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背景:牛磺酸是晶状体内重要的非酶系统抗氧化剂,其抗氧化效能的作用机制主要是通过其抗脂质过氧化反应,保护晶状体免受氧化损伤。目的:实验拟进一步观察体外条件下,外源性牛磺酸对过氧化氢所诱发的晶状体上皮细胞凋亡的影响。设计:随机对照动物实验。单位:青岛大学医学院附属医院眼科。材料:实验选用成年新西兰标准兔75只,雌雄不拘,体质量1.5~2.5kg,由青岛市实验动物中心提供。实验所用的原位细胞凋亡检测试剂盒由美国Sigma公司生产,牛磺酸和过氧化氢由上海光达化学试剂厂生产。方法:实验于2005-05/2007-06在青岛大学医学院附属医院眼科和中心实验室完成。取兔晶状体,并随机分为3组:对照组,无血清无酚红的MEM培养基培养;氧化损伤组,含1mmol/L过氧化氢溶液的无血清无酚红的MEM培养基培养,每6h再加入62μL过氧化氢溶液(30g/L);牛磺酸组,用含1mmol/L过氧化氢溶液和10g/L牛磺酸的无血清无酚红的MEM培养基培养。实验中对动物的处置符合动物伦理学标准。主要观察指标:观察培养6,12,24,48和72h后晶状体混浊情况;采用DNA缺口末端原位法及DNA片段分析法检测各组晶状体上皮细胞凋亡情况。结果:①晶状体混浊情况:氧化损伤组随氧化损伤作用时间延长,晶状体混浊度逐渐加重,而牛磺酸组晶状体混浊情况明显轻于氧化损伤组。②晶状体上皮细胞凋亡情况:对照组在培养的72h内一直未见有凋亡细胞。而氧化损伤组随氧化损伤作用时间延长,凋亡细胞逐渐增多,72h时全为凋亡细胞。自培养24h开始,牛磺酸组开始有少量凋亡细胞出现,随培养时间延长,凋亡细胞逐渐增多,72h时接近30%。各培养时间点牛磺酸组凋亡率都明显低于氧化损伤组,差异有显著性意义(q=8.6845,P<0.01),牛磺酸组和对照组比较,差异无显著性意义(P>0.05)。③DNA片段分析结果:培养24,36,48,72h氧化损伤组晶状体上皮细胞DNA琼脂糖凝胶电泳可见凋亡细胞典型的梯状条带;培养24h时对照组和牛磺酸组则不出现此变化而仅呈现正常细胞条带。结论:牛磺酸可抑制氧化损伤诱导的兔晶状体上皮细胞凋亡,减轻晶状体混浊。
BACKGROUND: Taurine is an important non-enzymatic antioxidant in the lens. Its anti-oxidant effect is mainly due to its anti-lipid peroxidation, which protects the lens from oxidative damage. OBJECTIVE: To further observe the effect of exogenous taurine on the apoptosis of lens epithelial cells induced by hydrogen peroxide in vitro. Design: Randomized controlled animal experiments. Unit: Affiliated Hospital of Qingdao University Medical College. MATERIALS: Seventy-five adult New Zealand rabbits were selected for testing, both male and female, with a body mass of 1.5-2.5 kg provided by Qingdao Experimental Animal Center. The in situ apoptosis assay kit used in the experiment was produced by Sigma Company in the USA. Taurine and hydrogen peroxide were produced by Shanghai Guangda Chemical Reagent Factory. Methods: The experiment was performed in the ophthalmology and central laboratory of Affiliated Hospital of Qingdao University from May 2005 to June 2007. The rabbits were randomly divided into 3 groups: control group, serum-free phenol red-free MEM culture medium, oxidative injury group, serum-free phenol red-free MEM culture medium containing 1 mmol / L hydrogen peroxide solution, After 6h, 62μL of hydrogen peroxide solution (30g / L) was added. Taurine group was cultured in serum-free phenol red-free MEM containing 1mmol / L hydrogen peroxide solution and 10g / L taurine. The disposal of animals in the experiment is in line with animal ethical standards. MAIN OUTCOME MEASURES: The opacification of lens was observed after 6, 12, 24, 48 and 72 hours. The apoptosis of lens epithelial cells in each group was detected by DNA nick end in situ and DNA fragment analysis. Results: (1) The opacity of lens: The oxidative damage was prolonged with the time of oxidative damage, and the degree of lens opacity increased gradually. However, the opacity of lens in taurine group was lighter than that of oxidative damage group. ② Apoptosis of lens epithelial cells: There was no apoptotic cells in the control group within 72 hours of culture. The oxidative injury group with the role of oxidative damage, the apoptotic cells gradually increased, all apoptotic cells 72h. Apoptotic cells began to appear in the taurine group starting from the beginning of culturing 24h. With the prolongation of culture time, apoptotic cells gradually increased, reaching nearly 30% at 72h. The apoptotic rate of taurine group was significantly lower than that of oxidative damage group at each culture time point (q = 8.6845, P <0.01), and there was no significant difference between taurine group and control group (P > 0.05). ③ DNA fragment analysis results: cultured 24,36,48,72 h oxidative damage lens epithelial cells DNA agarose gel electrophoresis typical apoptotic cells visible ladder striae; cultured 24h when the control group and the taurine group does not appear this change Only normal cell bands are present. Conclusion: Taurine can inhibit the apoptosis of rabbit lens epithelial cells induced by oxidative damage and reduce the lens opacity.