建立应激性溃疡细胞损伤模型，观察过氧化氢对人胃腺癌细胞株AGS的浓度及时间损伤效应，并探讨miRNA-146a对其调控作用。

(1)将人胃腺癌细胞株AGS分为5组，每组4孔：其中4组在200 μmol/L过氧化氢处理3、6、12、24 h，另1组为未处理组，分别检测人胃腺癌细胞株AGS脂质过氧化代谢产物丙二醛含量和超氧化物歧化酶(SOD)活力。(2)将人胃腺癌细胞株AGS分为6组，每组4孔，其中4组分别用50、100、200、400、600 μmol/L过氧化氢处理6 h，另1组为未处理组，检测其丙二醛含量和SOD活力。(3)将人胃腺癌细胞株AGS分为2组，实验组和对照组，每组4孔，对照组不做处理，实验组用200 μmol/L过氧化氢刺激6 h后，采用实时定量PCR法分别检测两组细胞miRNANA-146a的相对表达量。(4)将人胃腺癌细胞株AGS分为4组，每组4孔：未处理组、miRNANA对照组、miRNA-146a抑制组、miRNA-146a增强组。处理组分别转染miRNA-146a对照剂、miRNA-146a抑制剂和miRNA-146a模拟物，培养24 h后，用200 μmol/L过氧化氢刺激6 h，检测细胞miRNA-146a表达、丙二醛含量和SOD活力。多组间比较采用单因素方差分析，两组间比较采用LSD-t检验。

(1)在200 μmol/L过氧化氢刺激后，未处理组、不同时间培养组组间细胞丙二醛含量、SOD活力比较，差异均有统计学意义(F＝46.744、42.736，P值均小于0.01)；随着作用时间的延长，细胞丙二醛含量逐渐升高，在培养6 h时达到峰值(2.15±0.50)μmol/mg，然后逐渐降低；随着作用时间的延长，SOD活力逐渐降低，在培养6 h时达到最低(6.51±0.54)U/mg，然后逐渐升高。(2)用不同浓度的过氧化氢处理人胃腺癌细胞株AGS 6 h后，未处理组、不同浓度处理组组间细胞丙二醛含量、SOD活力比较，差异均有统计学意义(F＝152.786、129.231，P值均小于0.01)；随着浓度的增加丙二醛含量逐渐升高，600 μmol/L处理组时最高(2.46±0.07) μmol/mg；随着浓度的增加SOD活力逐渐降低，600 μmol/L处理组时最低(3.14±0.10) U/mg。(3)实验组人胃腺癌细胞株AGS miRNA-146a表达明显量高于对照组(t＝－17.398，P<0.01)。(4)不同转染组用200 μmol/L过氧化氢刺激6 h后，miRNA-146a增强组与未处理组、miRNA-146a对照组、miRNA-146a抑制组组间细胞miRNA-146a的相对表达量、SOD活力和丙二醛含量比较，差异均有统计学意义(F＝232.643、1 070.580、56.191，P值小于0.01)；miRNA-146a增强组数百倍高于其他3个小组，差异均有统计学意义(P值均小于0.01)；miRNA-146a增强组SOD活力均较miRNA-146a抑制组和miRNA-146a对照组高，差异均有统计学意义(P值均小于0.01)；miRNA-146a增强组丙二醛水平均较miRNA-146a抑制组和miRNA－146a对照组低，差异均有统计学意义(P值均小于0.01)。

200 μmol/L过氧化氢刺激人胃腺癌细胞株AGS 6 h能较好地建立应激性胃溃疡氧化损伤细胞模型，miRNA-146a可负性调控过氧化氢所致人胃腺癌细胞株AGS应激性溃疡的氧化应激反应。

To observe the concentration and time damage resulting from hydrogen peroxide on human gastric cancer cell line AGS and to investigate the effect regulated from microRNA-146a by constructing the cell model of stress ulcer.

(1)The AGS cells were divided into 5 groups, with 4 wells in each group. The content of malondialdehyde and the vigor of superoxide dismutase(SOD) were tested which were kind of lipid peroxidation products from AGS cells that stimulus with 200 μmol/L hydrogen peroxide concentration at time points of 3, 6, 12 and 24 h. (2)The AGS cells were divided into 6 groups, with 4 wells in each group. Then the content of malondialdehyde and the vigor of SOD were tested which were kind of lipid peroxidation products from AGS cells that stimulus with 50, 100, 200, 400 and 600 μmol/L hydrogen peroxide concentration all after 6 hours. (3) The AGS cells were divided into 2 groups, with 4 wells in each group. Then the AGS cells were excited 6 hours with 200 μmol/L hydrogen peroxide applying real-time PCR to detect gene expression of miRNA-146a of AGS cells. (4) The AGS cells were divided into 4 groups, with 4 wells in each group. Cells in miRNA-146a control group were transfected with microRNA control; cells in miRNA-146a enhancement group were transfected with miRNA-146a mimics; cells in miRNA-146a inhibition group were transfected with miRNA-146a inhibitor. After cultivating for 24 hours, miRNA-146a expression, the content of malondialdehyde and the vigor of SOD of the cells wer tested that have been stimulated 6 hours by hydrogen peroxide. Statistics were chose one-factor analysis of variance between groups and LSD-t test for the two groups.

(1)After 200 μmol/L hydrogen peroxide stimulation, comparing the malondialdehyde content and SOD vitality in cells between untreated group and different time training group, the differences were statistically significant(F=46.744, 42.736, P values were less than 0.01). As the extension of the time, the malondialdehyde content of cells was gradually increased, and peaked at(2.15±0.50)μmol/mg in the culturing 6 h, then gradually reduced. Contrarily, as the extension of the time, the SOD vitality of cells was gradually reduced, and footed at(6.51±0.54)μmol/mg in the culturing 6 h, then gradually increased. (2) After 6 hours treatment of different concentration hydrogen peroxide, comparing the malondialdehyde content and SOD vitality in cells between untreated group and different time training group, the differences were statistically significant(F=152.786, 129.231, P values were less than 0.01). With the increase of concentration, malondialdehyde content gradually increased, and highest at (6.51±0.54)U/mg dealing with 600 μmol/L treatment group, while the SOD vitality gradually decreased, and lowest at (3.14±0.10) U/mg dealing with 600 μmol/L treatment group. (3)The miRNA-146a expression of AGS cells of the experimental group was significantly higher than the normal group (t=-17.398, P<0.01). (4) After 6 hours stimulation of 200 μmol/L hydrogen peroxide, comparing the relative expression of miRNA-146a, malondialdehyde content and SOD vitality in cells among miRNA-146a enhancement group, untreated group, miRNA-146a control group and miRNA-146a inhibitor group, the differences were statistically significant (F=232.643, 1 070.580, 56.191, P values were less than 0.01). The miRNA-146a expression of the miRNA-146a enhancement group was hundreds of times than others, the differences were statistically significant(P values were less than 0.01), while the SOD vitality of the miRNA-146a enhancement group was higher than the miRNA-146a control group and the miRNA-146a inhibition group, the differences were statistically significant(P values were less than 0.01), at the same time, the malondialdehyde content of the miRNA-146a enhancement group was lower than the miRNA-146a control group and the miRNA-146a inhibition group, the differences were statistically significant(P values were less than 0.01).

Applying 200 μmol/L hydrogen peroxide to deal with AGS cells for 6 hours can better construct the cell model of stress ulcer. miRNA-146a can negatively regulate oxidative stress reaction of the AGS cells stress ulcer resulting from hydrogen peroxide.