成年及幼年大鼠硬脑膜差异蛋白质组学研究
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摘要
目的:幼年动物具有很强的颅骨缺损自行修复能力,当动物进入成年后这种能力很快消失。既往文献报道硬脑膜在颅骨再生中发挥了关键作用,其中幼年大鼠硬脑膜有一些独特的特点与颅骨缺损自行修复相关。但颅骨缺损自行修复过程中的分子生物学机制并不完全清楚,并且目前没有相关的蛋白质组学研究。
材料与方法:1、采用双向电泳技术,对样品的处理、上样量、等电聚焦、SDS-PAGE电泳等步骤进行优化。2、应用双向电泳、质谱及Western-blot技术筛选并鉴定成年及幼年大鼠硬脑膜中差异表达的蛋白质。3、对大鼠硬脑膜细胞原代培养,观察硬脑膜细胞体外培养的细胞形态和细胞增殖活性;应用透射电子显微镜,对硬脑膜细胞超微结构进行观察比较。
结果:1、获得重复性和分辨率比较好的成年及幼年大鼠硬脑膜蛋白双向电泳图谱;2、比较成年及幼年大鼠硬脑膜蛋白双向电泳图谱,发现24个差异表达蛋白质(丰度相差3倍以上),鉴定出11个蛋白质(均为首次报道),其中幼年大鼠硬脑膜蛋白上调的有:Cytoskeleton-associated protein 4,vimentin,typeⅡkeratin,β-actin,γ-actin,Apolipoprotein A-Ⅳprecursor,alpha-tropomyosin和TCTP1;成年大鼠硬脑膜蛋白上调的有:COMM domain containing 7,albumin和RAB5-interacting protein;3、Western-blot结果显示在幼年大鼠硬脑膜vimentin,tropomyosin,β-actin和γ-actin水平分别是成年大鼠硬脑膜的3.2,2.4,3.6和2.2倍,这与双向电泳/质谱结果一致;4、在体外培养中,成年硬脑膜细胞于10～15天后呈贴壁生长,25～30天出现细胞融合。幼年硬脑膜细胞于5～8天后呈贴壁生长,12～15天出现细胞融合。5、成年大鼠硬脑膜细胞呈梭形,细胞核狭长,细胞核质比约为1:2.5～3,细胞器比较发达,胶原排列整齐、致密;幼年大鼠硬脑膜细胞呈椭圆形,细胞核比较丰满,细胞核质比约为1:1.3～1.5,细胞器比较不发达,细胞外基质不丰富。
结论:1、.在标准操作流程和条件下,优化双向电泳关键技术是获得重复性和分辨率较好的双向电泳图谱的必要条件。2、成年及幼年大鼠硬脑膜存在11种差异蛋白,为从整体上认识幼年动物具有很强颅骨缺损自行修复能力的分子机制提供了实验和理论基础。3、成年及幼年大鼠硬脑膜细胞增殖能力以及成年及幼年大鼠硬脑膜超微结构差异较大。
Background:It has been widely observed that infants can reossify large calvarial defects,whereas adults older than 2 years of age lose this regenerative potential.Dura mater serves as a key regulator of calvarial regeneration.However,the molecular mechanism of calvarial reossification remains elusive.And few quantitative systematic studies by proteomics approaches have been performed.
Methods:1.A 2-DE technique was used.Sample preparation,rehydration, SDS-PAGE and other steps were established and optimized.2.Dura mater of juvenile and adult rats was excised.Total proteins were extracted from rat dura,and then analyzed by combination of 2-DE and MALDI-TOF/TOF MS.The differential expressional levels of dentified proteins were validated by Western blot analysis.3.In vitro,morphological changes of juvenile and adult rats dura cells cultured was investigated;and in vivo,ultrastructure of juvenile and adult rats dura cells was studied.
