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目的一氧化氮 NO 已被认为是生理和病理状态下一种重要生物分子.以氧化砷(As_2O_3)诱导食管癌细胞(SHEEC1)凋亡过程中检测细胞 NO,NO 合成酶(NOS)和一氧化氮合成酶mRNA(NOSmRNA),探索 NO 和细胞凋亡的关系.方法用 As_2O_3(5 μmol·L~(-1)和10μmol·L~(-1))诱导 SHEEC1凋亡,在加药后2,4,8,16,24h 取样检查,用分光光密度法检测细胞培养液的 NO 含量;用免疫组化检测诱导型 NO 合成酶(NOSⅡ);分子原位杂交(ISH)检测 NOSmRNA;实验终点细胞作透射电镜(TEM)检查细胞凋亡.结果在 As_2O_3诱导下,SHEEC1从 NO 基础量(0.68×10~(-2)μmol·L~(-1))逐渐增加,至加药后16h 达最高(2.38×10~(-2)μmol·L~(-1));NOSⅡ在加药后4h～16h 呈阳性反应;NOSmRNA 杂交信号位于胞质,4h 杂交点小而少,以后增加明显.24h,TEM可见 SHEEC1出现细胞凋亡.结论 As_2O_3 可诱导 SHEEC1释放 NO 和细胞凋亡,实验开始至16h NO 量不断增加.NOSⅡ表达和 NOSmRNA 转录上调.提出 NO 可能是 As_2O_3诱导细胞凋亡的介导和作用因子.
Objective Nitric oxide (NO) has been considered as an important biomolecule under physiological and pathological conditions.Apoptosis of esophageal cancer cells (SHEEC1) induced by arsenic oxide (As_2O_3) was measured during the apoptotic process of NO, NO synthase (NOS) (NOSmRNA), explore the relationship between NO and apoptosis.Methods SHEEC1 apoptosis was induced by As_2O_3 (5 μmol·L -1 and 10 μmol·L -1) , 4,8,16,24h samples were taken, and the content of NO in cell culture medium was detected by spectrophotometric method. The expression of NOS mRNA was detected by immunohistochemistry and NOS mRNA by in situ hybridization (ISH) The cells were examined for apoptosis by transmission electron microscopy (TEM) .Results SHEEC1 increased gradually from 0.68 × 10 ~ (-2) μmol·L ~ (-1) after induction with As_2O_3 until 16 h after dosing (2.38 × 10 ~ (-2) μmol·L -1). NOSⅡhad a positive response at 4h ~ 16h after the addition of the drug. The signal of NOSmRNA hybridization was located in the cytoplasm, and the hybridization point was smaller and smaller at 4h. TEM and TEM showed that SHEEC1 cells were apoptotic.Conclusion As2O3 can induce SHEEC1 to release NO and apoptosis, and the amount of NO began to increase after 16 hours.NOSⅡexpression and NOS mRNA transcription Proposed As_2O_3 NO may be mediated induction of apoptosis and the role of factors.