小窝蛋白-1mRNA在紫外线诱导的白内障的晶状体中的表达及意义
摘要
目的研究小窝蛋白-1(CAV-1)mRNA在正常及紫外线照射的白内障模型大鼠晶状体上的表达,探讨其在氧化性白内障发生过程中的作用。
方法取50只大鼠的晶状体于MEM培养液中培养8h后随机分为A组(对照组25只)B组(UV照射组25只)。实验组:紫外线照射晶状体30min,加入MEM培养液,于37℃下,置5%CO_2培养箱中培养,分别在加入培养液后0、6、12、24及48 h时,应用荧光realtime RT-PCR技术检测晶状体中小窝蛋白-1的表达量,对照组不照射,与同等条件下用同样的方法检测小窝蛋白-1的表达量。
结果紫外线照射30min后,采用RT-PCR技术测小窝蛋白-1mRNA表达水平随着时间的延长有逐渐下降的趋势。0h:实验组(0.5920±0.1254)对照组(1.0242±0.1117)(P＜0.05)两组相比差异有显著意义;6h:实验组(0.3914±0.2070)对照组(0.9662±0.1286)(P＜0.01)两组相比差异有显著意义;12h:实验组(0.2658±0.0938)对照组(0.9248±0.1545)(P＜0.01)两组相比差异有显著意义;24h:实验组(0.1244±0.0953)对照组(0.8840±0.0710)(P＜0.01)两组相比差异有显著意义;48h:实验组(0.0598±0.0414)对照组(0.8420±0.1023)(P＜0.01)两组相比差异有显著意义。
结论紫外线照射对鼠晶状体上皮细胞产生影响,并导致细胞小窝蛋白-1表达量减少,小窝蛋白-1的减少在氧化性白内障的发生中有一定作用。推测小窝蛋白-1对维持氧化损伤时晶状体透明具有保护意义。
Objective: To study the expression of caveolin-1 mRNA in lens of normal rats and rats radiation by ultraviolet radiation. In order to explore its role in development of oxidative cataract.
Methods 50 rat lens in Minimum Essential Media in the culture medium after 8 hours were divided into Group A (control group 25) and Group B (UV-irradiation 25). Experimental group: ultraviolet radiation lens 30 min after joining Minimum Essential Media medium at 37℃, 5% CO_2 incubator home in the culture, To detect the expression of caveolin-1 using fluorescence realtime RT -PCR in the control medium by adding 0, 6, 12, 24 and 48 hours. The methed and condition in control group were as same as the experimental group,but no ultraviolet radiation .
Results After ultraviolet irradiation 30 min, and by RT-PCR to determine, the caveolin-1 mRNA expression level had been a downward trend;with time extension after ultraviolet irradiation 30 min by RT-PCR ;0 hours: the experimental group (0.5920±0.1254) were different from control group (1.0242±0.1117) (P <0.05) significantly; 6 hours: the experimental group (0.3914±0.2070) were different from control group (0.9662±0.1286) (P <0.01) significantly; 12 hours: the experimental group (0.2658±0.0938) were different from control group (0.9248±0.1545) (P <0.01 ) significantly; 24 hours: the experimental group (0.1244±0.0953) were different from control group (0.8840±0.0710) (P <0.01) significantly; 48 hours: the experimental group (0.0598±0.0414) were different from control (0.8420±0.1023) (P <0.01) significantly.
Conclusions The rats lens lens epithelial cells were effected by ultraviolet irradiation lead to the expression reduction of caveolin-1. The expression reduction played a role in the incidence of cataract. We can speculate that caveolin-1 played a protective role in maintening lens transparent of oxidative damage.
方法取50只大鼠的晶状体于MEM培养液中培养8h后随机分为A组(对照组25只)B组(UV照射组25只)。实验组:紫外线照射晶状体30min,加入MEM培养液,于37℃下,置5%CO_2培养箱中培养,分别在加入培养液后0、6、12、24及48 h时,应用荧光realtime RT-PCR技术检测晶状体中小窝蛋白-1的表达量,对照组不照射,与同等条件下用同样的方法检测小窝蛋白-1的表达量。
结果紫外线照射30min后,采用RT-PCR技术测小窝蛋白-1mRNA表达水平随着时间的延长有逐渐下降的趋势。0h:实验组(0.5920±0.1254)对照组(1.0242±0.1117)(P＜0.05)两组相比差异有显著意义;6h:实验组(0.3914±0.2070)对照组(0.9662±0.1286)(P＜0.01)两组相比差异有显著意义;12h:实验组(0.2658±0.0938)对照组(0.9248±0.1545)(P＜0.01)两组相比差异有显著意义;24h:实验组(0.1244±0.0953)对照组(0.8840±0.0710)(P＜0.01)两组相比差异有显著意义;48h:实验组(0.0598±0.0414)对照组(0.8420±0.1023)(P＜0.01)两组相比差异有显著意义。
结论紫外线照射对鼠晶状体上皮细胞产生影响,并导致细胞小窝蛋白-1表达量减少,小窝蛋白-1的减少在氧化性白内障的发生中有一定作用。推测小窝蛋白-1对维持氧化损伤时晶状体透明具有保护意义。
Objective: To study the expression of caveolin-1 mRNA in lens of normal rats and rats radiation by ultraviolet radiation. In order to explore its role in development of oxidative cataract.
