【作者】 严京；

【导师】 祁明信； 黄秀榕；

【作者基本信息】 福建中医学院 ， 中西医结合基础， 2004， 硕士

【摘要】 目的 本研究旨在探讨细胞凋亡抑制剂阿魏酸钠、五味予乙素、菊花、车前子对氧化损伤的晶状体上皮细胞内Ca²⁺、环磷酸腺苷（cAMP）、环磷酸鸟苷（cGMP）的影响，从细胞信号转导角度揭示天然药物防治白内障的细胞和分子学机制。 方法 1．采用牛晶状体上皮细胞（LEC）进行原代和传代培养，取第三代细胞进行实验。 2．以含有过氧化氢（H₂O₂）的培养液孵育晶状体上皮细胞复制氧化损伤模型。以四种药物与损伤的LEC共同孵育，观察药物的剂量效应与时间效应。 3．采用四甲基偶氮唑兰（MTT）比色测定法观察在不同时间和浓度条件下LEC活性变化。 4．采用荧光分光光度计测定不同时间细胞内钙离子浓度([Ca²⁺]_i)。 5．采用放射免疫分析法测定不同时间LEC内cAMP、cGMP的含量。 结果 1．采用MTT比色法观察：H₂O₂组在不同时间可引起LEC吸光度值（A）明显下降，与空白对照组比较差异有非常显著意义（P＜0.01）。四种细胞凋亡抑制剂均明显增强LEC活性，同一时间点时A值的升高与浓度的增加呈依赖关系；同一作用浓度而时间不同时，A值随着时间的延长而增加。 2．采用荧光分光光度计测定[Ca²⁺]_i显示：氧化损伤的LEC[Ca²⁺]_i升高，与空白对照组比较差异有非常显著意义（P＜0.01）。不同时间药物组均可以降低由H₂O₂引起的细胞[Ca²⁺]_i的升高，并且表现出明显的时间依赖性。 3．放射免疫分析法测定cAMP的含量：不同时间H₂O₂组与空白对照组比较，LEC内cAMP浓度明显升高差异有非常显著意义（P＜0.01），而四种药物组与H₂O₂组相比较，cAMP浓度明显降低差异有非常显著意义（P＜0.0），并且随时间延长而降低。 4．放射免疫分析法测定cGMP的含量：不同时间H₂O₂组与空白对照组比较，LEC内cGMP浓度明显降低差异有非常显著意义（P＜0.01），而四种药物组与H₂O₂组相比细胞凋亡抑制剂对氧化损伤的品状体_!几皮细胞的信号转导作用较，cGMP浓度明显升高差异有非常显著意义（P<0.01），并且随时间延长升高。结论1.过氧化氢可造成LEc活性下降，使细胞[Ca勺*升高，。AMP水平上调，cGMP 水平下降。2.阿魏酸钠、五味子乙素、菊花、车前子可使HZOZ作用的LEC活性增强，细胞 [eaZ午]。降低。3.阿魏酸钠、五味子乙素、菊花、车前子可使HZO:作用的LEC内cAMP水平下 调，而使cGMP水平升高。4.阿魏酸钠、五味子乙素、菊花、车前子均可明显抑制LEC氧化损伤及凋亡。其 作用机制可能是通过钙信号系统、cAMP信号系统、。GMP信号系统及其相互 作用来调节生物学效应，从而抑制LEC凋亡，防止和延缓白内障的发生及发 展。更多还原

【Abstract】 ObjectiveTo investigate the prevention of anti-apoptosis drugs (sodium ferulate, SF; schisandrin B, Sch B; flos chrysanthemi, FC; semem planagines, SP) on lens epithelial cell (LEC) damaged by oxidative stress, and to study the changes of intracellular-free calcium, cyclic adenosine monophosphate (cAMP) and cyclic guanine monophosphate (cGMP), in order to disclose the mechanism of signal transduction in cellular and molecular level.Methods1. Bovine LEC were primarily cultured and sub-cultured, and used for experiment in generation 3.2. LEC were incubated with hydrogen peroxide (H2O2). Four different concentration drugs (SF 25 u g/ml, 50 u g/ml, 100 u g/mk Sch B 12.5 u g/ml, 25u g/ml, 50 u g/mk FC 0.2 mg/ml, 0.4 mg/ml, 0.8 mg/mk SP 2 mg/ml, 4 mg/ml, 8 mg/ml) were added into the LEC damaged by oxidative stress and incubated for 12, 24 and 36 hours respectively.3. The LEC activities were observed by 3-(4, 5-dimethy-thiazol- 2- yl)-2, 5-diphenyItetrazolium bromide(MTT) assay.4.1ntracellular-free calcium content of LEC were detected by spectrofluorophotometer using Fura-2/AM as an indicator.5. Intracellular cAMP and cGMP concentrations of LEC were determined by radioimmunoassay (RIA).Results1. The results of MTT test shown that the absorbance values(A values ) in LEC of H2O2 groups reduced obviously to compare with control group(P<0.01). The activities of LEC treated with different concentrations of four anti-apoptosisdrugs for different times increased, and showing obvious dose-dependent and time-dependent.2. The intracellular-free calcium contents of LEC in H2O2 group by spectrofluorophotometer increased significantly compared with control group (P <0.01); however, it decreased remarkably in the groups of four anti-apoptosis drugs, showing obvious time-dependent (P<0.01).3. The cAMP concentration of LEC in H2O2 group by radioimmunoassay increased significantly compared with control group (P<0.01); however, it decreased remarkably in the groups of four anti-apoptosis drugs, showing obvious time-dependent (P<0.01).4. The cGMP concentration of LEC in H2O2 group by radioimmunoassay decreased significantly compared with control group (P<0.01); however, it increased remarkably in the groups of four anti-apoptosis drugs, showing obvious time-dependent (P<0.01).Conclusion1. Hydrogen peroxide can restrain the activity of LEC, and increase the intracellular-free calcium content and cAMP level but decrease cGMP level.2. Four anti-apoptosis drugs (SF, Sch B, FC, SP,) can prevent activity of LEC from oxidative damage, decrease the intracellular-free calcium content.3. Four anti-apoptosis drugs (SF, Sch B, FC, SP,) down-regulate cAMP level and up-regulate cGMP level4. Four anti-apoptosis drugs protect LEC from oxidative damages and inhibit apoptosis of LEC. It is possible that the mechanisms of preventing and delaying cataract formation by four anti-apoptosis drugs may be the cross-talk among calcium signal transduction system, cAMP signal transduction system and cGMP signal transduction system.更多还原