Results:1.Well-resovled,reproducible 2-DE patterns of juvenile and adult rats dura proteins were established.2.According to the results of 2-DE patterns of juvenile and adult rats dura proteins,24 differential protein spots between juvenile and adult rats dura were identified by 2-DE.Among them,14 protein spots were identified by MALDI-TOF/TOF MS and database search,which represented 11 proteins identified with high statistical reliability.Cytoskeleton-associated protein 4,vimentin,typeⅡkeratin,β-actin,γ-actin,apolipoprotein A-Ⅳprecursor,alpha-tropomyosin and TCTP1 were upregulated,and Copper metabolism MURR1(COMM) domain containing 7,albumin and RAB5-interacting protein were downregulated in juvenile dura compared to adult dura.3.Western blot showed that vimentin,α-tropomyosin,β-actin,andγ-actin were expressed at higher levels in the juvenile dura(3.2 fold,2.4 fold,3.6 fold,and 2.2 fold higher,respectively),confirming the differential protein expressions determined electrophoretically and by mass spectrometry.4.As observed under inversion microscope,adult dura cells began to grow on 10-15 days after culture and to cell fusion on 25-30 days.Juvenile dura cells began to grow on 5-8 days after culture and to cell fusion on 12-15 days.5.As observed under transmission electron microscope,adult dura cells were fusiform with long,narrow nuclei. Nuclear-cytoplasmic ratio was 1:2.5-3;while juvenile dura cells tended to be elliptical with plump nuclei.Nuclear-cytoplasmic ratio was 1:1.3-1.5.
Conclusions:1.It is essential to obtained 2-DE profiles with high resolution and reproducible to optimize the technology of 2-DE.2.Most of these proteins were shown to be invovled in cell shape and size,translational control,cell proliferation, transport of special cellular organs,stress reaction etc.These data are valuable for studying molecular mechanism of calvarial reossification of juvenile animals.3. Ability of proliferation and ultrastructure of juvenile and adult rats dura cells was obviously different.
材料与方法:1、采用双向电泳技术,对样品的处理、上样量、等电聚焦、SDS-PAGE电泳等步骤进行优化。2、应用双向电泳、质谱及Western-blot技术筛选并鉴定成年及幼年大鼠硬脑膜中差异表达的蛋白质。3、对大鼠硬脑膜细胞原代培养,观察硬脑膜细胞体外培养的细胞形态和细胞增殖活性;应用透射电子显微镜,对硬脑膜细胞超微结构进行观察比较。
结果:1、获得重复性和分辨率比较好的成年及幼年大鼠硬脑膜蛋白双向电泳图谱;2、比较成年及幼年大鼠硬脑膜蛋白双向电泳图谱,发现24个差异表达蛋白质(丰度相差3倍以上),鉴定出11个蛋白质(均为首次报道),其中幼年大鼠硬脑膜蛋白上调的有:Cytoskeleton-associated protein 4,vimentin,typeⅡkeratin,β-actin,γ-actin,Apolipoprotein A-Ⅳprecursor,alpha-tropomyosin和TCTP1;成年大鼠硬脑膜蛋白上调的有:COMM domain containing 7,albumin和RAB5-interacting protein;3、Western-blot结果显示在幼年大鼠硬脑膜vimentin,tropomyosin,β-actin和γ-actin水平分别是成年大鼠硬脑膜的3.2,2.4,3.6和2.2倍,这与双向电泳/质谱结果一致;4、在体外培养中,成年硬脑膜细胞于10～15天后呈贴壁生长,25～30天出现细胞融合。幼年硬脑膜细胞于5～8天后呈贴壁生长,12～15天出现细胞融合。5、成年大鼠硬脑膜细胞呈梭形,细胞核狭长,细胞核质比约为1:2.5～3,细胞器比较发达,胶原排列整齐、致密;幼年大鼠硬脑膜细胞呈椭圆形,细胞核比较丰满,细胞核质比约为1:1.3～1.5,细胞器比较不发达,细胞外基质不丰富。
结论:1、.在标准操作流程和条件下,优化双向电泳关键技术是获得重复性和分辨率较好的双向电泳图谱的必要条件。2、成年及幼年大鼠硬脑膜存在11种差异蛋白,为从整体上认识幼年动物具有很强颅骨缺损自行修复能力的分子机制提供了实验和理论基础。3、成年及幼年大鼠硬脑膜细胞增殖能力以及成年及幼年大鼠硬脑膜超微结构差异较大。
Background:It has been widely observed that infants can reossify large calvarial defects,whereas adults older than 2 years of age lose this regenerative potential.Dura mater serves as a key regulator of calvarial regeneration.However,the molecular mechanism of calvarial reossification remains elusive.And few quantitative systematic studies by proteomics approaches have been performed.
