褪黑素对高糖条件下牛主动脉内皮细胞保护作用的实验研究

PROTECTIVE EFFECTS OF MELATONIN ON CALF AORTIC ENDOTHELIAL CELLS IN MEDIUM WITH HIGH CONCENTRATION OF GLUCOSE

观察褪黑素 (Mel)对牛主动脉内皮细胞分泌舒张因子前列环素 (PGI2 )、一氧化氮 (NO)及表达诱生型一氧化氮合酶(iNOS)基因的影响 ,以探讨Mel的血管内皮保护作用。原代培养牛胸主动脉内皮细胞 ,取 3～ 5代用于实验。实验分 5组 :A组为低糖对照组 ,培养液中葡萄糖浓度为 5 5mmol/L ;B组为高糖 ( 3 3mmol/L)对照组 ;C组为高糖 ( 3 3mmol/L)、亚生理浓度Mel( 10 -13mol/L)组 ;D组为高糖( 3 3mmol/L)、生理浓度Mel( 10 -9mol/L)组 ;E组为高糖、药理浓度Mel( 10 -5mol/L)组。体外孵育 2 4h后 ,用放免法检测培养液中PGI2 的代谢产物 6 酮 前列腺素F1α( 6 酮 PGF1α)浓度 ,比色法检测培养液中NOS活性 ,RT PCR法检测内皮细胞iNOSmRNA表达。A组 6 酮 PGF1α浓度、NOS活性及iNOSmRNA表达均显著低于B组(P <0 0 1) ;B组与C组比较差异均无显著性意义 (P >0 0 5 ) ;而D组、E组 6 酮 PGF1α浓度显著高于B组(分别为P <0 0 5和P <0 0 1) ;D组、E组NOS活性均显著低于B组 (P<0 0 5 ) ;D组、E组iNOSmRNA表达均显著低于B组 (P <0 0 1)。

To observe the effects of melatonin on postacyclin concentration, nitric oxidase activity(NOS) and inducible nitric oxidase(iNOS) mRNA expression in calf aortic endothelial cells in medium with high concentration of glucose, endothelial cells of calf thoracic arota were cultured and divided into five groups. Group A was a low-glucose control with 5.5mmol/L glucose in medium. Group B was a high-glucose control with 33mmol/L glucose in medium. Cells of group C was cultured with 33mmol/L glucose and 10 -13mol/L melatonin, those of group D was cultured with 33mmol/L glucose and 10 -9mol/L melatonin, and those of group E was cultured with 33mmol/L glucose and 10 -5mol/L melatonin. The concentration of 6-keto-PGF 1α, metabolizing product of PGI 2, in the culture medium was measured with radio-immunological assay. NOS activity was determined with colorimetry and iNOS mRNA was measured with semi-quantitative RT-PCR. 6-keto-PGF 1α concentration, NOS activity and iNOS mRNA expression in group A were significantly lower than those in group B(P<0.01), and there were no significant difference between group B and group C(P>0.05). 6-keto-PGF 1α concentration of group D and group E was significantly higher than that in group B(P<0.05 and P<0.01, respectively). NOS activity of group D and group E was significantly lower than that in group B(both P<0.05). The expression of iNOS mRNA of group D and group E was significantly lower than that in group B(both P<0.01). Melatonin at physiological and pharmacological concentration improves diastolic function of endothelial cells.