梅花鹿鹿茸再生干细胞蛋白质组双向电泳条件优化
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- 英文论文题名：The Optimization of two-dimensional electrophoresis for antler stem cells in sika deer(Cervus nippon)
- 论文作者：董振 ; 王权威 ; 刘振 ; 李春义
- 英文论文作者：Dong Zhen ; Wang Quanwei ; Liu Zhen ; Li ChunYi ; State Key Lab for Molecular Biology of Special Animals/Institute of Special Animal and Plant Sciences ; Chinese Academy of Agricultural Science
- 年：2016
- 作者机构：中国农业科学院特产研究所特种动物分子生物学国家重点实验室;
- 论文关键词：鹿茸再生干细胞 ; 蛋白质组 ; 双向电泳 ; 条件优化
- 英文论文关键词：Antler stem cells ; Proteome ; Two-dimensional electrophoresis ; Condition optimization
- 会议召开时间：2016-06-01
- 会议录名称：“十二五” 鹿科学研究进展
- 语种：中文
- 分类号：S825
- 学会代码：ZXMY
- 学会名称：中国畜牧业协会
- 页数：11
- 文件大小：1338k
- 原文格式：O
摘要
鹿茸是目前唯一可以完全再生的哺乳动物附属器官,这种再生基于鹿茸再生干细胞。本研究以梅花鹿(Gervus nippon)鹿茸再生干细胞为样品,在处理方法、染色方法、蛋白纯化和等电聚焦条件4个方面对蛋白质组双向电泳进行优化,为不同发育期梅花鹿鹿茸再生干细胞比较蛋白质组学研究奠定基础。结果显示,利用Bullet Blender细胞组织破碎仪处理细胞优于超声破碎;双染法染色能够得到更多且更清晰的蛋白点;等电聚焦总volt-hours在15 000V-hrs左右时竖条纹相对较少;通过比较6种不同的蛋白提取方法与纯化方法组合,发现采用自制裂解液与双向电泳纯化试剂盒纯化相结合的方式获得的电泳图谱较好。通过综合优化后的双向电泳技术所得到的蛋白图谱中蛋白点相对较多且圆滑,条纹现象较轻,重复性较好,满足后续软件分析以及数据处理的要求,适用于梅花鹿鹿茸再生干细胞蛋白质组学研究。
Deer antlers are the only known mammalian organs which can periodically regenerate from pedicles.Antler regeneration is known as a stem cell-based process and antler stem cells reside in the pedicle periosteum.Conditions of two-dimensional electrophoresis for antler stem cells were optimized in the following four aspects including cell processing,staining method,isoelectric focusing condition and protein purification method.All these researches could provide foundation for the study of comparative proteomics in the different developmental stages of antler stem cells.Results showed that comparing with the method of ultrasonic,Bullet Blender had a better effect on cell breaking;It could get more and clearer protein points by combining coomassie brilliant blue stain and silver stain;When the total volt-hoursof isoelectric focusing was around 15 000 v-hrs,the vertical stripes were relatively lighter;Through the comparison of six kinds of protein extraction and purification methods,the one combining the manual lysate and 2D cleanup kit could get more protein points,lighter background and clearer maps.After the comprehensive optimization of two-dimensional electrophoresis,the acquired maps had more and clearer protein spots,less stripes and better reproducibility,at the same time,they could meet the requirements of software analysis and experimental treatments.The optimization could apply to the study of antler stem cell proteome.
Deer antlers are the only known mammalian organs which can periodically regenerate from pedicles.Antler regeneration is known as a stem cell-based process and antler stem cells reside in the pedicle periosteum.Conditions of two-dimensional electrophoresis for antler stem cells were optimized in the following four aspects including cell processing,staining method,isoelectric focusing condition and protein purification method.All these researches could provide foundation for the study of comparative proteomics in the different developmental stages of antler stem cells.Results showed that comparing with the method of ultrasonic,Bullet Blender had a better effect on cell breaking;It could get more and clearer protein points by combining coomassie brilliant blue stain and silver stain;When the total volt-hoursof isoelectric focusing was around 15 000 v-hrs,the vertical stripes were relatively lighter;Through the comparison of six kinds of protein extraction and purification methods,the one combining the manual lysate and 2D cleanup kit could get more protein points,lighter background and clearer maps.After the comprehensive optimization of two-dimensional electrophoresis,the acquired maps had more and clearer protein spots,less stripes and better reproducibility,at the same time,they could meet the requirements of software analysis and experimental treatments.The optimization could apply to the study of antler stem cell proteome.