Methods:1.A 2-DE technique was used.Sample preparation,rehydration, SDS-PAGE and other steps were established and optimized.2.Dura mater of juvenile and adult rats was excised.Total proteins were extracted from rat dura,and then analyzed by combination of 2-DE and MALDI-TOF/TOF MS.The differential expressional levels of dentified proteins were validated by Western blot analysis.3.In vitro,morphological changes of juvenile and adult rats dura cells cultured was investigated;and in vivo,ultrastructure of juvenile and adult rats dura cells was studied.
Results:1.Well-resovled,reproducible 2-DE patterns of juvenile and adult rats dura proteins were established.2.According to the results of 2-DE patterns of juvenile and adult rats dura proteins,24 differential protein spots between juvenile and adult rats dura were identified by 2-DE.Among them,14 protein spots were identified by MALDI-TOF/TOF MS and database search,which represented 11 proteins identified with high statistical reliability.Cytoskeleton-associated protein 4,vimentin,typeⅡkeratin,β-actin,γ-actin,apolipoprotein A-Ⅳprecursor,alpha-tropomyosin and TCTP1 were upregulated,and Copper metabolism MURR1(COMM) domain containing 7,albumin and RAB5-interacting protein were downregulated in juvenile dura compared to adult dura.3.Western blot showed that vimentin,α-tropomyosin,β-actin,andγ-actin were expressed at higher levels in the juvenile dura(3.2 fold,2.4 fold,3.6 fold,and 2.2 fold higher,respectively),confirming the differential protein expressions determined electrophoretically and by mass spectrometry.4.As observed under inversion microscope,adult dura cells began to grow on 10-15 days after culture and to cell fusion on 25-30 days.Juvenile dura cells began to grow on 5-8 days after culture and to cell fusion on 12-15 days.5.As observed under transmission electron microscope,adult dura cells were fusiform with long,narrow nuclei. Nuclear-cytoplasmic ratio was 1:2.5-3;while juvenile dura cells tended to be elliptical with plump nuclei.Nuclear-cytoplasmic ratio was 1:1.3-1.5.
Conclusions:1.It is essential to obtained 2-DE profiles with high resolution and reproducible to optimize the technology of 2-DE.2.Most of these proteins were shown to be invovled in cell shape and size,translational control,cell proliferation, transport of special cellular organs,stress reaction etc.These data are valuable for studying molecular mechanism of calvarial reossification of juvenile animals.3. Ability of proliferation and ultrastructure of juvenile and adult rats dura cells was obviously different.
引文
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82、 Sun Z, Lee E, Herring SW. Cell proliferation and osteogenic differentiation of growing pig cranial sutures. J Anat. 2007; 211(3): 280-9.
83、 Schick B, Wolf G, Romeike BF, Mestres P, Praetorius M, Plinkert PK. Dural cell culture. A new approach to study duraplasty. Cells Tissues Organs. 2003; 173(3):129-37
84、 Zhou F, Chen G, Zhang JM, Huang ZS. An in vitro culturing model for rabbit dural cells. Ann Clin Lab Sci. 2006; 36(3): 341-4
85、 Thery M, Bornens M. Cell shape and cell division. Curr Opin Cell Biol. 2006;18(6): 648-57
86、 Thery M, Racine V, Pepin A, Piel M, Chen Y, Sibarita JB, et al. The extracellular matrix guides the orientation of the cell division axis. Nat Cell Biol. 2005; 7(10):947-53
87、 Thery M, Racine V, Piel M, Pepin A, Dimitrov A, Chen Y, et al. Anisotropy of cell adhesive microenvironment governs cell internal organization and orientation of polarity. Proc Natl Acad Sci U S A. 2006; 103(52): 19771-6
88、 Parker KK, Brock AL, Brangwynne C, Mannix RJ, Wang N, Ostuni E, et al. Directional control of lamellipodia extension by constraining cell shape and orienting cell tractional forces. FASEB J. 2002; 16(10): 1195-204
89、 Novak IL, Slepchenko BM, Mogilner A, Loew LM. Cooperativity between cell contractility and adhesion. Phys Rev Lett. 2004; 93(26 Pt 1): 268109
90、 Wang N, Ostuni E, Whitesides GM, Ingber DE. Micropatterning tractional forces in living cells. Cell Motil Cytoskeleton. 2002; 52(2): 97-106
91、 Yamakita Y, Totsukawa G, Yamashiro S, Fry D, Zhang X, Hanks SK, et al.Dissociation of FAK/p130(CAS)/c-Src complex during mitosis: role of mitosis-specific serine phosphorylation of FAK. J Cell Biol. 1999; 144(2): 315-24.
