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物理化学学报  2017, Vol. 33 Issue (12): 2446-2453    DOI: 10.3866/PKU.WHXB201706133
论文     
软骨细胞体外增殖去分化的拉曼光谱分析
金璐頔1,徐晶晶2,张勇3,余跃洲2,刘畅3,赵东平3,叶安培1,3,*()
1 北京大学前沿交叉学科研究院,北京100871
2 北京大学-清华大学生命科学联合中心,北京100871
3 北京大学信息科学技术学院,纳米器件物理与化学教育部重点实验室,北京100871
Raman Spectroscopic Analysis of Chondrocyte Dedifferentiation during in vitro Proliferation
Lu-Di JIN1,Jing-Jing XU2,Yong ZHANG3,Yue-Zhou YU2,Chang LIU3,Dong-Ping ZHAO3,An-Pei YE1,3,*()
1 Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, P. R. China
2 Peking-Tsinghua Center for Life Sciences, Beijing 100871, P. R. China
3 Key Laboratory for the Physics & Chemistry of Nano-devices, School of Electronics Engineering & Computer Science, Peking University, Beijing 100871, P. R. China
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摘要:

基于显微拉曼光谱技术,对组织工程的软骨种子细胞在传代增殖过程中的去分化进行单细胞分析。首先,对体外单层培养的第1-4代(P1-P4)大鼠软骨细胞样本进行了单细胞拉曼光谱检测,由此识别出软骨细胞中各种碱基、糖基、氨基酸等主要物质分子结构的特征峰集合。随后,分析拉曼光谱中若干重点特征峰强度随细胞传代次数的变化,发现软骨细胞体外增殖过程中核酸(789、1094、1576 cm-1)含量降低、Ⅱ型胶原(特异组分为羟脯氨酸,1207 cm-1)和蛋白聚糖(特异组分为糖胺聚糖,1042、1063、1126、1160 cm-1)合成下降、脂质(1304 cm-1)及磷酸盐(957 cm-1)含量增加等分子水平变化,从而在活体单细胞层次初步揭示了去分化引起软骨细胞增殖变缓、分泌减弱、形态纤维化等现象的分子机制。

关键词: 软骨细胞去分化单细胞分析显微拉曼光谱分子机制    
Abstract:

Seeded chondrocytes play a crucial role in current cartilage tissue engineering, yet both the quality and quantity of these cells could be impaired owing to cell dedifferentiation during in vitro proliferation. Here, we used micro-Raman spectroscopy to investigate changes in cellular components upon monolayer culturing of primary rat chondrocytes through multiple passages. Based on the average spectral profiles, we detected a series of Raman peaks and recognized related radicals such as nucleobases, pyranose rings, sulfate, tyrosine, proline, and amides at the single-chondrocyte level. Quantitative analysis of the Raman peak intensities showed that nucleic acids (at 789, 1094, 1576 cm-1) decreased significantly from passage 1 (P1) to passage 4 (P4), whereas lipids (at 1304 cm-1) and phosphate (at 957 cm-1) increased significantly. Moreover, the syntheses of two major hyaline cartilage-associated proteins, aggrecan and type-2 collagen, were impeded, as indicated by the marked decline in the levels of their specific components (glycosaminoglycan at 1042, 1063, 1126, 1160 cm-1, and hydroxyproline at 1207 cm-1). Taken together, these features reveal the diminished propagation and secretion abilities of passaged chondrocytes needed for matrix-induced implantation, and shed light on the molecular mechanism of chondrocyte dedifferentiation.

Key words: Chondrocyte    Dedifferentiation    Single-cell analysis    Micro-Raman spectroscopy    Molecular mechanism
收稿日期: 2017-05-02 出版日期: 2017-06-13
中图分类号:  O641  
基金资助: 国家自然科学基金(U1636110);科技部“科技支撑计划”(2012BAF14B14);北京大学“医学-信息”交叉研究种子基金(2014-MI-19)
通讯作者: 叶安培     E-mail: yap@pku.edu.cn
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引用本文:

金璐頔,徐晶晶,张勇,余跃洲,刘畅,赵东平,叶安培. 软骨细胞体外增殖去分化的拉曼光谱分析[J]. 物理化学学报, 2017, 33(12): 2446-2453.

Lu-Di JIN,Jing-Jing XU,Yong ZHANG,Yue-Zhou YU,Chang LIU,Dong-Ping ZHAO,An-Pei YE. Raman Spectroscopic Analysis of Chondrocyte Dedifferentiation during in vitro Proliferation. Acta Phys. -Chim. Sin., 2017, 33(12): 2446-2453.