Methods 50 rat lens in Minimum Essential Media in the culture medium after 8 hours were divided into Group A (control group 25) and Group B (UV-irradiation 25). Experimental group: ultraviolet radiation lens 30 min after joining Minimum Essential Media medium at 37℃, 5% CO_2 incubator home in the culture, To detect the expression of caveolin-1 using fluorescence realtime RT -PCR in the control medium by adding 0, 6, 12, 24 and 48 hours. The methed and condition in control group were as same as the experimental group,but no ultraviolet radiation .
Results After ultraviolet irradiation 30 min, and by RT-PCR to determine, the caveolin-1 mRNA expression level had been a downward trend;with time extension after ultraviolet irradiation 30 min by RT-PCR ;0 hours: the experimental group (0.5920±0.1254) were different from control group (1.0242±0.1117) (P <0.05) significantly; 6 hours: the experimental group (0.3914±0.2070) were different from control group (0.9662±0.1286) (P <0.01) significantly; 12 hours: the experimental group (0.2658±0.0938) were different from control group (0.9248±0.1545) (P <0.01 ) significantly; 24 hours: the experimental group (0.1244±0.0953) were different from control group (0.8840±0.0710) (P <0.01) significantly; 48 hours: the experimental group (0.0598±0.0414) were different from control (0.8420±0.1023) (P <0.01) significantly.
Conclusions The rats lens lens epithelial cells were effected by ultraviolet irradiation lead to the expression reduction of caveolin-1. The expression reduction played a role in the incidence of cataract. We can speculate that caveolin-1 played a protective role in maintening lens transparent of oxidative damage.
引文
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5、Ottonello S,Foroni C,Carta A,et al,Oxidative stress and age-related cataract[J]Ophthalmologica,2000,214(1):78-85
6、Rujoi M,Jin J,Bo.rchman D,et al.Isolation and lipid characterization of cholesterol-enriched fractions in cortical and nuclear human lens fibers[J].Invest Ophthalmol Vis Sci,2003,44:163-164
7、钟一声,李浒源,金婉容,等.维拉帕米在控制半乳糖性白内障中的作用[J].眼科研究,2000,18(2):117.
8、MichaeL R,Vrensen G F,Van Marl E J,et all Repair in the rat lens after threshold ultraviolet radiation in jury[J],Invest Ophthalmol Vis Sci,2000,41(1):20-41
9、Ultraviolet Action Spectrum for Cell Killing in a Human Lens Epithelial Cell Line Industrial Health.2007 Jan;45(1):137-142.
10、Engelman JA,Zhang XL,Galbiati F,et al.Chromosomal localization,genomic organization,and developmental expression of the murine caveolin gene family(Cav-1,-2,and-3):Cav-1 and Cav-2 genes map to a known tumor suppressor locus(6-A2/7q31).FEBS Lett,1998,429:330-336
11、Rosenthal,et al.The effects of sunglasses on ocular exposure to ultraviolet radiation[J].American Journal of Public Health,1988,78:72-74
12、唐建,刘谊.紫外线对晶状体上皮细胞损伤的研究进展[J].眼视光学杂志2003;5(3):187-189.
13、陈莺 陈大本白内障发病机制及预防治疗的研究进展.眼科新进展2005;25(2):190一193
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2、zhang SY,Zhou LH,Gao YQ,et al.National epidemiological survery of blindness and low vision in china.Chun Med J.1992;105:603~608
3、郭秉宽 中国医学百科全书--眼科学[M].上海:上海科学技术出版社,1985.9.
4、布娟 赵堪兴先天性白内障分子发病机制的研究进展 国际眼科杂志2005;5(4)973-977
5、Ottonello S,Foroni C,Carta A,et al,Oxidative stress and age-related cataract[J]Ophthalmologica,2000,214(1):78-85
6、Rujoi M,Jin J,Bo.rchman D,et al.Isolation and lipid characterization of cholesterol-enriched fractions in cortical and nuclear human lens fibers[J].Invest Ophthalmol Vis Sci,2003,44:163-164
7、钟一声,李浒源,金婉容,等.维拉帕米在控制半乳糖性白内障中的作用[J].眼科研究,2000,18(2):117.
