线粒体在高糖损伤人腹膜间皮细胞中的作用

The role of mitochondria in progression of high-glucose-inducing injury to human peritoneal mesothelial cells

目的:探讨高浓度葡萄糖损伤人腹膜间皮细胞的机制。方法:(1)分离培养人腹膜间皮细胞,以细胞形态、免疫细胞化学染色作细胞鉴定;(2)第2代人腹膜间皮细胞分别经含1.5%、2.5%、4.25%葡萄糖的M199培养基培养48h后(以正常M199培养基和含4.25%甘露醇的M199培养基为对照),检测间皮细胞线粒体膜电位、凋亡率和caspase-3活性;(3)分别用0.1μmol/L和0.01μmol/L环孢菌素A(CsA)与4.25%葡萄糖共同刺激体外培养的人腹膜间皮细胞(以4.25%葡萄糖为对照),检测间皮细胞凋亡率和caspase-3活性。结果:(1)1.5%、2.5%、4.25%葡萄糖组线粒体膜电位丧失的细胞百分率显著高于M199对照组(P<0.05)且随着葡萄糖浓度增加,线粒体膜电位丧失的细胞百分率增加,4.25%葡萄糖组线粒体膜电位丧失的细胞百分率显著高于甘露醇对照组;(2)2.5%葡萄糖组和4.25%葡萄糖组间皮细胞caspase-3活性和凋亡率显著高于M199对照组(P<0.05),而1.5%葡萄糖组及4.25%甘露醇组间皮细胞caspase-3活性和凋亡率差异无统计学意义(P>0.05)。且随着葡萄糖浓度增加,间皮细胞caspase-3活性和凋亡率上升,4.25%葡萄糖组caspase-3活性和凋亡率显著高于4.25%甘露醇组(P<0.05);(3)随着葡萄糖浓度增加,线粒体膜电位丧失的间皮细胞百分率与间皮细胞凋亡率、caspase-3活性呈显著正相关(P<0.01);(4)0.1μmol/L CsA组间皮细胞凋亡率和caspase-3活性显著低于高糖对照组(P<0.05)。结论:(1)高浓度葡萄糖能以剂量依赖的方式诱导人腹膜间皮细胞线粒体膜电位丧失、caspase-3活化和凋亡;(2)高糖诱导人腹膜间皮细胞caspase-3活化和凋亡可能依赖于线粒体活化。

Objective：To investigate mechanism of high -glucose- inducing injury to human peritoneal mesothelial cells. Methods ： （ 1 ） The cultured human mesothelial cells were exposed to culture medium containing different concentrations of glucose （1.5% ,2.5% ,4.25% ）, with 4.25% mannitol and normal culture medium as control for 48 hours. Then mitochondrial membrane potential（ψ） and the apoptotic rate were measured by flow cytometry, and caspase -3 activity was measured by ApoAlert（tm） CPP33/Caspase -3Assay kits. （2） The cultured human mesothelial cells were exposed to 4.25% glucose cul- ture medium containing different concentrations of mitochondrial transition pore inhibitor - cyclosporin A （0.01, 0.1 μmol/L） for 24 hours, with 4.25% glucose culture medium as control, Then the apoptotic rate was measured by flow cytometry and caspase -3 activity was measured by ApoAlert（tm） CPP33/Caspase -3Assay kits. Results：（ 1 ） The percentage of mesothelial cells with loss of mitochondrialψ was significantly higher in high glucose groups than that in normal culture medium group, which was increasing as glucose concentration increases. The percentage of mesothelial cells with loss of mitoehondrialψ was also significantly higher in 4.25% glucose groups than that in 4.25% mannitol group. （2） Caspase -3 activity and the apoptotic rate of mesothelial cells was significantly higher in 4.25% glucose group and 2.5% glucose group than that in normal culture medium group, but no significant difference in 1.5% glucose group and 4.25% mannitol group compared to in normal culture medium group. Caspase - 3 activity and the apoptotic rate of mesothelial cells was also significantly higher in 4.25% glucose group than that in 4.25% mannitol group. Caspase - 3 activity and the apoptotic rate of mesothelial cells was increasing as glucose concentration increases. （3） There was significantly positive correlation among the apoptotic rate of mesothelial cells, caspase - 3 activity of mesothelial cells and the percentage of mesothelial cells with loss of mitochondrialψ. （4） Apoptotic rate and easpase - 3 activity of mesothelial cells was significantly lower in 0.1 μmol/L CsA group than that in control. Conclusion ： （ 1 ） High - glucose can induce loss of mitochondrial ψ , apoptosis and caspase - 3 activation of human peritoneal mesothelial cells in dose - dependent manner. （2） Caspase - 3 activation in progression of high - glucose - inducing apoptosis of human peritoneal mesothelial cells by mitochondria activation.