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激光照射对中华大蟾蜍原肠胚蛋白质表达的影响

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维普资讯 http://www.cqvip.com 第16卷第1期 2007年2月 激光生物学报 V01.16 No.1 Feb.2007 ACTA LASER BIOLOGY SINICA ・基础研究・ Effects of Laser Irradiation on the Protein Expression Patterns of Gastrula of Bufo ZHANG Nan。,SHI Yong.xiang。 ,BAI Zeng.1iang。 ,ZHANG Shao-jun ,L1 Li-li (Shandong University a.School of Life Science;b.State Key Laboratory of Crystal Materials; e.State Key Laboratory of Microbiotechnology,Jinan 250100,Shandong,China) Abstract:A YAG double- ̄equency laser microbeams was used to irradiate bufo gargarizens at the gastrula stage of its early development.The patterns of protein expression were investigated in gastrula by two・dimensional electrophore— sis after irradiation with a laser.PDQuest sotfware was used to examine the distirbution of protein spots in two-dimension— al gels after scanning with a GS・800 protein densitometer.The results demonstrated that protein expression was influ— eneed by laser iradiation and the total number of protein spots was related to the intensity of the laser beam.There were some proteins obviously changed in laser treatment groups compared to the control and some spots visible in the treatment groups but not observed in the conto1.r Key words:laser;patterns of protein;two dimensional electrophoresis;sodium dodecyl sylfate.polyaerylamide gel electrophresis(SDS-PAGE);embryo; 6咖gargarizens CLC number:Q631 Document code:A Arcticle ID:1007-7146(2007)O1-0006-06 激光照射对中华大蟾蜍原肠胚蛋白质表达的影响 张楠 ,时永香”,白增亮 ,张少军 ,李丽莉 (山东大学a.生命科学学院;b.晶体材料国家重点实验室;e.微生物技术国家重点实验室,山东济南250100) 摘 要:用YAG倍频激光器选择不同能量密度对中华大蟾蜍原肠胚进行照射,对各组样品进行双向电泳,凝 胶成像后用PDQuest软件进行分析,结果表明:激光照射对蛋白质点数及其分布影响显著,影响程度与照射激光 密度有关。各激光照射组与对照组相比,有些蛋白质点变化明显,有些蛋白质点在对照组未发现,而在各激光照 射组皆新出现。结论:激光照射可对原肠胚一些蛋白质的表达产生明显的影响。 关键词:激光;蛋白质谱;双向电泳;十二烷基硫酸钠.聚丙烯酰胺凝胶电泳;胚胎;中华大蟾蜍 中图分类号:Q631 文献标识码:A 文章编号:1007-7146(2007)01-0006-06 Low-energy laser irradiation(LELI)has been found to modulate various biological processes .such as in. Received date:2006.1 0-24 Foundation item:National Natural Science Foundation of China(30570199);Scientiifc Research Encouragement Founda. tion for Excellence Meddleaged and Youthful Scientists of Shandong Province(2006BS03043) Biography:ZHANG Nan(1981一),female,research fields:developmental biology. }Communication author:(Te1)053 1-88364889;(E-mail)shiyx@sdu.edu.ca;zengliangbai@sdu.edu.ca 维普资讯 http://www.cqvip.com No.1 ZHANG Nan et a1:Eriects 0f Laser Irradiation on the Protein Expression Patterns of Gastrula of Bufo bufo gargarizens 7 creasing mitochondrial respiration and ATP synthesis , facilitating wound healing[ ]and promoting the process of ergeneration and angiogenesis[ 。7。. In recent years. we have irradiated early stage embryos of Bufo bufo gargari一 , with YAG double—frequency laser microbeam and g0t some significant results.Firstly,certain kinds of abnor- malities were produced in the embryos by the laser iradia— tion.Secondly,laser irradiation had no influence on lac— tate dehydrogenase,but it increased easterase activity and caused a doubling or elimination of chromosomes.Thim— ly,protein expression was ilfnuenced by lsaer irradiation in neurula and blsatula[。一 2。. In this paper we analyzed the changes in protein expression in gastmla after irradia— tion with laser microbeams of differing intensities by two— dimensional gel electrophoresis to study the effects of laser biology on the embryos’development. 