维生素C抑制晶状体上皮细胞凋亡(英文)

Inhibiting apoptosis of lens epithelial cells by vitamin C

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： Oxidation injury can lead to the apoptosis of lens epithelial cells. Some researches have found that body supplement of vitamin C is beneficial to inhibit oxidation injury by increasing the level of vitamin C in aqueous humor. OBJECTIVE： To observe the inhibitive effect of vitamin C on H2O2 induced apoptosis of lens epithelial cell. DESIGN： Randomized controlled trial taking animal＇s lens as subject. SETTING： Department of Ophthalmology, Medical College of Qingdao University. MATERIALS： 120 adult New Zealand rabbits of both genders, weighing 3-5 kg, were selected. Vitamin C and 300 g/L H2O2 were purchased from Shanghai Guangda Chemical Reagent Factory. METHODS： The experiment was carried out in the Central Laboratory, the Affiliated Hospital of Medical College of Qingdao University from January 2002 to January 2004. ①Culture of lens： 240 lenses were isolated from 120 rabbits after being killed. Among them, 192 clear lenses were selected and divided randomly to three groups with 64 lenses in each group： control group, H2O2 group and H2O2＋vitamin C group. Lenses in the control group were incubated in non-serum and non- phenolsulfonphthalein MEM medium, those in the H202 group incubated in nonserum and non- phenolsulfonphthalein MEM medium containing 1 mmol/L H2O2 （62μL of 30 g/L H2O2 was added into the medium every 6 hours to keep H2O2 level maintain 1 mmol/L in the medium）, and those in the H2O2＋vitamin C group incubated in non-serum and non- phenolsulfonphthalein MEM medium containing 1 mmol/L H2O2 and 1 mmol/L vitamin C. Under the same culture condition, the lenses in each group were detected at hours 6, 12, 18, 24, 36, 48, 72 ＂and 108 after culture, respectively.②Measurement of the opacity of lens： Under the white background, two black lines 0.5 mm in width, which were mutually vertical with an interval of 10 mm. The lenses were placed above the crossing to measure the opacity. ③Detecting the apoptosis of lens epithelial cells： The apoptosis of lens epithelial cells was examined using TUNEL method and DNA fragmentation assay under light microscope to calculate the apoptotic rate of epithelial ceils. MAIN OUTCOME MEASURES： ①Opacity of the lenses in each group; ②apoptotic rate of lens epithelial cells in each group. RESULTS： ①After 108 hours of culture, the opacity of the lenses in the H2O2＋vitamin C group was obviously lower than that in the H2O2 group. ② The apoptotic rates in the control group were lower that those in the H2O2 group at different time point and those in the H2O2＋vitamin C group at hours 24, 48 and 72 after culture（P 〈 0.05-0.01）. The apoptotic rates in the H2O2＋vitamin C group were significantly lower than those in the H2O2 group at different time points after culture （P 〈 0.01）. ③The findings of DNA fragment analysis showed that the DNA ＂ladder＂ was found in the H2O2 group during 24-72 hours, while no DNA ＂ladder＂ but only normal electrophoresis strap were present in the other two groups 24 hours after culture. CONCLUSION： Lens can take refuge from the oxidative injury of H202 with the addition of vitamin C. As a result, the apeptosis rate of lens epithelial cells and further, cataract formation can be restrained.