高糖诱导PC12细胞损伤的影响因素及梓醇的保护作用

Influencing factors of PC12 cell injury induced by high glucose and the protective effects of catalpol

目的 探讨不同条件下高浓度葡萄糖(高糖)对大鼠肾上腺嗜铬细胞瘤细胞系PC12细胞的损伤作用,分析建立高糖损伤细胞模型的影响因素,并用确定的细胞模型观察梓醇的保护作用。方法 PC12细胞种植密度为1×10^(3)、1×10^(4)、1×10^(5)个/mL,高糖加入时间点分别为细胞种植后24 h和48 h,葡萄糖终浓度为30、40、50和75 mmol/L,据此分为HG30、HG40、HG50、HG75组,另外对照组细胞加入等容积生理盐水。分别作用24、48、72 h,MTT法检测细胞存活率。用确定条件建立的细胞损伤模型观察梓醇的保护作用,细胞分为对照组、模型组、梓醇低剂量组(1×10^(-5)mol/L)和梓醇高剂量组(1×10^(-4)mol/L),ELISA法和荧光探针检测细胞活性氧(ROS)含量,ELISA测定乳酸脱氢酶(LDH)的释放量。结果 不同葡萄糖作用条件下,与对照组比较,HG30组细胞存活率不变或降低(P>0.05或P<0.05),HG40、HG50和HG75组细胞存活率降低(P<0.05或P<0.01)。与对照组比较,模型组的细胞ROS含量增加(P<0.01),LDH释放量升高(P<0.01);与模型组比较,梓醇低剂量组和梓醇高剂量组细胞存活率升高(P<0.01),细胞ROS含量和LDH释放量降低(P<0.01)。结论 高糖诱导的PC12细胞损伤受细胞种植密度、葡萄糖浓度和作用时间等因素影响;以1×10^(4)个/mL密度种植细胞、种植后24 h加入终浓度50 mmol/L葡萄糖作用时长24 h,可成功建立PC12细胞高糖损伤模型,梓醇对该细胞模型有保护作用。

Objective To investigate the damaging effects of high glucose(HG) on rat adrenal pheochromocytoma PC12 cells under different conditions,to analyze the influencing factors of establishing a model of cell injury induced by HG,and to explore the protective effects of catalpol.Methods PC12 cells were seeded with a density of 1×10^(3)、1×10^(4)、1×10^(5) cells/mL.HG was added into the medium at 24 and 48 hours after seeding.The final concentrations of glucose were 30,40,50 and 75 mmol/L.PC12 cells damaged by HG were divided into HG30,HG40,HG50 and HG75 groups accordingly.Control cells were treated with equal volume of physiological saline.After HG acted for 24,48,and 72 hours,respectively,the cell survival rate was measured with MTT.After the cell injury model was established,cells were divided into control group,model group,low catalpol dose group(1×10^(-5)mol/L) and high catalpol dose group(1×10^(-4)mol/L).The reactive oxygen species(ROS) content was detected with ELISA and fluorescent probe.The lactate dehydrogenase(LDH) content was measured with ELISA.Results Under different glucose conditions,compared with the control group,the cells in HG30 group had unchanged(P<0.05) or decreased survival rate(P<0.05),while the cells in HG40,HG50,and HG75 groups had decreased survival rate(P>0.05 or P<0.01).Compared with the control group,the model group had increased ROS and LDH contents(both P<0.01).Compared with model group,the low and high catalpol dose groups had increased survival rate(P<0.01),and decreased ROS and LDH contents(P<0.01).Conclusion Injury in PC12 cells induced by HG is influenced by factors such as cell planting density,glucose concentration and action duration.The HG induced PC12 cell injury model can be established stably by a final concentration of 50 mmol/L glucose acting on cells seeded with a density of 1×10^(4) cells/mL for 24 hours.Catalpol plays a protective role on the model.