1 Materials and Methods 1.1 Materiais Healthy Bufo bufo gargarizens were injected into LRH—A and each female received 5 U while males re— ceived 2 U.Within 24 hoursthey mated and the eggs .were fertilized as soon as it were laid. Immediately,the fertilized eggs were cultured in 1/10 Holfreter’s fluids.Gastrula were gathered in Petri dishes and divided into four groups,one of them was served as a contro1. 1.2 Laser irradiation Three groups of gastrula stage embryos were irradia— ted with a laser microbeam( =532 nm).Light doses were 100 pJ/cm ,300 pJ/cm and 600 pJ/cm in five pulses respectively.Embryos of all treatment groups and control were cultured in 1/10 Holfreter’s fluids in Petri dishes. 1.3 Sample preparation and IEF/SDS-PAGE two-dimensional electrophoresis All were done as we have reported[ . 2 Results The two dimensional electrophoretic patterns of gas— trula proteins of the control and the three laser treatment rgoups were displayed in Fig.1—4.To some extent,dis— tribution of protein spots were similar in the four groups. 4—-m●‘l---・---- IEF 7‘ ●●●一 ..L, 97.4 kD--- 66.2 kDgp ∽ 口 ∽ I 43.0 kD- 》 0 ● 31.0 k ・- ■ . t・ I B0 \ 。 20.1 kD hO 14.4 kD 一 ..| Fig.1 Two—dimensional electorphoretic map of gastrula proteins from the contorl group ・ 97.4 kD 66・ kD- * . . 43.0 kD一-- 嘻 ◆ ● C 、 31.0 kD‘_-0 气 ’ 20.1 kD・ 。\ A E1 14.4 kD一 Fig.2 Two—dimensional electrophoretic map of gastrnla proteins after laser iradiation with 100 to/ca 4 7.5 97.4 kD一 66・2 kJ)● 43.0 kD‘_I ,t ,一 31.0 kD・~ L‘一● ・ . ・ j2 ・ ・ 20.1 kD●I \\A2 14.4 kD 眩 Fig.3 Two—dimensional electorphoretic map of gastrula proteins after lsaer iradiation with 300 /cm 维普资讯 http://www.cqvip.com 8 ACTA LASER BIOLOGY SINICA Vo1.16 The densities of some protein spots were increased or de- 4...|—一’~● IEF 7.5 creased relative to the contro1.Some protein spots disap— peared completely,while new protein spots which did not exist in the controls appeared in the laser treatment ,97.4 kD・・一 .一  66.2 kD●● 43.0 kD’ 喀 .= 。 T・, . groups・ After further analyse of the protein spot patterns by PDQuest software,the following r values were produced by Bio—Rad Laboratories Scatter Plot soOware:0797919 .31・0 k1),_. 卜 (control group VS.100 I ̄J/cm group),0.801290 (control group VS.300 I ̄J/cm group),0.834022 (control group VS.600 pA/cm group),0.801030 (100 VS.300 I ̄J/cm group),0.886671(100 VS.600 VS.600 I ̄J/cm group),0.862823(300 20・1 kD.- E。 14.4 kD 。 Fig.4 Two-dimensional electrophoretic map of gastrula proteins after laser irradiation with 6O0 pj/cm pA/cm group).An r value more close to 1 indicates more similarity in the distibution of protrein spots be— tween the two groups. Tab・1 Distibution of gastrrula proteins with different pI Tab.2 Changes in protein spots at different pI Spots disappeared( ) Range ofpI 100 >6.60 6.30-6.60 6.o0 .30 5.70-6.O0 5.40-5.70 5.10-5.40 4.80-5.10 <4.80 Spots new added(oJ) 100 4 2 1 13 12 18 12 1 Spots that increased 2 fold( )Spots that decreased 2 fold( ) 100 300 600 100 300 600 300 1 3 9 28 26 36 16 0 600 0 2 10 32 32 35 18 1 300 5 3 3 18 30 38 30 1 600 13 2 5 8 6 1 1 9 0 1 3 11 34 33 32 17 1 Total 132 119 130 63 128 54 维普资讯 http://www.cqvip.com Tab.4 Changes in protein spots at different molecular weights Comparing the distribution pattern of proteins based 300 cm group had the most new protein spots ap- on their pI and molecular weight,there were diferences pearance,while the group treated with 600 l ̄l/cm had among the four groups as shown in table 1 to table 4.The the fewest new protein spots.The number of protein spots data in tbale 1 shows that the soluble gastrula proteins that decreased by two fold gradually increased with in- distributed mainly in a wide