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目的研究丹那唑对大鼠原代肝细胞的损伤作用与机制。方法原代大鼠肝细胞贴壁培养4 h,加入丹那唑0,50,100,200μmol/L,4 h和24 h后采用乳酸脱氢酶法测定细胞活力并检测三磷酸腺苷(ATP)水平,同时提取线粒体,分别测5个呼吸链复合酶(complex Ⅰ~V)活性。培养液中加入20 mmol/L果糖促进糖酵解,观察果糖对丹那唑细胞毒性的影响。结果丹那唑50,100μmol/L处理4 h和24 h对细胞几乎没有杀伤作用,但可致ATP显著降低;200μmol/L处理4 h和24 h均可导致细胞坏死。果糖可对抗丹那唑4 h具杀伤作用,但对24 h杀伤作用无效。丹那唑(50~200μmol/L)显著抑制线粒体呼吸链复合酶Ⅰ活性,但不影响complexⅡ~V活性。结论丹那唑选择性抑制线粒体呼吸链复合酶Ⅰ活性而导致ATP生成减少并诱发细胞坏死。
Objective To study the effect and mechanism of Danazol on rat primary hepatocytes. Methods Primary rat hepatocytes were adherently cultured for 4 h. The cells were stained with 0, 50, 100, 200 μmol / L of Danazol for 4 h and 24 h, and the activity of ATP was measured by lactate dehydrogenase assay. Meanwhile, the mitochondria, Five complexes of respiratory chain enzyme (complex Ⅰ ~ V) were measured respectively. Addition of 20 mmol / L fructose to the culture medium promoted glycolysis and observed the effect of fructose on the cytotoxicity of Danazol. Results Danazol at 50 and 100 μmol / L for 4 h and 24 h had almost no cytotoxic effect on cells, but significantly decreased ATP. At 200 μmol / L for 4 h and 24 h, cell necrosis was induced. Fructose 4 h anti-danazol can kill, but no effect on 24 h killing. Danazol (50 ~ 200μmol / L) significantly inhibited mitochondrial respiratory enzyme complex Ⅰ activity, but did not affect the activity of complex Ⅱ ~ V. Conclusion Danazol can selectively inhibit the activity of mitochondrial respiratory chain enzyme Ⅰ, resulting in the decrease of ATP production and cell necrosis.