引文
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[3]林冬云,黄晓南,柯李晶,等.鹿茸中促大鼠成骨样细胞增殖活性组分的纯化与表征[J].中国中药杂志,2005,30(11):851-855.(Lin D Y,Huang X N,Ke L J,et al.Purification and characterization of the proliferation of rat osteoblast-like cells ITMR-106 from Pilose Antler[J].China Journal of Chinese Materia Medica,2005,30(11):851-855.)
[4]李丽梅,王秀梅.HepG2细胞蛋白质组双向电泳条件的优化与图谱的建立[J].内蒙古医学志,2011,43(5):527-529.(Li L M,Wang X M.Establishment and Optimization of Twodimensional Gel Electrophoresis Technique for Proteomics of HepG2 Cells[J].Inner Mongolia Medical Journal,2011,43(5):527-529.)
[5]赖童飞,董薇,贾银华,等.棉花胚珠蛋白三氯乙酸丙酮法与酚抽提法的比较分析[J].农业生物技术学报,2009,17(2):317-322.(Lai T F,Dong W,Jia Y H,et al.Evaluations of Trichloroacetic Acid-acetone Method and Phenol Method for Protein Extraction from Cotton Ovule[J].Journal of Agricultural Biotechnology,2009,17(2):317-322.)
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[7]王雪,权春善,王建华,等.不同细胞破碎方法对无细胞蛋白表达系统细胞抽提物活性的影响[J].中国生物工程杂志,2011,31(1):46-50.(Wang X,Quan C S,Wang J H,et al.The Influence of Different Cell Disruption Methods on the Activity of the Extract in Cell-free Protein Synthesis System[J].China Biotechnology,2011,31(1):46-50.)
[8]徐代勋.梅花鹿鹿茸角柄骨膜不同部位差异蛋白的筛选[D].镇江:江苏科技大学,2011.(Xu D X.Screening of Differential Proteins In Different Parts of Pedicle Periosteum of Sika Deer[D].Zhen Jing;Jiangsu University of Science and Technology,2011.)
[9]徐代勋,王桂武,赵海平,等.梅花鹿角柄骨膜蛋白质组学初步研究[J].特产研究,2011(01):5-8.(Xu D X,Wang G W,Zhao H P,et al.Proteomics Study On Pedicle Periosteum of Sika Deer[J].Special Wild Economic Animal Plant Research,2011,(01):5-8.)
[10]姚宏亮.胃癌和胃正常粘膜组织差异蛋白质组学和免疫组化研究[D].长沙:中南大学,2009.(Yao H L.Study on Differential Proteome and Immunohistochemistry in Adenocarcinoma and Normal Mucosal Tissues of Stomach[D].Chang sha:Middle and Southern University,2009.)
[11]赵东.梅花鹿鹿茸总蛋白提取方法对双向电泳图谱的影响[J].中国畜牧兽医,2012,39(7):25-28.(Zhao D.Methods of Antler Total Protein Extraction on the Effect of Map Quality of Two-dimensional Electrophoresis in Sika Deer[J].China Animal Husbandry and Veterinary Medicine,2012,39(7):25-28.)
[12]C.Li,A.Harper,J.Puddick,et al.Proteomes and Signalling Pathways of Antler Stem Cells[Jj.PloS ONE,2012,7(1):e30026.DOI:10.1371/journal.pone.0030026.
[13]C.Li,et al.Morphogenetie Mechanisms in the Cyclic Regeneration ofHair Follicles and Deer Antlers from Stem Cells[J].BioMed Research International,http://dx.doi.org/10.1155/2013/643601.
[14]C.Li,et al.Deer antler-A novel model for studying organ regeneration in mammals[J].The International Journal of Biochemistry&Cell Biology,http://dx.doi.org/10.1016/j.biocel.2014.07.007.
[15]H.J.Park,et al.Proteome analysis of red deer antlers[J].Proteomics,2004(4):3642-3653.DOI:10.1002/pmic.200401027
[16]O'FARRELL P H.High resolution two-dimensional electrophoresis of proteins[J].JOURNAL OF BIOLOGICAL CHEMISTRY,1975,250(10):4 007-4 021.
[17]Marouga R,David S,Hawkins E.The development of the DIGE system;2D fluorescence difference gel analysis technology[J].Analytical and Bioanalytical Chemistry,2005,382:669_678.DOI:10.1007A00216-005-3126-3.
[2]柯李晶,聂毅磊,叶秀云,等.梅花鹿鹿茸中促PCI2细胞增殖蛋白的分离和活性研究[J].中草药,2009,40(5):715-718.(Ke L J,Nie Y L,Ye X Y,et al.Isolation and PC 12 cell proliferative protein fraction from pilose antler and its activity[J].Chinese Traditional and Herbal Drugs,2009,40(5):715-718.).DOI:10.7501/j.issn.0253-2670.
