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目的研究甲醛诱导肾上腺嗜铬细胞损伤特征及机制。方法将肾上腺嗜铬细胞瘤细胞与不同浓度甲醛(20,40,80,160μmol/L)共培养,24h以后用四甲基偶氮噻唑蓝比色法检测细胞活力;Hoechst33258荧光染色法检测细胞凋亡;分光光度法检测乳酸脱氢酶漏出量;硝酸还原酶法检测细胞上清液一氧化氮含量;生化法检测总一氧化氮合酶活性和诱导型一氧化氮合酶活性;磷酯酰丝氨酸结合蛋白-异硫氢酸荧光素/碘化丙啶双染法、流式细胞仪分析细胞死亡;免疫着色实验技术检测半胱氨酸蛋白酶-3前体表达。结果20～160μmol/L甲醛诱导细胞死亡,低剂量甲醛(<80μmol/L)主要诱导凋亡,高剂量甲醛(>80μmol/L)主要诱导细胞坏死;细胞乳酸脱氢酶漏出量具有甲醛浓度依赖性,从(374.04±15.64)U/L增加至(800.18±24.84)U/L,且一氧化氮含量逐渐升高,与对照组比较,差异有统计学意义(P<0.01)。甲醛浓度依赖性诱导总一氧化氮合酶及型一氧化氮合酶活性表达增加,半胱氨酸蛋白酶-3前体蛋白酶原表达随甲醛浓度逐渐增强;氨基胍保护组细胞损伤明显减轻。结论甲醛呈浓度依赖性诱导肾上腺嗜铬细胞瘤细胞死亡;机制与半胱氨酸蛋白酶激活有关;氨基胍对细胞损伤有保护作用。
Objective To study the characteristics and mechanism of formaldehyde induced chromaffin cell injury in adrenal gland. Methods Adrenal pheochromocytoma cells were co-cultured with different concentrations of formaldehyde (20, 40, 80 and 160μmol / L), and cell viability was detected by MTT assay after 24h. Apoptosis was detected by Hoechst33258 fluorescence staining , The amount of leakage of lactate dehydrogenase was detected by spectrophotometry, the content of nitric oxide in supernatant was detected by nitrate reductase method, the activity of nitric oxide synthase and inducible nitric oxide synthase were detected by biochemical method, Binding protein-fluorescein isothiocyanate / propidium iodide double staining, cell death was analyzed by flow cytometry, and the expression of caspase-3 precursor was detected by immunohistochemistry. Results Formaldehyde induced cell death at 20 ~ 160μmol / L, low level of formaldehyde (<80μmol / L) induced apoptosis, and high dose of formaldehyde (> 80μmol / L) induced cell necrosis. Lactate dehydrogenase (374.04 ± 15.64) U / L increased to (800.18 ± 24.84) U / L, and the content of nitric oxide increased gradually. Compared with the control group, the difference was statistically significant (P <0.01). The formaldehyde concentration-dependently induced the expression of nitric oxide synthase and nitric oxide synthase, and the expression of caspase-3 precursor protease gradually increased with the formaldehyde concentration. The aminoguanidine protective group significantly reduced the cell injury. Conclusions Formaldehyde can induce the apoptosis of adrenal pheochromocytoma cells in a concentration-dependent manner. The mechanism is related to caspase activation. Aminoguanidine has a protective effect on cell injury.