链接本文:

http://www.whxb.pku.edu.cn/CN/10.3866/PKU.WHXB201706133        http://www.whxb.pku.edu.cn/CN/Y2017/V33/I12/2446

图1  本工作所用的共聚焦显微拉曼实验平台
图2  P1-P4组细胞归一化后的均值拉曼光谱
图3  P1-P4组软骨细胞均值拉曼光谱的共有特征峰
Raman peak wavenumbers/cm-1Wavenumbers in references/cm-1Peak assignments
P1P2P3P4
789787791789787DNA/RNA (O-P-O stretch, cytosine, uracil, thymine)
861860860857858Proline
935933942942937Ring breathing mode of tyrosine, and C-C stretch of proline ring
954-955954957Phosphate (hydroxyapatite)
10011000100110001001Symmetric ring breathing mode of phenylalanine
10441044104410441042Pyranose ring
106110591063-1063Sulfate
10881091109310911094DNA/RNA (O-P-O stretch)
11211123112311261126Pyranose ring
11631161116111631160Pyranose ring
12071204120512021207Aromatic ring stretch
12451247124412451245Amide Ⅲ (CH2 wagging, C-N stretch)
1303-130613041304Lipids (CH2/CH3 twisting)
14421439144414441446Lipids/proteins (CH2 bending, amide Ⅰ)
--1515-1515C=C stretch of carotenoid
15721574157615771576Guanine-N3
16521654165516561656Amide Ⅰ (C=O stretch)
表1  P1-P4组软骨细胞拉曼光谱特征峰位置及其归属24-35
图4  P1–P4组软骨细胞若干拉曼特征峰的相对强度比较
1 von der Mark K. ; Gauss V. ; von der Mark H. ; Muller P. Nature 1977, 267, 531.
doi: 10.1038/267531a0
2 Freed L. E. ; Marquis J. C. ; Nohria A. ; Emmanual J. ; Mikos A. G. ; Langer R. J. Biomed. Mater. Res. 1993, 27, 11.
doi: 10.1002/jbm.820270104
3 Vacanti C. A. ; Kim W. S. ; Schloo B. ; Upton J. ; Vacanti J. P. Am. J. Sports Med. 1994, 22, 485.
doi: 10.1177/036354659402200408
4 Freed L. E. ; Marquis J. C. ; Langer R. ; Vunjak-Novakovic G. ; Emmanual J. Biotechnol. Bioeng. 1994, 43, 605.
doi: 10.1002/bit.260430710
5 Sato T. ; Chen G. P. ; Ushida T. ; Ishii T. ; Ochiai N. ; Tateishi T. Mater. Sci. Eng. C-Biomimetic Supramol. Syst. 2001, 17, 83.
doi: 10.1016/S0928-4931(01)00313-7
6 Mayne R. ; Vail M. S. ; Mayne P. M. ; Miller E. J. Proc. Natl. Acad. Sci. U. S. A. 1976, 73, 1674.
doi: 10.1073/pnas.73.5.1674
7 Benya P. D. ; Padilla S. R. ; Nimni M. E. Cell 1978, 15, 1313.
doi: 10.1016/0092-8674(78)90056-9
8 Benya P. D. ; Shaffer J. D. Cell 1982, 30, 215.
doi: 10.1016/0092-8674(82)90027-7
9 Dominice J. ; Levasseur C. ; Larno S. ; Ronot X. ; Adolphe M. Mech. Ageing Dev. 1987, 37, 231.
doi: 10.1016/0047-6374(86)90040-0
10 Knutsen G. ; Drogset J. O. ; Engebretsen L. ; Gr ntvedt T. ; Isaksen V. ; Ludvigsen T. C. ; Roberts S. ; Solheim E. ; Strand T. ; Johansen O. J. Bone Joint Surg.-Am. Vol. 2007, 89, 2105.
doi: 10.2106/JBJS.G.00003
11 Shelbourne K. D. ; Jari S. ; Gray T. J. Bone Joint Surg.-Am. Vol. 2003, 85 (Suppl. 2), 8.
doi: 10.2106/00004623-200300002-00002
12 Binette F. ; McQuaid D. P. ; Haudenschild D. R. ; Yaeger P. C. ; McPherson J. M. ; Tubo R. J. Orthop. Res. 1998, 16, 207.
doi: 10.1002/jor.1100160208