range of the acidic region creasing laser’S intensity. with only a few proteins in the alkaline region.The data We found that there were 70 consensus protein spots in table 3 shows the distribution pattern of molecular in the four groups.There were some proteins obviously weight.Proteins from the four groups mainly distributed changed in laser treatment groups compared to the control in the range of20.1—97.4 kD with only a small amount and some spots visible in the treatment groups but not ob- of low and hi gh molecular weight gastrula proteins in all served in the contor1.One protein,Wp(24.10 kD, four groups.Comparing the proteins of laser treatment 6.06,see A0一A3 in Fig.1—4)was increased 16 fold in groups with those of the controls,we found a great in- the laser treatment goups.The proteins Wp(32.53 kD, crease in the 300 p ̄l/cm treatment,while there was no 5.60,see BO—B3 in Fig.1—4)had no obvious change at obviously change in the 100 tzJ/cm group.The 600 / 600 izJ/cm but was increased 2 fold in the 100 and 300 cm treatment group had an obvious loss.It was the laser ,cmL laser treatment goups compared to the contro1. treatment group of 600 ̄J/cm in which protein spots One protein(39.46 kD,5.02,see CO—C3.in Fig.1—4) disappeared the most,while the group treated wiht 300 Was decreased 7 fold in all three laser treatment groups td/cm ̄had the fewest spots disappearance.In fact the compared to the contro1.The proteins Wp(56.65 kD, 维普资讯 http://www.cqvip.com ACTA LASER BIOLOGY SINICA Vol_l6 5.04.see DO—D3 in Fig.1—4)were found the lower laser energy density the lower protein density.The protein spots Wp(39.54 kD,5.10,see EO—E3 in Fig.1—4) was obviously visible in the treatment groups but not ob— served in the contorls. 3 Discussion Currently,it.is known that the biological effects of laser treatment are thermal,pressure,light and electro— magnetic.Thermal effects could lcad enzyme to inactiva— tion and protein to denaturation and result in physiologic and genetic variation.Pressure effect could lcads to tis— sue deformation and disruption and results in physiologic and genetic variation.Light effects could cause changes in biomolecules through the absorption of photons of ifxed wavelengths and transition to speciifc energy states.E・ lectromagnetic effects could lcad to the creation of free radicals and result in DNA damage E .In【he search for a mechanism by which LELI manifests muscle cell re— sponses,Shefer et a1.[14]demonstrated that LELI specif- ically activated the mitogen・-activated protein kinase/ex・- tracellular signal—regulated protein kinase(MAPK/ ERK)pathway,probably by phosphorylation of speciifc receptors.They showed that LELI promoted the cell cy— cle entry and accumulation of satellite cells around fibers grown under serum starvation and that these effects were synergistic with serum addition.The inhibitory effect of LELI on cell apoptosis went hand in hand with increasing Bcl一2 and decreasing BAX expression in both these fibers and myogenic cultured cells.Applying low—energy laser to irradiate skeletal mUSCle cells.Shefer et a1 E ]disc0v— ered that low・・energy laser treatment could facilitate pro・- tein synthesis and cyclin D1,which plays a key role in cell proliferation and was produced in 6 minutes after la— ser treatment.Analyzing the physical mechanism of 532 nm laser—induced erythrocyte fluorescence.Ga0 et a1 E ] discovered that when erythrocytes were irrradiated with a low・lever frequency・doubled Nd:YAG laser with wave— length 532 nm,C・C bonds or C—N bonds on membrane of the erythrocyte would absorb the energy by non—resonance and the bonds broken to produce lone—pairs of electrons。 