【摘要】 目的:研究原花青素(procyanidins,PC)对紫外线诱导的人晶状体上皮细胞(len epithelial cells,LECs)DNA氧化损伤的保护作用。方法:采用照射剂量为15mJ/cm2的中波紫外线(UVB)分别照射用0(对照组),0.05,0.1,0.2mg/mL的PC,牛磺酸(TAU),维生素C(VC)预处理的LECs,未处理组未给予紫外线和任何抗氧化剂处理,用单细胞凝胶电泳法(SCGE)检测分析各PC组LECs DNA单链断裂的程度,并与其他各组的检测结果相比较。结果:各PC组的尾长、尾部DNA百分比、尾力矩在数值上均明显小于对照组,且与对照组相比,差异均有统计学意义(P<0.01)。各PC实验组的尾部DNA百分比、尾力矩与未处理组相比差异均无统计学意义(P>0.05)。相同浓度下,PC,TAU和VC组的各检测指标数值逐渐增大。结论:外源性PC对UVB照射诱导损伤的人LECs DNA有保护作用。PC的保护作用明显强于TAU和VC,并且在一定浓度范围内随着浓度的增加其抗氧化作用逐渐增强。
【关键词】 原花青素;紫外线;晶状体上皮细胞;DNA损伤
Study on protection of procyanidins against UVinduced oxidative damage of lens epithelial cells
Pan Yu,XinLing Wang,QiChang Yan
Foundation items: Liaoning Province Natural Science Foundation of China (No.20092101); Key Laboratory Foundation of Liaoning Provincial Department of Education, China (No.2009S111, LS2010177)
Department of Ophthalmology, the Fourth Affiliated Hospital of China Medical University, Eye Hospital of China Medical University, the Key Laboratory of Lens of Liaoning Province, Shenyang 110005, Liaoning Province, China
AbstractAIM: To determine the protection of procyanidins(PC) against UVBinduced oxidative damage of lens epithelial cells (LECs) with alkaline single cell gel electrophoresis, in order to provide an experimental foundation for cataract clinical treatment.METHODS: LECs lines of generation 4 were divided randomly into 5 groups. As untreated group: normal cultured LECs; control group: LECs + UVB; three treated groups: PC treated groups: LECs + PC + UVB; taurine (TAU) treated groups: LECs + TAU + UVB; VC treated groups: LECs+VC+UVB. All groups of antioxidants were divided into 3 groups according to the final concentration from low to high (0.05, 0.1, 0.2mg/mL). Each treated group was treated by antioxidants 2 hours before irradiated under UVB, respectively, then conventional cultivation. The LECs of treated groups and control group were all irradiated at UVBdose 15mJ/cm2. All of LECs in every group were collected after treatment an hour respectively and analyzed with alkaline single cell gel electrophoresis.RESULTS: The differences of tail length, percentage of tail DNA and tail moment between PC treated groups and control group were statistically significant (P<0.01). The differences of percentage of tail DNA, tail moment in each PC treated group and untreated group were not significant (P>0.05). The results of PC treated groups were significantly smaller than those of TAU treated groups and VC treated groups, The differences of tail length, percentage of tail DNA and tail moment in various antioxidant treated groups were statistically significant (P<0.01).CONCULSION: Exogenetic PC has protective effect to UVBinduced LECs DNA damage. The protective effect of PC is obviously stronger than those of TAU and VC, and in a certain range of concentration, the protective effect of PC has the positive relationship of dose effect.