13 Claus S. ; Aubert-Foucher E. ; Demoor M. ; Camuzeaux B. ; Paumier A. ; Piperno M. ; Damour O. ; Duterque-Coquillaud M. ; Galéra P. ; Mallein-Gerin F. J. Cell. Biochem. 2010, 111, 1642.
doi: 10.1002/jcb.22897
14 Liu G. Y. ; Kawaguchi H. ; Ogasawara T. ; Asawa Y. ; Kishimoto J. ; Takahashi T. ; Chung U. I. ; Yamaoka H. ; Asato H. ; Nakamura K. ; Takato T. ; Hoshi K. J. Biol. Chem. 2007, 282, 20407.
doi: 10.1074/jbc.M608383200
15 Feng S. Y. ; Wang W. B. ; Tai I. T. ; Chen G. N. ; Chen R. ; Zeng H. S. Biomed. Opt. Express 2015, 6, 3494.
doi: 10.1364/BOE.6.003494
16 Wu L. ; Li F. ; Jin Z. Y. ; Li Y. T. ; Hu W. Prog. Biochem. Biophys. 2016, 43, 281.
doi: 10.16476/j.pibb.2015.0341
吴雷; 李菲; 金周雨; 李雨婷; 胡薇. 生物化学与生物物理进展, 2016, 43, 281.
doi: 10.16476/j.pibb.2015.0341
17 Vo-Dinh T. ; Wang H. N. ; Scaffidi J. J. Biophotonics 2010, 3, 89.
doi: 10.1002/jbio.200910015
18 Sirimuthu N. M. ; Syme C. D. ; Cooper J. M. Anal. Chem. 2010, 82, 7369.
doi: 10.1021/ac101480t
19 Dochow S. ; Krafft C. ; Neugebauer U. ; Bocklitz T. ; Henkel T. ; Mayer G. ; Albert J. ; Popp J. Lab Chip 2011, 11, 1484.
doi: 10.1039/C0LC00612B
20 Banerjee H. N. ; Zhang L. Mol. Cell. Biochem. 2007, 295, 237.
doi: 10.1007/s11010-006-9278-4
21 Stone N. ; Kendall C. ; Shepherd N. ; Crow P. ; Barr H. J. Raman Spectrosc. 2002, 33, 564.
doi: 10.1002/jrs.882
22 Kunstar A. ; Leijten J. ; van Leuveren S. ; Hilderink J. ; Otto C. ; van Blitterswijk C. A. ; Karperien M. ; van Apeldoorn A. A. J. Biomed. Opt. 2012, 17, 116012.
doi: 10.1117/1.JBO.17.11.116012
23 Kumar R. ; Singh G. P. ; Gr nhaug K. M. ; Afseth N. K. ; de Lange Davies C. ; Drogset J. O. ; Lilledahl M. B. Int. J. Mol. Sci. 2015, 16, 9341.
doi: 10.3390/ijms16059341
24 Cheng W. T. ; Liu M. T. ; Liu H. N. ; Lin S. Y. Microsc. Res. Techniq. 2005, 68, 75.
doi: 10.1002/jemt.20229
25 Faoláin E. . ; Hunter M. B. ; Byrne J. M. ; Kelehan P. ; McNamara M. ; Byrne H. J. ; Lyng F. M. Vib. Spectrosc. 2005, 38, 121.
doi: 10.1016/j.vibspec.2005.02.013
26 Ellis R. ; Green E. ; Winlove C. P. Connect. Tissue. Res. 2009, 50, 29.
doi: 10.1080/03008200802398422
27 Bonifacio A. ; Beleites C. ; Vittur F. ; Marsich E. ; Semeraro S. ; Paoletti S. ; Sergo V. Analyst 2010, 135, 3193.
doi: 10.1039/C0AN00459F
28 Pudlas M. ; Brauchle E. ; Klein T. J. ; Hutmacher D. W. ; Schenke-Layland K. J. Biophotonics 2013, 6, 205.
doi: 10.1002/jbio.201200064
29 Frank C. J. ; McCreery R. L. ; Redd D. C. Anal. Chem. 1995, 67, 777.
doi: 10.1021/ac00101a001
30 Lakshmi R. J. ; Kartha V. B. ; Krishna C. M. ; Solomon J. G. R. ; Ullas G. ; Devi P. U. Radiat. Res. 2002, 157, 175.
doi: 10.1667/0033-7587(2002)157[0175:TRSFTS]2.0.CO;2
31 Notingher I. ; Green C. ; Dyer C. ; Perkins E. ; Hopkins N. ; Lindsay C. ; Hench L. L. J. Roy. Soc. Interface 2004, 1, 79.
doi: 10.1098/rsif.2004.0008
