较低剂量甲醛诱导人脐静脉内皮细胞损伤的时相特征及机制

Time-effect of Damage on Human Umbilical Vein Endothelial Cell Lines Induced by Low Dose Formaldehyde and its Mechanism

目的观察较低剂量甲醛(formaldehyde,FA,浓度为40μmol/L)诱导人脐静脉内皮细胞损伤时相特征及一氧化氮(NO)含量变化的时相规律,分析甲醛在诱导内皮细胞损伤中的作用机制。方法将人脐静脉内皮细胞(HU-VEC-12株)与甲醛浓度为40μmol/L的DMEM培养基孵育。分别处理一定时间后(0,4,12,24,48,72h)H-E染色-光学显微镜观察细胞形态变化;MTT法测定细胞的活性;丙酮酸二硝基苯腙比色-分光光度法检测细胞上清液乳酸脱氢酶(LDH)漏出量;硫代巴比妥法检测细胞上清液中丙二醛(MDA)浓度;硝酸还原酶法测定培养液中NO浓度;生化分析法测定内皮细胞一氧化氮合酶活性;免疫细胞化学法测定内皮型一氧化氮合酶(eNOS)和诱生型一氧化氮合酶(i NOS)的蛋白表达;RT-PCR法测定eNOS和i NOS的mRNA表达。结果较低浓度甲醛呈时间依赖性地促进细胞LDH漏出,以及内皮细胞上清液MDA和NO的产生;降低eNOS的活性,抑制eNOS在蛋白和基因水平的表达;增加i NOS活性,促进i N-OS在蛋白和基因水平的表达。结论甲醛诱导HUVEC-12细胞损伤,机制可能与其抑制eNOS的活性及表达,诱导i NOS的活性及表达,增加病理性NO产生,从而导致内皮细胞损伤。

Objective To explore the time - effect of damage in human umbilical vein endothelial cell lines induced by low dose formaldehyde and to analyze its mechanism. Methods HUVECs - 12 were cultured in DMEM containing 10% FCS. At 80% confluence, the cells washed with D- Hank＇s fluid and cultured in DMEM containing 1% FCS for 24 h again. Cells were incubated in low concentration formaldehyde （40 μmol/L） to test the time - effect of damage in human umbilical vein endothelial ceils induced by low dose formaldehyde. The results were examined at the 0, 4, 12, 24, 48,and 72 h time spots respectively. The transudation content of lactate dehydregenase （LDH） was monitored colorimetricafly, and the level of malondialde - hyde （MDA） in supernatant- conditioned media was monitored by thiobituric acid reaction （TBA）. The level of NO in the culture medium was monitored to evaluate the influence of formaldehyde by nitrate reductase method, the nitric oxide synthase （NOS） activity was monitored biochemically, the protein and mRNA levels of iNO＇S and eNOS were monitored by immunohistochemistry and RT - PCR approach respectively to elucidate the injuring mechanism. Results Formaldehyde conspicuously inhibited the activity of eNOS, expression of eNOS protein and eNOS mRNA. Formaldehyde induced the activity of iNOS, expression of iNOS protein and iNOS mRNA, and increased the production of nitric oxide （NO）, MDA and the transudation content of LDH on HUVECs - 12 in a time - dependent manner. Conclusions Low dose formaldehyde can induce damage in human umbilical vein endothelial cells. This change is related to reduce the activity and expression of eNOS, increase the activity and expression of iNOS, and elevate the production of nitric oxide.