摘要目的 观察过氧化氢酶、N-乙酰-L-半胱氨酸(NAC)、二碘基苯(DPI)、AACOCF3四种氧化还原信号转导抑制剂对人晶状体上皮细胞(HLEC)SRA 01/04细胞增殖的作用.方法 为实验研究.取5～10代SRA 01/04细胞逐步降低血清浓度,在无血清培养液中培养2 h,分别用过氧化氢酶、NAC、DPI、AACOCF3预处理细胞30 min后,给予EGF 20μg/L或bFGF 20μs/L,48 h后检测3H-胸腺嘧啶脱氧核苷掺入量,计数活细胞数.在短期实验中,抑制剂只作用于细胞30 min;长期实验中,抑制剂共作用48.5 h.采用单因素方差分析对不同浓度EGF、bFGF对细胞增殖作用的样本均数进行统计.抑制剂对细胞增殖作用的实验设计属于非平衡多因素组合设计,采用拆分组别法进行统计分析.其中对照组与生长因子组间比较采用独立样本t检验,生长因子组与不同抑制剂组间比较采用单因素方差分析,不同抑制剂的不同浓度间比较采用嵌套设计定量资料方差分析.结果 作用30 min(短期实验)和作用48.5 h(长期实验),四种抑制剂都能显著抑制EGF或bFGF引起的细胞增殖.在EGF刺激下的短期实验中,105U/L过氧化氢酶,0.5 mmol/L NAC,0.1 izmoL/L DPI或0.5μmol/LAACOCF3预处理细胞30 min可以使EGF引起的细胞DNA合成分别减少18.0%(t=6.132,P<0.01),24.6%(t=6.188,P<0.01),28.5%(t=6.386,P<0.01)和16.4%(t=3.705,P=0.001).在EGF刺激下的长期实验中,能抑制细胞增殖的最低浓度低于短期实验需要的最低浓度,分别为5×104U/L过氧化氢酶,0.2 mmol/L NAC,0.01 μmol/L DPI和0.1μmol/L AACOCF3.短期实验及长期实验中抑制剂的作用强度均有剂量依赖性.相同浓度下,长期实验的抑制效果强于短期实验.在bFGF刺激下的短期实验及长期实验中得到了相似的结果.结论 氧化还原信号转导对HLEC SRA01/04增殖有重要的调控作用,阻断活性氧的产生或清除细胞内的活性氧能显著抑制LEC增殖.

abstractsObjective Physiological level of reactive oxygen species (ROS) has been shown to play an important role in mitogen-stimulated cell signaling in many cell types. Both EGF and bFGF can induce ROS generation in human lens epithelial cells. But the role of ROS and Redox signaling on EGF and bFGF-stimulated cell proliferation is not clear. This study was to investigate the control of EGF and bFGF-induced cell proliferation by Redox signaling in human lens epithelial cells (SRA 01/04), using specific inhibitors to Redox signaling. Methods EGF and bFGF-induced cell proliferation was measured by [methyl-<'3>H]thymidine incorporation assay. In some experiments, cell proliferation was also measured by typan blue negative cell counting parallel with <'3>H-thymidine incorporation assay. The inhibitors used in this study include: catalase (specific enzyme to detoxify hydrogen peroxide ), N-acetyl-L-cysteine (free radical scavenger), DPI (inhibitor for NADPH oxidase ) and AACOCF3 (specific inhibitor for cytosolic phosphohpase A2, which had been shown to play important role in ROS generation in our previous study). Serum starved SRA 01/O4 cells were pretreated with these inhibitors for 30 minutes before exposure to EGF or bFGF (20 μg/L). In short term study, all these inhibitors were removed before adding growth factor,while in long term study, inhibitars ware maintained in the medium along with growth factor. Cells were kept growing in the medium with 20 μg/L EGF or 20 μg/L bFGF for 48 hours. Then cell proliferation was quantified by [ methyl-<'3>H ] thymidine incorporation assay or by cell counting. Results We found that catalase, NAC, DPI and AACOCF3 were able to suppress EGF and bFGF-induced cell proliferation in both short term and long term study. In EGF study, 20 μg/L EGF produced about 26% (t =7.093,P <0.01 )increase in DNA synthesis after 48 hours. Pretreatment of the cells for 30 minutes with 1×10<'5> U/L catalase,0. 5 mmol/L NAC, 0.1 μmol/L DPI or 0.5 μmol/L AACOCF3 inhibited EGF-stimulated DNA synthesis by 18.0% (t =6.132,P<0.01), 24.6% (t =6.188,P<0.01), 28.5%(t=6.386,P<0.01) and 16.4% (t = 3.705, P = 0.001 ) respectively. The inhibition was dose-dependent and was proved by typan-blue negative cell counting. If the cells were treated with inhibitors for 48.5 hours ( long term study), the lowest concentrations to inhibit cell proliferation were much lower than those used in short term study. Treatment of the cells with 0.5 × 10<'5> U/L catalase, 0.2 mmol/L NAC, 0.01 μmol/L DPI and 0.1 μmol/L AACOCF3 led to suppression on DNA synthesis significantly. Similar results were detected in bFGF study. 48 hours treatment with 20 μg/L bFGF induced about 28.8% (t =9. 523 ,P <0. 01 ) increase in cell proliferation. If the cells were pretreated with 1 × 10<'5> U/L catalase, 0. 5 mmol/L NAC, 0. 1μmol/L DPI or 0.5 μmol/L AACOCF3 for 30 minutes, bFGF-stimulated cell proliferation was suppressed by 24.5% (t = 6.697,P <0.01), 22.2% (t =6.693,P<0.01), 23.9% (t =6.661,P <0.01) and 30.5% (t =8.959,P <0.01)respectively. If cells were treated with inhibitors for 48. 5 hours, the lowest concentration of catalase, NAC,DPI and AACOCF3 to inhibit cell proliferation significantly was 0.5 × 10<'5> U/L(t =21.641 ,P <0.01 ), 0.2mmol/L(t = 11.218,P <0.01), 0.01 μmol/L(t =4.570,P <0.01 ) and 0. 1 μmol/L(t =5.426,P <0.01 ) respectively, lower than those used in short term study. Conclusions We conclude that mitogenic stimulus function of EGF and bFGF in human lens epithelial cells appears to be mediated via ROS to activate cell proliferation. Inhibition of Redox signaling, either by removal of ROS ( the role of catalase and NAC) or blocking ROS generation ( the role of DPI and AACOCF3 ), eradicate EGF and bFGF-stimulated cell proliferation. It is proposed that Redox signaling may play an important role in cell proliferation in human lens epithelial cells.