阿司匹林预处理对原代培养大鼠Ⅱ型肺泡上皮细胞抗氧化损伤的影响研究

目的 观察阿司匹林对原代培养大鼠Ⅱ型肺泡上皮细胞(AEC Ⅱ)的保护效应,并探讨其抗氧化损伤的机制.方法 将原代分离、纯化、培养的离体大鼠AEC Ⅱ分为5组.过氧化氢损伤(H2O2)组在培养40 h后加入0.5 mmol/L H2O2,建立细胞氧化损伤模型;生理盐水(NS)组则加入NS ;阿司匹林预处理1、2、3(A1～3)组在加入H2O2前给予阿司匹林50、100、200μmol/L预处理.3 h后观察细胞形态学变化和贴壁细胞计数;采用四甲基偶氮唑盐(MTT)比色法检测细胞存活率;采用免疫组化法和聚合酶链反应法检测NS组、A1～3组在培养20、40、60 h AEC Ⅱ中血红素氧合酶-1(HO-1)的蛋白及mRNA表达.结果 采用胰蛋白酶消化、免疫黏附法每只鼠可收获(2.0～2.5)×107个AEC Ⅱ,纯度和活性均＞90%.与NS组比较,H2O2组细胞间隙增宽,贴壁细胞数减少,细胞皱缩,细胞存活率(A值)明显下降(0.054 6±0.004 0比0.103 8±0.009 9,P＜0.01);与H2O2组比较,A1～3组贴壁细胞数增多,细胞形态较完整,无明显皱缩,细胞存活率(A值)明显增加(0.066 9±0.003 9、0.071 0±0.006 5、0.078 7±0.009 2比0.054 6±0.004 0,均P＜0.01).与NS组比较,A1～3组培养20、40、60 h时HO-1的蛋白及mRNA表达均明显增加,60 h时达峰值蛋白(积分A值):1.59±0.12、1.60±0.09、1.61±0.08比1.25±0.11;mRNA(Ct值比值):24.31±1.74、30.45±2.53、32.63±3.74比22.99±1.95,均P＜0.05];但A1～3组间HO-1蛋白表达无明显差异.结论 阿司匹林通过上调HO-1表达对离体培养氧化损伤的大鼠AEC Ⅱ起保护效应;HO-1可能是其中重要的保护调节因子.

Abstract：

Objective To investigate the protective effect of aspirin on primary cultured type Ⅱ alveolar epithelial cell (AEC Ⅱ ), and the mechanism of its effect on anti-oxidation damage. Methods The original generation of adult rat AEC Ⅲ were cultured and purified. They were divided into normal saline (NS) group,hydrogen peroxide injury group (H2O2 group), and 1, 2, 3 aspirin pretreatment groups (Al -3 groups). In H2O2 group, 0. 5 mmol/L H2O2 was added to AEC Ⅱ after 40 hours of culture to reproduce a cell oxidative injury model. In NS group, only NS was added to AEC Ⅱ culture. To the A1 - 3 groups aspirin 50, 100 and 200 μmol/L were added respectively. Cell form, cell count and cell survival rate were observed at 3 hours after H2O2 was given. Immunohistochemical and polymerase chain reaction (PCR) methods were used for the determination of heme oxygenase-1 (HO-1) protein and HO-1 mRNA (20, 40, 60 hours of culture). Results With trypsin digestion and immune adherence method AEC Ⅱ could be harvested (2.0- 2. 5)× 107, and the purity and activity were both over 90%. Compared with NS group, gaps between cells were widened in H2O2 group, cell account was reduced, and the survival rate (A value) was reduced significantly (0. 054 6±0. 004 0 vs. 0. 103 8±0. 009 9, P＜0. 01). Compared with H2O2 group, in A1 - 3 groups the number of adherent cells was increased, cell morphology was intact, and no obvious cell shrinkage was found. Higher survival rate (A value) was found in A1 - 3 groups than that of H2O2 group (0. 066 9±0. 003 9, 0. 071 0±0. 006 5,0. 078 7±0. 009 2 vs. 0. 054 6±0. 004 0, all P＜0. 01). Compared with NS group, HO-1 protein and HO-1 mRNA expression in AEC Ⅱ after 20, 40 and 60 hours of culture reached peak level at 60 hours, and they were increased significantly in A1 - 3 groups protein (A value) : 1.59±0. 12, 1.60±0. 09, 1.61±0. 08 vs.1.25±0. 11; mRNA (the ratio of Ct value: 24.31±1.74, 30. 45±2. 53, 32. 63±3. 74 vs. 22.99±1.95, all P＜0. 05]. There was no significant difference in HO-1 protein expression among A1 - 3 groups. Conclusion There are significant protective effects of aspirin against anti-oxidative damage in cultured AEC Ⅱ cell.As expression of HO-1 is increased in aspirin groups, it may be considered as a protective factor agninst anti-oxidative damage in AEC Ⅱ cell culture.