钙通道阻滞剂对环孢素A所致内皮细胞损伤的保护作用

PROTECTIVE EFFECTS OF CALCIUM CHANNEL BLOCKER ON CYCLOSPORIN A INDUCED ENDOTHELIAL CELL INJURY

目的：研究钙通道阻滞剂异搏定对环孢素Ａ（ＣＳＡ）所致内皮细胞（ＥＣ）损伤的保护作用。方法：在培养的ＥＣ中加入ＣＳＡ１００μｇ／Ｌ的同时加入异搏定１００μｇ／Ｌ作用２４ｈ，以直接计数粘附细胞数观察细胞分离；光镜、电镜观察细胞形态；Ｈ３－ＴｄＲ掺入法及流式细胞仪观察ＤＮＡ抑制；Ｆｕｒａ－２ＡＭ荧光负载法检测细胞内游离钙（［Ｃａ２＋］ｉ）；放射免疫法检测细胞上清中内皮素（ＥＴ）的含量；斑点杂交检测ＰｒｅｐｒｏＥＴ－１ｍＲＮＡ水平。结果：异搏定可减轻ＣｓＡ对内皮细胞的损伤，加入异搏定的内皮细胞，细胞内游离钙下降及细胞分离减少，超微结构损伤减轻，ＤＮＡ合成抑制改善，异搏定还可明显降低ＣＳＡ介导的前原内皮１ｍＲＮＡ（ＰｒｅｐｒｏＥＴ－１ｍＲＮＡ）水平的升高，使细胞上清中ＥＴ的含量减少。结论：异搏定可有效地改善ＣＳＡ１００μｇ／Ｌ作用２４ｈ所致的ＥＣ细胞毒作用，从而防治其所致的肾毒性和高血压。

OBJECTIVE To study the protective effects of calcium channel blocker on cyclosporin A induced endothelial cell injury. METHODOLOGY Cultured human umbilical vein endothelial cells(HUVEC) were incubated with medium containing 100 μg/L CsA and/or 100 μg/L verapamil for 24 hours. Cell detachment was determined by counting adherent cells; Morphological changes of the cells were observed under light microscope and electronic microscope; Release of 3H thymidine by pre labeled cells after exposure to CsA and verapamil was used as marker of cell disruption; Free intracellular calcium concentration ([Ca 2+ ]i) was measured fluorometrically in cells loaded with the intracellular probe fura 2; Endothelin was detected by radioimmunologic technique; PreproET 1 mRNA was detected by using digoxigenin labeled preproET 1 oligonuclectide probe. RESULTS After 24 hours' incubation with 100 μg/L CsA, a significant increase in the percentage of detached cell(29 8% vs 5 2%), [Ca 2+ ]i(505 9 vs 102 5 nmol/L), the inhibition percentage of DNA replication(32% vs 6 7%) and the concentration of ET(735 5 vs 56 9 pg/L) were found in HUVEC as compared to the control. Swelling and vascuolation were observed in CsA treated HUVEC under light microscope, and injuries of mitochondria and endoplasmic reticulum under electronic microscope. In verapamil and CsA treated HUVEC, all the CsA caused damage mentioned above were attenuated. CsA produced a significant increase in preproET 1 mRNA expression (optical density × blot area: 63 8 vs 19 5), however, the mRNA expression was decreased when verapamil was added into the CsA treated HUVEC in culture(optical density × blot area:40 5 vs 63 8). CONCLUSION Verapamil can ameliorate CsA caused HUVEC injury in culture and might be used to prevent CsA caused nephrotoxicity and hypertension in vivo.