92、 Sunada R, Gorzer I, Oma Y, Yoshida T, Suka N, Wintersberger U, et al. The nuclear actin-related protein Act3p/Arp4p is involved in the dynamics of chromatin-modulating complexes.Yeast.2005;22(10):753-68
93、陈强,史俊南,任雨笙。牛血小板衍化尘长因子对人牙周膜成纤维细胞超微结构的影响。牙体牙髓牙周病学杂志1998,8(1):29-31
94、韦长元,陈建思,刘剑仑,杨南武,李挺。应用顺铂后肝癌细胞超微结构的研究。中华肝脏病杂志,2000,8(2):89-90
1.Dominguez DC,Lopes R,Torres ML.Introduction to proteomics.Clin Lab Sci.2007 Fa11;20(4):234-8
2.Wittmann-Liebold B,Graack HR,Pohl T.Two-dimensional gel electrophoresis as tool for proteomics studies in combination with protein identification by mass spectrometry.Proteomics.2006 Sep;6(17):4688-703
3.Chambers G,Lawrie L,Cash P,Murray GI.Proteomics:a new approach to the study of disease.J Pathol.2000 Nov;192(3):280-8
4.Swinbanks D.Government backs proteome proposal.Nature.1995 Dec 14;378(6558):653.
5.Pandey A,Mann M.Proteomics to study genes and genomes.Nature.2000 Jun 15;405(6788):837-46
6.G(o|¨)rg A,Weiss W,Dunn MJ.Current two-dimensional electrophoresis technology for proteomics.Proteomics.2004 Dec;4(12):3665-85
7.Fountoulakis M,Lahm HW.Hydrolysis and amino acid composition of proteins.J Chromatogr A.1998 Nov 27;826(2):109-34.
8.马海蓉,乍维琪。大规模的酵母双杂交系统在蛋白质相互作用组图谱中的应用。微生物学通报,2003,30(6):119-123
9.López JL.Two-dimensional electrophoresis in proteome expression analysis.J Chromatogr B Analyt Technol Biomed Life Sci.2007 Apr 15;849(1-2):190-202
10.Barnouin K.Two-dimensional gel electrophoresis for analysis of protein complexes.Methods Mol Biol.2004;261:479-98.
11.Jorrín JV,Maldonado AM,Castillejo MA.Plant proteome analysis:a 2006 update.Proteomics.2007 Aug;7(16):2947-62
12.Hoorn EJ,Hoffert JD,Knepper MA.The application of DIGE-based proteomics to renal physiology.Nephron Physiol.2006;104(1):p61-72
13.Wheelock AM,Goto S.Effects of post-electrophoretic analysis on variance in gel-based proteomics.Expert Rev Proteomics.2006 Feb;3(1):129-42
14.Woodson SA.Structure and assembly of group I introns.Curr Opin Struct Biol.2005 Jun;15(3):324-30.
15.Gygi SP,Rist B,Griffin TJ,Eng J,Aebersold R.Proteome analysis of low-abundance proteins using multidimensional chromatography and isotope-coded affinity tags.J Proteome Res.2002 Jan-Feb;1(1):47-54
16.雷红灵,吴永尧。蛋白质组学研究策略及质谱技术的应用。湖北民族学院学报:自然科学版,2007,25(3):346-349
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4、 Dominguez DC, Lopes R, Torres ML. Proteomics technology. Clin Lab Sci. 2007;20(4): 239-44
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82、 Sun Z, Lee E, Herring SW. Cell proliferation and osteogenic differentiation of growing pig cranial sutures. J Anat. 2007; 211(3): 280-9.