[3]林冬云,黄晓南,柯李晶,等.鹿茸中促大鼠成骨样细胞增殖活性组分的纯化与表征[J].中国中药杂志,2005,30(11):851-855.(Lin D Y,Huang X N,Ke L J,et al.Purification and characterization of the proliferation of rat osteoblast-like cells ITMR-106 from Pilose Antler[J].China Journal of Chinese Materia Medica,2005,30(11):851-855.)
[4]李丽梅,王秀梅.HepG2细胞蛋白质组双向电泳条件的优化与图谱的建立[J].内蒙古医学志,2011,43(5):527-529.(Li L M,Wang X M.Establishment and Optimization of Twodimensional Gel Electrophoresis Technique for Proteomics of HepG2 Cells[J].Inner Mongolia Medical Journal,2011,43(5):527-529.)
[5]赖童飞,董薇,贾银华,等.棉花胚珠蛋白三氯乙酸丙酮法与酚抽提法的比较分析[J].农业生物技术学报,2009,17(2):317-322.(Lai T F,Dong W,Jia Y H,et al.Evaluations of Trichloroacetic Acid-acetone Method and Phenol Method for Protein Extraction from Cotton Ovule[J].Journal of Agricultural Biotechnology,2009,17(2):317-322.)
[6]唐秀英,邵彩虹,谢金水.双向电泳中4种常用染色方法的比较[J].江西农业学报,2010,22(7):100-102.(Tang X Y,Shao C H,Xie J S.Comparison of Four Protein Staining Methods in Common Use in Two-dimensional Electrophoresis[J].Acta Agriculturae Jiangxi,2010,22(7):100-102.)
[7]王雪,权春善,王建华,等.不同细胞破碎方法对无细胞蛋白表达系统细胞抽提物活性的影响[J].中国生物工程杂志,2011,31(1):46-50.(Wang X,Quan C S,Wang J H,et al.The Influence of Different Cell Disruption Methods on the Activity of the Extract in Cell-free Protein Synthesis System[J].China Biotechnology,2011,31(1):46-50.)
[8]徐代勋.梅花鹿鹿茸角柄骨膜不同部位差异蛋白的筛选[D].镇江:江苏科技大学,2011.(Xu D X.Screening of Differential Proteins In Different Parts of Pedicle Periosteum of Sika Deer[D].Zhen Jing;Jiangsu University of Science and Technology,2011.)
[9]徐代勋,王桂武,赵海平,等.梅花鹿角柄骨膜蛋白质组学初步研究[J].特产研究,2011(01):5-8.(Xu D X,Wang G W,Zhao H P,et al.Proteomics Study On Pedicle Periosteum of Sika Deer[J].Special Wild Economic Animal Plant Research,2011,(01):5-8.)
[10]姚宏亮.胃癌和胃正常粘膜组织差异蛋白质组学和免疫组化研究[D].长沙:中南大学,2009.(Yao H L.Study on Differential Proteome and Immunohistochemistry in Adenocarcinoma and Normal Mucosal Tissues of Stomach[D].Chang sha:Middle and Southern University,2009.)
[11]赵东.梅花鹿鹿茸总蛋白提取方法对双向电泳图谱的影响[J].中国畜牧兽医,2012,39(7):25-28.(Zhao D.Methods of Antler Total Protein Extraction on the Effect of Map Quality of Two-dimensional Electrophoresis in Sika Deer[J].China Animal Husbandry and Veterinary Medicine,2012,39(7):25-28.)
[12]C.Li,A.Harper,J.Puddick,et al.Proteomes and Signalling Pathways of Antler Stem Cells[Jj.PloS ONE,2012,7(1):e30026.DOI:10.1371/journal.pone.0030026.
[13]C.Li,et al.Morphogenetie Mechanisms in the Cyclic Regeneration ofHair Follicles and Deer Antlers from Stem Cells[J].BioMed Research International,http://dx.doi.org/10.1155/2013/643601.
[14]C.Li,et al.Deer antler-A novel model for studying organ regeneration in mammals[J].The International Journal of Biochemistry&Cell Biology,http://dx.doi.org/10.1016/j.biocel.2014.07.007.
[15]H.J.Park,et al.Proteome analysis of red deer antlers[J].Proteomics,2004(4):3642-3653.DOI:10.1002/pmic.200401027
[16]O'FARRELL P H.High resolution two-dimensional electrophoresis of proteins[J].JOURNAL OF BIOLOGICAL CHEMISTRY,1975,250(10):4 007-4 021.
[17]Marouga R,David S,Hawkins E.The development of the DIGE system;2D fluorescence difference gel analysis technology[J].Analytical and Bioanalytical Chemistry,2005,382:669_678.DOI:10.1007A00216-005-3126-3.