钙对人腹膜间皮细胞损伤、增殖及纤维连接蛋白合成的影响

目的研究不同浓度钙对体内外人腹膜间皮增殖、损伤和间质纤维化的影响。方法体外试验:以人腹膜间皮细胞永生化细胞株(HMrSV5)为研究对象,予不同浓度钙(0、0.25、0.75、1.25、1.75、2.25mmol/L)处理该细胞12、24h。采用四甲基偶氮唑盐(methyl thiazolyl tetrazolium,MTT)比色法和乳酸脱氢酶(lactate dehydrogenase,LDH)评估细胞的增殖能力和损伤。蛋白免疫印迹法检测纤维连接蛋白(fibronectin,FN)的表达。临床试验:随机选择47例既往使用低钙透析液(Baxter PD4,含钙1.25mmol/L)的稳定维持性腹膜透析患者,平均透析时间为(18.5±11.7)个月。采用前后自身对照法进行研究。以患者刚入选本研究时作为对照组,入选后换用含钙1.75mmol/L的PD2进行透析(其他成份均与PD4相同)4周,其他治疗(降压、降磷、活性维生素D3等)不变。患者使用PD2透析后的每一周末作为一组(分别为第1、2、3、4周组)。酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测腹膜透析引流液中的FN和LDH,电化学发光法(electrochemiluminescence assay,ECLA)检测CA125。同时检测血清钙、磷及全段甲状旁腺激素(intact parathyroid hormone,iPTH)水平。结果体外试验:钙呈时间及浓度依赖性促进HMrSV5增殖,含钙1.75mmol/L组最高;不同浓度钙呈时间依赖性显著诱导LDH上调,其中钙1.25mmol/L组LDH最低(P<0.05);FN亦呈钙浓度及时间依赖性表达上调。临床试验:4个试验组腹膜透析引流液中LDH值均与对照组有显著差异且呈时间依赖性升高。第4周组的FN和LDH水平显著高于其他组,CA125水平明显低于其他组,差异均有统计学意义(均P<0.01)。第4周组的血清钙高于对照组,血磷低于对照组差异均有统计学意义(均P<0.05),iPTH与对照组差异无统计学意义(P>0.05)。结论在体内外试验中,高钙(1.75mmol/L)可促进人腹膜间皮细胞增殖,同时加重细胞损伤,促进FN合成,而生理浓度钙(1.25mmol/L)对人腹膜间皮细胞有一定保护作用并可能预防腹膜纤维化。

The influences of calcium on proliferation,daminification and fibronectin synthesis in human peritoneal mesothelial cells

Objective To investigate the influences of various concentrations of calcium on human peritoneal mesothelial cells （HPMCs） proliferation, damnification and interstitial fibrosis in vitro and in vivo. Methods HPMCs were cultured in the media containing calcium 0, 0.25, 0.75, 1.25, 1.75 or 2.25mmol/L for 12h or 48h. The proliferation of HPMCs was assessed by tetrazolium salt colorimetry assay （MTT assay）, and the damage of HPMCs was evaluated by measuring LDH in the medium. Fibronectin （FN） in cytoplasm of HPMCs was determined by western blotting. For clinical observations, a self-controlled study was conducted in 47 patients on peritoneal dialysis using the dialysis fluid PD4 （1.25mmol/L calcium, Baxter） for （18.5±11.74） months. The patients were then treated with the dialysis fluid PD2 （1.75mmol/L calcium, other components are the same as PD4, Baxter） for 4 weeks without any other changes in their therapies. Patients were examined after the dialysis using PD2 fluid for 1, 2, 3 and 4 weeks. FN and LDH in peritoneal effluent were measured by enzyme linked immunosorbent assay （ELISA）, and CA125 was detected by electrochemiluminescence assay （ECLA）. We also assayed patients？serum Ca, P, and intact parathyroid hormone （iPTH）. Results Calcium in culture media correlated to the proliferation of HPMCs in concentration- and time- dependent manners, with the most promotion effect at 1.75mmol/L calcium. Calcium in culture media also correlated to the increase of LDH in peritoneal effluents in a time-dependent manner, with the least impact on LDH at 1.25mmol/L calcium （P0.05）, and to the increase of FN in cells in concentration- and time-dependent manners. In peritoneal dialysis patients after switch to PD2 fluid, LDH in peritoneal effluents increased significantly in a time-dependent manner. In those after the switch for 4 weeks, LDH and FN reached to the highest values （P0.01）, CA125 decreased to the lowest level （P0.01）, serum calcium was higher than that of control group （P0.05）, serum phosphorus was lower than that of control group （P0.05）, and iPTH remained unchanged （P0.05）. Conclusions Our in vivo and in vitro studies demonstrate that higher calcium concentration in peritoneal dialysis fluid at 1.75mmol/L stimulates HPMCs proliferation, injures HPMCs, and up-regulates FN expression in HPMCs. physiological calcium concentration in peritoneal dialysis fluid at 1.25 mmol/L protects the peritoneum from fibrosis.