83、 Schick B, Wolf G, Romeike BF, Mestres P, Praetorius M, Plinkert PK. Dural cell culture. A new approach to study duraplasty. Cells Tissues Organs. 2003; 173(3):129-37
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85、 Thery M, Bornens M. Cell shape and cell division. Curr Opin Cell Biol. 2006;18(6): 648-57
86、 Thery M, Racine V, Pepin A, Piel M, Chen Y, Sibarita JB, et al. The extracellular matrix guides the orientation of the cell division axis. Nat Cell Biol. 2005; 7(10):947-53
87、 Thery M, Racine V, Piel M, Pepin A, Dimitrov A, Chen Y, et al. Anisotropy of cell adhesive microenvironment governs cell internal organization and orientation of polarity. Proc Natl Acad Sci U S A. 2006; 103(52): 19771-6
88、 Parker KK, Brock AL, Brangwynne C, Mannix RJ, Wang N, Ostuni E, et al. Directional control of lamellipodia extension by constraining cell shape and orienting cell tractional forces. FASEB J. 2002; 16(10): 1195-204
89、 Novak IL, Slepchenko BM, Mogilner A, Loew LM. Cooperativity between cell contractility and adhesion. Phys Rev Lett. 2004; 93(26 Pt 1): 268109
90、 Wang N, Ostuni E, Whitesides GM, Ingber DE. Micropatterning tractional forces in living cells. Cell Motil Cytoskeleton. 2002; 52(2): 97-106
91、 Yamakita Y, Totsukawa G, Yamashiro S, Fry D, Zhang X, Hanks SK, et al.Dissociation of FAK/p130(CAS)/c-Src complex during mitosis: role of mitosis-specific serine phosphorylation of FAK. J Cell Biol. 1999; 144(2): 315-24.
92、 Sunada R, Gorzer I, Oma Y, Yoshida T, Suka N, Wintersberger U, et al. The nuclear actin-related protein Act3p/Arp4p is involved in the dynamics of chromatin-modulating complexes.Yeast.2005;22(10):753-68
93、陈强,史俊南,任雨笙。牛血小板衍化尘长因子对人牙周膜成纤维细胞超微结构的影响。牙体牙髓牙周病学杂志1998,8(1):29-31
94、韦长元,陈建思,刘剑仑,杨南武,李挺。应用顺铂后肝癌细胞超微结构的研究。中华肝脏病杂志,2000,8(2):89-90
1.Dominguez DC,Lopes R,Torres ML.Introduction to proteomics.Clin Lab Sci.2007 Fa11;20(4):234-8
2.Wittmann-Liebold B,Graack HR,Pohl T.Two-dimensional gel electrophoresis as tool for proteomics studies in combination with protein identification by mass spectrometry.Proteomics.2006 Sep;6(17):4688-703
3.Chambers G,Lawrie L,Cash P,Murray GI.Proteomics:a new approach to the study of disease.J Pathol.2000 Nov;192(3):280-8
4.Swinbanks D.Government backs proteome proposal.Nature.1995 Dec 14;378(6558):653.
5.Pandey A,Mann M.Proteomics to study genes and genomes.Nature.2000 Jun 15;405(6788):837-46
6.G(o|¨)rg A,Weiss W,Dunn MJ.Current two-dimensional electrophoresis technology for proteomics.Proteomics.2004 Dec;4(12):3665-85
7.Fountoulakis M,Lahm HW.Hydrolysis and amino acid composition of proteins.J Chromatogr A.1998 Nov 27;826(2):109-34.
8.马海蓉,乍维琪。大规模的酵母双杂交系统在蛋白质相互作用组图谱中的应用。微生物学通报,2003,30(6):119-123
9.López JL.Two-dimensional electrophoresis in proteome expression analysis.J Chromatogr B Analyt Technol Biomed Life Sci.2007 Apr 15;849(1-2):190-202
10.Barnouin K.Two-dimensional gel electrophoresis for analysis of protein complexes.Methods Mol Biol.2004;261:479-98.
11.Jorrín JV,Maldonado AM,Castillejo MA.Plant proteome analysis:a 2006 update.Proteomics.2007 Aug;7(16):2947-62
12.Hoorn EJ,Hoffert JD,Knepper MA.The application of DIGE-based proteomics to renal physiology.Nephron Physiol.2006;104(1):p61-72
13.Wheelock AM,Goto S.Effects of post-electrophoretic analysis on variance in gel-based proteomics.Expert Rev Proteomics.2006 Feb;3(1):129-42
14.Woodson SA.Structure and assembly of group I introns.Curr Opin Struct Biol.2005 Jun;15(3):324-30.
15.Gygi SP,Rist B,Griffin TJ,Eng J,Aebersold R.Proteome analysis of low-abundance proteins using multidimensional chromatography and isotope-coded affinity tags.J Proteome Res.2002 Jan-Feb;1(1):47-54
16.雷红灵,吴永尧。蛋白质组学研究策略及质谱技术的应用。湖北民族学院学报:自然科学版,2007,25(3):346-349
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