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背景:氧化损伤可诱发晶状体上皮细胞凋亡。研究已经发现,全身补充维生素C,可以使房水维生素C浓度随之增高,以避免上述氧化损伤发生。目的:观察维生素C对氧化损伤所诱发的晶状体上皮细胞凋亡的抑制作用。设计:以动物晶状体为观察对象,随机对照实验。单位:青岛大学医学院附属医院眼科。材料:选用成年新西兰标准兔120只,雌雄不拘,体质量3～5kg。维生素C及300g/L过氧化氢(上海光达化工试剂厂)。方法:实验于2002-01/2004-01在青岛大学医学院附属医院中心实验室完成。①晶状体培养:处死动物,取晶状体240只,培养后,将其中透明晶状体192只按随机抽签法分为3组,每组晶状体64只。对照组采用无血清无酚红的MEM培养基。过氧化氢组采用含1mmol/L过氧化氢的无血清无酚红的MEM培养基;每6h另加30g/L过氧化氢62μL,调整浓度至1mmol/L。过氧化氢+维生素C组采用含1mmol/L过氧化氢和1mmol/L维生素C的无血清无酚红的MEM培养基。每组在同等培养条件下,在培养6,12,18,24,36,48,72和108h8个时间点进行观察。②晶状体混浊情况观察方法:在白色背景下设计一宽0.5mm,间距10mm的垂直交叉的黑色线条,将被检晶状体置于“+”字交叉的黑色线条之上,观察透过晶状体所能看到的清晰度。③晶状体上皮细胞凋亡情况检测:用原位末端标记法及DNA片段分析检测各组各时间点晶状体上皮细胞凋亡情况。光镜下观察原位末端标记法染色标本,记录各组晶状体上皮细胞凋亡的发生率。计算凋亡细胞所占百分比即为上皮细胞凋亡率。主要观察指标:①各组晶状体混浊情况。②各组晶状体上皮细胞凋亡情况。结果:①晶状体混浊情况:培养108h后过氧化氢+维生素C组晶状体混浊情况明显轻于过氧化氢组。②晶状体上皮细胞凋亡情况:过氧化氢组各培养时间点和过氧化氢+维生素C组培养24,48,72h后晶体上皮细胞凋亡率明显高于对照组(P<0.05～0.01);过氧化氢+维生素C组各培养时间点晶体上皮细胞凋亡率明显低于过氧化氢组(P<0.01)。③DNA片段分析结果:过氧化氢组24,36,48及72h各时限均呈现凋亡细胞典型梯状条带,而培养24h的对照组和维生素C组均未出现此变化而仅呈现正常电泳条带。结论:维生素C可抑制过氧化氢氧化损伤所诱导的晶状体上皮细胞凋亡和白内障形成。
Background: Oxidative damage induces lens epithelial cell apoptosis. Studies have found that systemic vitamin C supplementation can increase the concentration of aqueous vitamin C in the aqueous humor to prevent the above-mentioned oxidative damage. Objective: To observe the inhibitory effect of vitamin C on the apoptosis of lens epithelial cells induced by oxidative damage. Design: The animal lens for the observation of randomized controlled trials. Unit: Affiliated Hospital of Qingdao University Medical College. MATERIALS: 120 adult New Zealand standard rabbits were chosen, both male and female, body weight 3 ~ 5kg. Vitamin C and 300g / L hydrogen peroxide (Shanghai Guangda Chemical Reagent Factory). Methods: The experiment was performed at the Central Laboratory of Affiliated Hospital of Qingdao University from January 2002 to January 2004. ① lens culture: animals were sacrificed, the lens 240, after culture, the transparent lens 192 of which were randomly selected by lottery method divided into three groups, each group of 64 lenses. The control group using serum-free phenol red-free MEM medium. Hydrogen peroxide group with serum-free phenol red-free MEM medium containing 1mmol / L hydrogen peroxide; every 6h plus 62g of 30g / L hydrogen peroxide, adjust the concentration to 1mmol / L. Hydrogen peroxide + vitamin C group using serum-free phenol red-free MEM medium containing 1mmol / L hydrogen peroxide and 1mmol / L vitamin C. Each group under the same culture conditions, cultured at 6, 12, 18, 24, 36, 48, 72 and 108h 8 time points were observed. Â ’¢ lens opacity observation method: in a white background design a width of 0.5mm, 10mm vertical spacing of the black lines intersect, will be inspected lens placed in the “+” cross on the black lines above, can be seen through the lens can see To the clarity. (3) Detection of lens epithelial cell apoptosis: The apoptosis of lens epithelial cells in each group at each time point was detected by in situ terminal labeling and DNA fragment analysis. The specimens were stained by light microscopy in situ and the incidence of apoptosis of lens epithelial cells in each group was recorded. Calculated the percentage of apoptotic cells is the rate of apoptosis of epithelial cells. MAIN OUTCOME MEASURES: ① The opacity of lens in each group. ② the incidence of lens epithelial cell apoptosis in each group. Results: ① Lens opacity: After 108 hours of culture, the opacity of lens in hydrogen peroxide + vitamin C group was obviously lighter than that in hydrogen peroxide group. ② Apoptosis of lens epithelial cells: The apoptotic rate of lens epithelial cells after 24, 48 and 72h incubation in H2O2 group and hydrogen peroxide + vitamin C group was significantly higher than that in control group (P <0.05 ~ 0.01). Hydrogen peroxide + vitamin C group cultured at each time point apoptosis rate of lens epithelial cells was significantly lower than the hydrogen peroxide group (P <0.01). ③ DNA fragment analysis results: 24, 36, 48 and 72h hydrogen peroxide group showed a typical apoptotic cell ladder, while the control group and the vitamin C group cultured 24h did not appear this change but only showed normal electrophoresis strips band. Conclusion: Vitamin C can inhibit lens epithelial cell apoptosis and cataract formation induced by hydrogen peroxide oxidative damage.