KEYWORDS: procyanidins; ultraviolet rays; len epithelial cells;DNA damage
0引言
原花青素(procyanidins,PC)是植物中广泛存在的天然抗氧化物质,具有很强的抗氧化活性,能够清除自由基
图1 倒置荧光显微镜下细胞形态学观察 A:未处理组;B:对照组;C:PC组;D:TAU组;E:VC组。
以及抑制脂质过氧化反应,是低毒高效热门的抗氧化剂。自从氧化损伤被认为是一个包括白内障在内的多种年龄相关疾病的共同启动因子,用化学方法延缓白内障的发生、发展就显得非常有价值[1]。紫外线可诱发白内障已得到广泛的流行病学证实[2,3]。晶状体上皮细胞(lens epithelial cells,LECs)是晶状体代谢最活跃的部位,可能是白内障发生的最初位置[2],而DNA是LECs受紫外线损伤最薄弱的靶子之一[4],因此,寻求高效的抗氧化剂拮抗紫外线对LECs DNA的损伤将成为预防白内障的重要手段之一。在到达眼部的紫外线中,UVB被认为生物损伤性最强,可以独立致眼部白内障[5]。我们采用UVB复制的人LECs损伤模型,采用单细胞凝胶电泳法(single cell gel electrophoresis,SCGE)研究原花青素对紫外线所致LECs DNA氧化损伤的直接保护作用,以期为白内障的临床药物治疗提供新的途径。
1材料和方法
1.1材料 细胞:人晶状体上皮细胞系SRA01/04(购自中国科学院肿瘤研究所)。主要试剂和仪器:MEM培养液、胰蛋白酶、非必需氨基酸、胎牛血清(美国Hyclone公司)、双抗(青霉素、链霉素)、正常熔点琼脂糖凝胶、低熔点琼脂糖凝胶(美国sigma公司)、溴化乙锭(Ethidium bromide,EB)、PC、牛磺酸、VC(成都生物制剂公司)、Olympus倒置荧光显微镜(日本Olympus公司BX51型)、UVB段紫外灯(美国SP公司xx15N/F型)、电泳仪(Tanon公司HE120型)。
1.2方法
1.2.1细胞培养 使用含100g/L胎牛血清、非必需氨基酸、50g/L双抗的MEM完全培养液,在37℃,50mL/L CO2,90%湿度的孵箱中人LECs单层贴壁生长。
1.2.2细胞分组及抗氧化剂处理 将LECs传代培养取第4代细胞随机分为5组。未处理组:正常培养的LECs;对照组:正常培养的LECs+UVB;实验组:PC组:LECs+PC+UVB;TAU组:LECs+TAU+UVB;VC组:LECs+VC+UVB。各抗氧化剂组按终浓度从低到高(0.05,0.1,0.2mg/mL)再分为3个亚实验组。各实验组于UVB照射前2h加药,常规培养。
1.2.3紫外线照射 紫外灯照射强度为1.5mW/cm2(每次使用前都预先经紫外线仪器测量,避免由于衰减造成的强度不一致),照射时间为10s,即采用的照射剂量为15mJ/cm2。取对数生长期的细胞置于暗室中紫外光源下10cm处,照射前将培养液弃去,用PBS轻柔冲洗2次,照射时打开培养皿。照射后加入完全培养液,培养箱内继续培养1h,收集细胞,消化后制备单细胞悬液(稀释成约2×104/mL),用于检测DNA。
1.2.4碱性SCGE检测 100μL 8g/L正常熔点琼脂糖凝胶溶解后滴加在经多聚赖氨酸预处理的载玻片上,用盖玻片使胶均匀展开,在酒精灯上略过火,使正常熔点琼脂糖完全凝固于载玻片上,即第1层胶。再次滴加与第一次相同的正常熔点琼脂糖,盖玻片使胶均匀展开后4℃下固化10min,为第二层胶。取混有新鲜制备细胞悬液的低熔点琼脂糖(按1∶3混合)100μL加到第2层胶上,立即盖上盖玻片,4℃固化10min,即第3层胶。轻轻揭去盖玻片待裂解,将凝胶载玻片浸没于新配置的裂解液中(pH=10)裂解2h,除去细胞质。从裂解液中取出载玻片,用蒸馏水浸泡5min后移入水平电泳槽,4℃冰箱内避光解旋20min(1mmol/L Na2EDTA, 300mmol/L NaOH, pH=13)。电压25V,4℃冰箱内避光条件下电泳15min。然后用PBS漂洗2次,每次5min。用5μg/mL的EB避光染色10min(每张2~3滴),染色后放入避光的密闭湿盒内,24h内进行图像分析。染色后的凝胶板,置于倒置荧光显微镜下可清晰的观察到核DNA和迁移的DNA。每个处理组随机保存200个细胞,用单细胞凝胶电泳分析软件TriTek CometScore进行图像分析,计量每个细胞的尾长、尾部DNA百分比和尾力矩。每一实验条件至少重复1次。
统计学分析:数据资料以±s表示,应用SPSS 16.0统计学软件进行随机样本的单因素方差分析,P<0.05为差异有统计学意义。
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