradiation group was higher than that in control group (P <0.05), and the apoptosis rate of HaCaT cells in combination group was higher than that in the other three groups (P <0.05). The expression of P53 and Caspase-3 in HaCaT cells of UVB-irradiated group and HaCaT-treated group were higher than that of control group. The expressions of P53 and Caspase-3 in HaCaT cells were higher than those in the other three groups. Conclusion UVB combined with calcium carbonate treatment of HaCaT cells damage more serious damage than single factor, showing inhibition of cell proliferation, induction of apoptosis, and promote P53 and Caspase-3 protein expression.