8、MichaeL R,Vrensen G F,Van Marl E J,et all Repair in the rat lens after threshold ultraviolet radiation in jury[J],Invest Ophthalmol Vis Sci,2000,41(1):20-41
9、Ultraviolet Action Spectrum for Cell Killing in a Human Lens Epithelial Cell Line Industrial Health.2007 Jan;45(1):137-142.
10、Engelman JA,Zhang XL,Galbiati F,et al.Chromosomal localization,genomic organization,and developmental expression of the murine caveolin gene family(Cav-1,-2,and-3):Cav-1 and Cav-2 genes map to a known tumor suppressor locus(6-A2/7q31).FEBS Lett,1998,429:330-336
11、Rosenthal,et al.The effects of sunglasses on ocular exposure to ultraviolet radiation[J].American Journal of Public Health,1988,78:72-74
12、唐建,刘谊.紫外线对晶状体上皮细胞损伤的研究进展[J].眼视光学杂志2003;5(3):187-189.
13、陈莺 陈大本白内障发病机制及预防治疗的研究进展.眼科新进展2005;25(2):190一193
14、Richer SP,Rose RC,Gogla R.An apparatus for ultraviolet B irradiation of small volumes of biological fluid under controlled oxygen tension.Optom Vis Sci.1996Nov;73(11):683-688.
15、Abrami L,FivazM,Kobayashi T,et al.Cross2talk between caveolae and glycosylphosphatidylinositol2rich domains[J].J B iol Chem,2001,276(33):30729-30736.
16、Simons K,Ehehalt R.Cholesterol,lipid rafts,and disease[J].J Clin Invest,2002,110(5):597-603.
17、Jeong J,McMahon AP.Cholesterolmodification of Hedgehog family proteins[J].J Clin Invest,2002,110(5):591-596.
18、Anderson RG.A nnu Rev Biochem,1998;67:199
19、Smart EJ,Ying Y,Donzell WC,et al.A role for caveolin in transport of cholesterol from endoplasmic reticulum to plasma membrane[J].J Biol Chem,1996,271(46):29427-29435.
20、Amino K,Honda Y,Ide C,et al.Distribution of plas malemmal Ca2+-pump and caveolin in the corneal epithelium during the wound healing process[J].Curr Eye Res,1997,16:1088-1095
21、Bridges CC,EI-Sherbeny A,Roon P,Ola MS,Kekuda R,Ganapathy V,Camero RS,Cameron PL,Smith SB.A comparison of caveolae and caveolin-1 to folate receptor alpha in retina and retinal pigment epithelium.Histochem[J]2001;33:149-158.
22、 Sexton PS,Neely AR,Cenedella RJ.Distribution of caveolin-1 in bovine lens and redistribution in cultured bovine lens epithelial cells upon confluence. ExpEyeRes, 2004, 78:75 -82
23、 Galbiati F, Razani B, Lisanti MP. Emerging themes in lip id rafts and caveolae[ J ]. Cell, 2001,106 : 403 - 411
24、 Krajewska WM,Maslowska I. Caveolins: structure and function in signal, transduction[ J ]. CellMol Biol Lett, 2004, 9 : 195 - 220
25、 ShaulPW,AndersonRG.AmJPhysiol, [J]1998;275(5Pt1):L843
26、 Luetterforst R, Stang E, ZorziN, et al. Molecular characterization of caveolin association with the Golgi comp lex: identification of acis - Golgi targeting domain in the caveolin molecule [ J ]. J Cell Biol, 1999,145:1443-1459.
27、 Simons K, Ikonen E. Functional rafts in cell membranes [ J ]. Nature,1997, 387 : 569 - 572
28、 Engelman JA,Zhang XL,Lisanti MP.Genes encoding human caveolin-1 and -2 are co-localized to the D7S522 locus (7q31 1),a known fragile site (FRA7G) that is frequently deleted in human cancers. FEBS Lett, 1998,436:403- 410
29、 Scherer PE,Tang Z,Chun M,et al. Caveolin isoforms differ in their N-terminal protein sequence and subcellular distribution:identification and epitope mapping of an isoform-specific mono-clonal antibody probe.J Biol Chem,1995,270:16395 -16401
30、 Wary K K,Mariotti A, Zurzolo C,et al.A requirement for caveolin-1 and associated kinase Fyn in integrin signaling and anchorage dependent cell growth.Cell, 1998,94:625 -634
31、Pelkmans L,Zerial M. Kinase-regulated quantal assemblies and kiss-and- run recycling of caveolae.Nature,2005,436:128 -133
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