【作者】 吴志鸿；

【导师】 张劲松；

【作者基本信息】 中国医科大学 ， 眼科学， 2005， 博士

【摘要】 目的 流行病学证据表明紫外线辐射是白内障形成的环境因素之一,长期慢性的紫外线辐射被认为是老年性白内障形成的主要原因,但是,有关紫外线辐射导致白内障的机制还不太清楚。自然光中的紫外线来自太阳光辐射。紫外线对晶状体的影响是长期的,慢性蓄积的氧化损伤过程,透明晶状体存在的防御系统虽然有足够的保护作用,但当积累的效应超过一个临界水平时,不可逆转的损伤就发生了。紫外线对晶状体的损伤机制为:(1)直接光化学损伤。(2)光氧化损伤。光的辐射能导致哺乳动物细胞DNA的损伤,损伤的程度是由波长和细胞的外在环境决定的。UV-C(100～290)射线无生态相关性,因为它能在数量上被地球大气层的氧和臭氧所吸收。而长波UV-B(290～320)和UV-A(320～400)射线可以明显作用于生物。在相对早期与年龄增加有关的氧化损伤,晶状体上皮细胞是一个很好的模型。氧化损伤显示与DNA损伤有关。在光诱导的氧化损伤中,代偿增多的抗氧化系统,如谷光苷肽、过氧化氢酶和超氧化物歧化酶的释放导致大分子化合物的损伤(DNA,晶状体球蛋白)。光的辐射能诱导多种DNA修复酶的产生来维持晶状体上皮细胞的长期功能。抗氧化防御系统能导致晶状体上皮细胞DNA的额外损伤。最丰富的损伤产物是DNA单链断裂,而碱性彗星法对检测DNA单链断裂是敏感的。普遍认为紫外线辐射产生活性氧,包括过氧化氢和超氧离子。这些活性氧诱导真核细胞各种反应,包括DNA和蛋白质的损害,启动信号传导通路,激活基因的表达。最近研究表明晶状体上皮细胞可能是白内障发生的最初位置,而DNA是晶状体上皮细胞受紫外线损伤最薄弱的靶子之一。因此,探讨紫外线诱导的晶状体上皮细胞DNA的损伤和修复机制及端粒酶活性和氧化损伤蛋白在DNA的损伤和修复中的变化和作用,寻求高效的抗氧化剂,拮抗紫外线诱导的DNA损伤,阻止和延缓白内障的发生、进展,对白内障的防治是非常有价值的。更多还原