32 Ruiz-Chica A. J. ; Medina M. A. ; Sánchez-Jiménez F. ; Ramírez F. J. J. Raman Spectrosc. 2004, 35, 93.
doi: 10.1002/jrs.1107
33 Fung M. F. K. ; Senterman M. K. ; Mikhael N. Z. ; Lacelle S. ; Wong P. T. T. Biospectroscopy 1996, 2, 155.
doi: 10.1002/(SICI)1520-6343(1996)2:3<155::AID-BSPY2>3.0.CO;2-7
34 Tarnowski C. P. ; Ignelzi M. A. ; Morris M. D. J. Bone Miner. Res. 2002, 17, 1118.
doi: 10.1359/jbmr.2002.17.6.1118
35 Verrier S. ; Notingher I. ; Polak J. M. ; Hench L. L. Biopolymers 2004, 74, 157.
doi: 10.1002/bip.20063
36 Vacanti C. A. ; Langer R. ; Schloo B. ; Vacanti J. P. Plast. Reconstr. Surg. 1991, 88, 753.
doi: 10.1097/00006534-199111000-00001
37 Rosenberg L. J. Bone Joint Surg.-Am. Vol. 1971, 53, 69.
doi: 10.2106/00004623-197153010-00007
38 Huang Z. W. ; Lui H. ; Chen X. K. ; Alajlan A. ; McLean D. I. ; Zeng H. S. J. Biomed. Opt. 2004, 9, 1198.
doi: 10.1117/1.1805553
39 Potter K. ; Kidder L. H. ; Levin I. W. ; Lewis E. N. ; Spencer R. G. S. Arthritis Rheum. 2001, 44, 846.
doi: 10.1002/1529-0131(200104)44:4<846::AID-ANR141>3.0.CO;2-E
40 Anderson D. E. J. ; Athanasiou K. A. Ann. Biomed. Eng. 2008, 36, 1992.
doi: 10.1007/s10439-008-9572-2
41 Yoon Y. M. ; Kim S. J. ; Oh C. D. ; Ju J. W. ; Song W. K. ; Yoo Y. J. ; Huh T. L. ; Chun J. S. J. Biol. Chem. 2002, 277, 8412.
doi: 10.1074/jbc.M110608200
42 Mogilner I. G. ; Ruderman G. ; Grigera J. R. J. Mol. Graph. 2002, 21, 209.
doi: 10.1016/S1093-3263(02)00145-6
43 Dehring K. A. ; Crane N. J. ; Smukler A. R. ; McHugh J. B. ; Roessler B. J. ; Morris M. D. Appl. Spectrosc. 2006, 60, 1134.
doi: 10.1366/000370206778664743
44 Yin J. ; Yang Z. ; Cao Y. P. ; Ge Z. G. Chin. Med. J. 2011, 124, 4245.
doi: 10.3760/cma.j.issn.0366-6999.2011.24.022
45 Takahashi Y. ; Sugano N. ; Takao M. ; Sakai T. ; Nishii T. ; Pezzotti G. J. Mech. Behav. Biomed. Mater. 2014, 31, 77.
doi: 10.1016/j.jmbbm.2013.02.014
46 Dehring K. A. ; Smukler A. R. ; Roessler B. J. ; Morris M. D. Appl. Spectrosc. 2006, 60, 366.
doi: 10.1366/000370206776593582
47 Kang S. W. ; Yoo S. P. ; Kim B. S. Bio-Med. Mater. Eng. 2007, 17, 269.
48 Gremlich H. U. ; Yan B. Infrared and Raman Spectroscopy of Biological Materials;CRC Press: Boca Raton, 2000, pp421- 476.
49 Kumar R. ; Gr nhaug K. M. ; Afseth N. K. ; Isaksen V. ; de Lange Davies C. ; Drogset J. O. ; Lilledahl M. B. Anal. Bioanal. Chem. 2015, 407, 8067.
doi: 10.1007/s00216-015-8979-5
50 Chua K. H. ; Aminuddin B. S. ; Fuzina N. H. ; Ruszymah B. H. Eur. Cells Mater. 2005, 9, 58.
doi: 10.22203/eCM.v009a08
51 Kino-Oka M. ; Yashiki S. ; Ota Y. ; Mushiaki Y. ; Sugawara K. ; Yamamoto T. ; Takezawa T. ; Taya M. Tissue Eng. 2005, 11, 597.
doi: 10.1089/ten.2005.11.597
52 Furukawa K. S. ; Imura K. ; Tateishi T. ; Ushida T. J. Biotechnol. 2008, 133, 134.
doi: 10.1016/j.jbiotec.2007.07.957
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