摘要
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 effect and mechanism of apoptosis of pheochromocytoma cells (PC12)induced by formaldehyde. Methods The viability of PC12 cells was detected by MTT. The apoptosis was measured by Hoechst 33258 fluorescence staining. The transudation content of LDH was determined by colormetry reaction. The nitrite content of the cul- ture medium was measured by nitrate reductase method. The apoptosis of NOS and iNOS were measured by biochemical method. The apoptosis of cells was determined by double-staining(Annexin-FITC/PI) and flow cytometry, The procaspase-3 expression level in PC12 cells treated by formaldehyde was detected by westen blot. Results The content of 20-160 ixmol/L formaldehyde induced apoptosis in PC12 cell. The low dose of formaldehyde( 〈 80p.mol/L)mainly induced apopto- sis, while the high dose of formaldehyde ( 〉 801xmoL/L) mainly caused necrosis. The content of LDH in supernatant and the amount of NO were gradually increased with the increment of the formaldehyde concentration . Formaldehyde promoted the activities of NOS and iNOS in a dose-dependent manner. With the increment of the formaldehyde concentration, the expres- sion level of procaspase-3 was increased gradually. The damage of PC12 cell was abated significantly in AG-treated group. Conclusion Formaldehyde could induce the apoptosis of PC12 cells in a dose-dependent manner via caspase pathway. AG could protect PC12 cells against the damage induced by formaldehyde.
出处
《中国公共卫生》
CAS
CSCD
北大核心
2009年第6期733-735,共3页
Chinese Journal of Public Health
基金
国家自然科学基金(30572191)
南华大学科研基金(DS02224)
南华大学博士启动基金(5-2005-XQD-001)