【摘要】：目的探討構(gòu)建過氧化氫(H_2O_2)誘導(dǎo)人卵巢顆粒細(xì)胞氧化應(yīng)激模型的方法。方法不同濃度H_2O_2處理人卵巢顆粒細(xì)胞COV434,采用Western blot檢測Caspase-3表達(dá),流式細(xì)胞儀和DAPI/TUNEL雙染色法檢測細(xì)胞凋亡情況,H2DCF-DA檢測細(xì)胞內(nèi)活性氧族(ROS)水平,通過量效關(guān)系和時(shí)效關(guān)系來確定H_2O_2的最佳作用條件。結(jié)果 1~1.5 mmol/L濃度H_2O_2處理COV434細(xì)胞2~4h后Caspase-3表達(dá)量升高最明顯。隨著H_2O_2濃度增加及作用時(shí)間的延長細(xì)胞凋亡率不斷升高。細(xì)胞內(nèi)ROS水平隨著H_2O_2濃度的增加而增加。1.5mmol/L濃度H_2O_2處理1h后細(xì)胞內(nèi)ROS水平上升最明顯。結(jié)論 H_2O_2誘導(dǎo)人卵巢顆粒細(xì)胞氧化應(yīng)激模型的最適宜濃度為1~1.5 mmol/L,采用細(xì)胞凋亡情況評(píng)價(jià)作用時(shí)間是2~4h,采用ROS評(píng)價(jià)作用時(shí)間為1h。
[Abstract]:Objective to establish a oxidative stress model induced by hydrogen peroxide (H_2O_2) in human ovarian granulosa cells. Methods the expression of Caspase-3 in human ovarian granulosa cells treated with different concentrations of H_2O_2 was detected by Western blot, the apoptosis was detected by flow cytometry and DAPI/TUNEL double staining, the level of intracellular active oxygen group (ROS) was detected by H2DCF-DA, and the optimum action conditions of H_2O_2 were determined by dose-effect relationship and time-effect relationship. Results 1 the expression of Caspase-3 in COV434 cells increased most significantly after treatment with H_2O_2 at the concentration of 1.5 mmol/L for 2 h. The apoptosis rate increased with the increase of H_2O_2 concentration and the prolongation of treatment time. The intracellular ROS level increased with the increase of H_2O_2 concentration. 1.5 mmol/L H_2O_2 treatment for 1 hour increased the intracellular ROS level most significantly. Conclusion the most suitable concentration of H_2O_2 induced oxidative stress model of human ovarian granulosa cells is 1 鈮，

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