【Abstract】 ObjectivesEpidemiological evidence has implicated ultraviolet light ( UV) irradiation as one of the environmental factors in human cataractogenesis. Long - term chronic ultraviolet exposure is believed to be the major cause of senile cataract. But till today, the mechanism by which ultraviolet induces cataract remains unknown. UV is one kind of electromagnetic wave. UV mainly come from nature light of solar radiation. The effect of UV - irradiation on crystalline lens is long - term chronic cumulative process of oxidative stress, it is reasonable to assume that the defense system in the crystalline lens remains sufficiently protective until the cumulative effects exceed a critical level. Then irreversible damage occurs. The mechanisms of UV - irradiation on crystalline lens are direct photochemistry damage and light oxidative stress. Exposure to light in mammalian cells can lead to DNA damage, the severity of which is dependent on the wavelength and the internal or external cellular environment. UV - C ( 100 ~ 290nm) radiation is ecologically not relevant since it is quantitatively absorbed by oxygen and ozone in the Earth’s atmosphere, the longer wavelength UV - B (290 ~320nm) and UV - A (320 ~400nm) radiation can have significant effects on the biota. Lens epithelial cells provide a good model for relatively early age - related increase in oxidative stress. Stress has been shown to be associated with DNA damage. Compensatory antioxidant defenses, such as glutathione, catalase and superox-ide dismutase are released in response to light - induced oxidative stress that would result in macromolecular damage ( DNA, crystalline). Also, it is likely that several DNA repair enzymes are induced by exposure to light, in order to maintain the long - term functionality of the lens epithelial cells. Compromisedanti oxidant defenses can lead to excessive DNA damage in lens epithelial cells. The most abundant lesion produced is DNA single strand breakage, which can be sensitively detected by the alkaline microgel electrophoresis (COMET) assay. It is generally accepted that UV irradiation generates active oxygen species including hydrogen peroxide and superoxide ion. These reactive oxygen species induces various cellular responses in eukaryotes, which includes damage of DNA and proteins, triggering of signal transduction pathways, and activation of gene expression. Recent studies have suggested that the lens epithelium may be the site of initiation for cataract development. DNA is obviously one of the most sensitive target for UV - induced damage in lens epithelial cells. It is therefore important to study the mechanism of UV - induced DNA damage and repair in human lens epithelial cell and to investigate the change and role of telomerase and other stress - related proteins. Powerful antioxidants are searched to defense DNA damage UV - induced. To delay the onset and retard the progression of cataract is valuable.Methods1. Human lens epithelial cells were irradiated at UV - doses 0. 0( control group) and 2.5 >5.0 ^7. 5 N10.0 mj/cm2(treated 1 ~4group). With the alkaline comet assay, the amounts of DNA single strand breaks ( SSB) were quantified, the spontaneous repair of DNA SSB after exposure to 10. OmJ/cm2 was also determined in human lens epithelial cells. Human lens epithelial cells were irradiated at UV - doses 0.0( control group) and 2.5,5.0,10.0 mj/cm (treated 1 ~ 3group) ,the distribution of cell cycles UV - induced DNA damage and repair in lens epithelial cells was assayed by flow cytometry(FCM).2. The degree of DNA damage was examined with the alkaline comet assay. Human lens epithelial cells were treated with different concentration of Vi-taminC, taurineN Superoxide dismutase ( SOD) and epigallocatechin gallate ( EGCG) before and after ultraviolet radiation ,the effect of antioxidants on DNA damage degree was examined with alkaline comet assay.3. Human lens epithelial cells were irradiated at UV - doses 0. 0( controlgroup) and 0.5,1. 5,2. 5,3. 5,5. 0,7. 5,10.0 mj/cm2(treated 1 ~7group). telomerase activity was determined by Telomeric Repeat Amplification Protocol -Enzyme Linked Immunosorbent Assay (TRAP -ELJSA) ,P53,P21, growth arrest and DNA damage inducible ( GADD45), proliferating cell nuclear antigen (PCNA) and PI 6 protein levels were analyzed by Western blotting.Results1. The amount of DNA SSB in control group and treated 1 ~ 4group showed increased tendency. The differences of DNA SSB were significantly ( P < 0. 01). UV - induced DNA SSB at UV - doses 10. 0 mj/cm2 in human lens epithelial cells, the half repair time was 60 minute. Human lens epithelial cells UV- induced were arrested at Gj phases of the cell cycle, the percentage in control and experimental guoup of G, phases cell were 51. 5% ,52. 9% ,52. 5% and 50. 0% immediately after UV irradiation; were 43. 7% ,45. 8% ,50. 0% and 51. 1% 2h after UV irradiation; were 38. 7,43. 1% ,47. 1% and 50. 8% 4h after UV irradiation; were 29.80% ,39. 8% ,48.5% and 50.4% 8h after UV irradiation ; were 56.6% ,48.0% ,30.0% and 26.3% 24h after UV irradiation;2. Human lens epithelial cells were treated with different concentration of taurine, SOD and EGCG before ultraviolet radiation, the differences of antioxida-nts effect on DNA in control and treated group were significantly (P <0. 01). Human lens epithelial cells were treated with different concentration of VitC, taurine>,SOD and EGCG after ultraviolet radiation, the differences of antioxida-nts effects on DNA between the control and treated group were significantly (P<0.01). The differences of effect on DNA in four kinds of antioxidants before UV irradiation were significantly (P <0.01); The differences of effect on DNA in four kinds of antioxidants after UV irradiation were not significantly (P >0. 05).3. Telomerase activity in control group and treated 1 ~ 7 group showed increased tendency, the differences of telomerase activity in 8 groups were significantly (P < 0.01). The expression of P53, P21, GADD45, PCNA, P16 proteins showed increased tendency in experimental group, compairing with the control更多还原