Thus,the new fluorophores from the lone—pairs of elec・ trons under the】aser excimti0n would emit fluorescence. Based on the patterns of protein spots produced by two—dimentional gel electrophoresis,most of the expres’ sion in the laser treatment groups were similar to that of the controls,though there were differences and a few specific proteins produced by the laser.When the low— energy laser with wavelength 5 32 nm irradiated early stage embryos,C—N bonds with resonance wavelength 5 34.2 nm may be broken because of non—resonance ab— sorbanee.Thus,the molecular structure may be changed and gene expression influenced.On the other hand,the low・-energy laser may cause variation in expression pat・- tern of the key proteins which regulate the cell cycle and influence protein synthesis. The biological effects of lasers are complex and many different genes are expressed during development. Next step we want to examine the physical and chemical character of changes proteins and detect the damniifcation of nuclear DNA and mtDNA and the changes of total mR— NA in early stage embryos after being iradiated by laser to study signal transduction pathway of laser effects on or・ ganism in detail. References [1]CONLAN M J,REPLEY J W,COBB C M.Binslimulalion of Wound Healing by Low—energy laser Irradiation[J].J Clin Peri— odontol,1996,23(5):492-496. [2] KARU T.Primal3,and Seenndary Mechanisms of Aelion of Visi— ble 1o Near—IR Radialion on Cells[J].J Pholo( hem Photobiol B, 1999.49(1):1—17. [3]MORIMOTO Y,ARAI T,KIKUCHI M H,et a1.Effeel of Low lnlensity Argon Laser Irradiation Oll Mitoehondria Respiration [J].Lasers Surg Med,1994,15(2):191—199. [4] YU W,MCGOWN M,IPPOI ITO K,el a1.Pholostimulalion of Oxidalive Melabolism anti Eleelron Chain Enzymes in Ral Liver Miloehondria[J].Photochem Pholobiol,1997,66(6):866. 871. [5] BIBIKOVA A,ORON U.Promotion of Muscle Regeneration in the Toad(8l加virdis)Gastrocnemius Muscle by Low—energy aLser Irradiation[J].Ana Ree,1993,235(3):374-380. [6] BIBIKOVA A,ORON U.Atlenuation of the Process of Muscle Regeneration in the Toad Gastorenemius Muscle by Low Energy aLser Iradiation[J].Lasers Surg Med,1994,14(4):355. 361. [7] BIBIKOVA A,BELKIN A,ORON U.Enhancement of Anglo— genesis in Regenerating Gastrocnemlus Muscle of the Toad(Bufo virdis)by Low Energy Laser Iradiation[J].Anat Embryo1. 1994.190(6):597-602. [8] 时永香,翟玉梅,孙洁,等.激光照射对中华大蟾蜍早期胚胎 发育的影响[J].应用激光,2000,20(1):32-34. 维普资讯 http://www.cqvip.com No.1 ZHANG Nan et al:Effects of Laser Irradiation on the Protein Expression Patterns of Gastrula of Bufo bufo gargarizens 1 1 欢迎订阅《激光生物学报》 《激光生物学报》的前身是《激光生物学》杂志,创刊于1992年,1997年经国家科委、新闻出版署批准改为现刊名,是由 中国科学技术协会主管、中国遗传学会主办、湖南师范大学承办、华南师范大学激光生命科学研究所、安徽农业大学生命科 学学院、中国海洋大学物理系、福建师范大学物理与光电信息科技学院、亚盛集团博士后科研工作站北京分站等协办,由国 内外有关专家、学者组成的《激光生物学报》编委会编辑部编辑、激光生物学报杂志社出版的学术性刊物,是目前国际上唯 一的一份激光生物学科的专业性学术刊物,已逐渐发行到中国、美国、英国、日本、俄罗斯、法国、德国、意大利、奥地利、瑞典 报道内容:以激光(光)生物学、生物光子学、激光(光)生物医学(含光子中医学、光动力疗法、激光整形美容)、辐射生 诸国;双月刊,每逢双月25日出版发行,国外发行代号:DK43001,国内发行代号:42—194,欢迎订阅。 物学(含激光育种、辐射育种、空间育种等)、离子束生物工程及其相关的激光生物技术(含微束照射技术、光镊技术、成像 技术、光谱技术、共聚焦扫描显微技术、细胞分流技术等)、仪器研制诸领域的学术论文,适量兼登生物物理学、生物化学、遗 传学、医学、农学等基础学科方面的学术论文。 本刊栏目:《激光生物学报》包含以下栏目:基础研究、应用研究、技术研究、仪器研制、专题综述、科技信息等。 读者对象:凡从事上述领域的研究人员、高等院校教师、研究生、医务工作者、育种工作者、激光仪器研制者等,均有裨 益。 订阅方法:(1)国内可以到全国各地邮局直接订阅,亦可通过邮局直接汇款至编辑部订阅。邮编:410081,地址:长沙市 岳麓山湖南师大生命科学学院内;或由银行转账,开户行:中行湖南师范大学支行,账号:46106572408091001,户名:长沙市 岳麓区激光生物学报杂志社。国内定价:16元/期,96元/年。(2)国外直接向中国出版对外贸易总公司(北京782信箱)订 阅。欢迎国内外读者订阅本刊! 《激光生物学报》编辑部 

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