神经酰胺在葡萄糖腹膜透析液诱导的腹膜间皮细胞凋亡中的作用

目的 观察葡萄糖腹膜透析液(PDS)对人腹膜间皮细胞(PMC)凋亡的影响和细胞内神经酰胺在PMC凋亡中的作用,初步探讨神经酰胺信号通路是否参与高浓度PDS诱导的PMC凋亡.方法 PMC分别在正常对照、1.5％ PDS、4.25％ PDS条件下培养,以4.25％甘露醇作为高渗对照.高压液相色谱串联质谱法(LC-MS-MS)检测细胞内神经酰胺的变化.Annexin - FITC- PI双染流式细胞术检测细胞凋亡.Western印迹法检测bax、p53、bcl-2蛋白表达.结果 (1)PDS可上调PMC细胞内神经酰胺表达,正常对照组、高渗对照组对细胞内神经酰胺均无明显影响.酸性鞘磷脂酶抑制剂地昔帕明可显著抑制高浓度PDS诱导的神经酰胺生成[(56.08±12.24)μg/L比(91.25±15.89) μg/L,P＜0.01].(2)与1.5％ PDS组相比,4.25％PDS可显著诱导PMC细胞凋亡[(26.65±6.21)％比(4.04±1.86)％,P＜0.01],上调bax、p53蛋白表达(P＜0.01),下调bcl-2蛋白表达(P＜0.05).地昔帕明可明显抑制高浓度PDS诱导的PMC细胞凋亡,bax、p53上调以及bcl-2下调(均P＜0.05),外源性神经酰胺则可明显逆转地昔帕明的此类作用(P＜0.05).正常对照组、高渗对照组对细胞内神经酰胺表达及细胞凋亡均无明显影响.结论 细胞内神经酰胺增加可能参与了高浓度PDS诱导的PMC凋亡.

Effect of ceramide on peritoneal mesothelial cells apoptosis induced by peritoneal dialysis solution

Objective To explore the effect of ceremide on process of peritoneal mesothelial cells(PMCs) apoptosis induced by peritoneal dialysis solution(PDS). Methods PMCs were cultured with normal DMEM, 1.5% PDS and 4.25% PDS. 4.25% mannitol was used as high osmotic pressure control. Ceremide were detected by LC-MS-MS. Flow cytometry was used in apoptosis analysis. Bax, p53 and bcl-2 protein expressions were detected by Western blotting. Results (1) PDS caused the increase of intracellular ceremide in PMCs, and normal and high osmotic pressure controls had no such effect. As the acidic sphigomyelinase inhibitor, desipramine significantly inhibited the production of ceramide induced by 4.25% PDS[(56.08±12.24) μg/L vs (91.25±15.89) μg/L, P<0.01]. (2) Compared with 1.5% PDS, 4.25% PDS stimulated PMCs apoptosis(26.65%±6.21% vs 4.04%±1.86%, P<0.01), up-regulated bax and p53 proteins expression(P<0.01), and down-regulated bcl-2 protein exprssion (P<0.05). Desipramine obviously inhibited the apoptosis induced by 4.25% PDS, decreased bax and p53 proteins expression, increased bcl-2 protein expression(P<0.05). Exogenous C2-ceremide reversed the effect of desipramine(P<0.05). Conclusion The increase of intracellular ceremide may play an important role in the PMCs apoptosis induced by high glucose PDS.