软骨细胞体外增殖去分化的拉曼光谱分析

doi:10.3866/PKU.WHXB201706133

物理化学学报 >> 2017, Vol. 33 >> Issue (12): 2446-2453.doi: 10.3866/PKU.WHXB201706133

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

收稿日期:2017-05-02 发布日期:2017-09-05
通讯作者: 叶安培 E-mail:yap@pku.edu.cn
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)

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 YE^1,^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

Received:2017-05-02 Published:2017-09-05
Contact: An-Pei YE E-mail:yap@pku.edu.cn
Supported by:
the National Natural Science Foundation of China(U1636110);the National Key Technologies R & D Program of China(2012BAF14B14);the Medicine-Informatics Interdisciplinary Project of Peking University, China(2014-MI-19)

摘要/Abstract

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

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. doi: 10.3866/PKU.WHXB201706133

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[J]. Acta Phys. -Chim. Sin. 2017, 33(12), 2446-2453. doi: 10.3866/PKU.WHXB201706133

链接本文: https://www.whxb.pku.edu.cn/CN/10.3866/PKU.WHXB201706133

https://www.whxb.pku.edu.cn/CN/Y2017/V33/I12/2446

参考文献

1	von der Mark K. ; Gauss V. ; von der Mark H. ; Muller P. Nature 1977, 267, 531.
2	Freed L. E. ; Marquis J. C. ; Nohria A. ; Emmanual J. ; Mikos A. G. ; Langer R. J. Biomed. Mater. Res. 1993, 27, 11.
3	Vacanti C. A. ; Kim W. S. ; Schloo B. ; Upton J. ; Vacanti J. P. Am. J. Sports Med. 1994, 22, 485.
4	Freed L. E. ; Marquis J. C. ; Langer R. ; Vunjak-Novakovic G. ; Emmanual J. Biotechnol. Bioeng. 1994, 43, 605.
5	Sato T. ; Chen G. P. ; Ushida T. ; Ishii T. ; Ochiai N. ; Tateishi T. Mater. Sci. Eng. C-Biomimetic Supramol. Syst. 2001, 17, 83.
6	Mayne R. ; Vail M. S. ; Mayne P. M. ; Miller E. J. Proc. Natl. Acad. Sci. U. S. A. 1976, 73, 1674.
7	Benya P. D. ; Padilla S. R. ; Nimni M. E. Cell 1978, 15, 1313.
8	Benya P. D. ; Shaffer J. D. Cell 1982, 30, 215.
9	Dominice J. ; Levasseur C. ; Larno S. ; Ronot X. ; Adolphe M. Mech. Ageing Dev. 1987, 37, 231.
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.
11	Shelbourne K. D. ; Jari S. ; Gray T. J. Bone Joint Surg.-Am. Vol. 2003, 85 (Suppl. 2), 8.
12	Binette F. ; McQuaid D. P. ; Haudenschild D. R. ; Yaeger P. C. ; McPherson J. M. ; Tubo R. J. Orthop. Res. 1998, 16, 207.
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.
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.
15	Feng S. Y. ; Wang W. B. ; Tai I. T. ; Chen G. N. ; Chen R. ; Zeng H. S. Biomed. Opt. Express 2015, 6, 3494.
16	Wu L. ; Li F. ; Jin Z. Y. ; Li Y. T. ; Hu W. Prog. Biochem. Biophys. 2016, 43, 281.
16	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.
17	Vo-Dinh T. ; Wang H. N. ; Scaffidi J. J. Biophotonics 2010, 3, 89.
18	Sirimuthu N. M. ; Syme C. D. ; Cooper J. M. Anal. Chem. 2010, 82, 7369.
19	Dochow S. ; Krafft C. ; Neugebauer U. ; Bocklitz T. ; Henkel T. ; Mayer G. ; Albert J. ; Popp J. Lab Chip 2011, 11, 1484.
20	Banerjee H. N. ; Zhang L. Mol. Cell. Biochem. 2007, 295, 237.
21	Stone N. ; Kendall C. ; Shepherd N. ; Crow P. ; Barr H. J. Raman Spectrosc. 2002, 33, 564.
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.
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.
24	Cheng W. T. ; Liu M. T. ; Liu H. N. ; Lin S. Y. Microsc. Res. Techniq. 2005, 68, 75.
25	Faoláin E. . ; Hunter M. B. ; Byrne J. M. ; Kelehan P. ; McNamara M. ; Byrne H. J. ; Lyng F. M. Vib. Spectrosc. 2005, 38, 121.
26	Ellis R. ; Green E. ; Winlove C. P. Connect. Tissue. Res. 2009, 50, 29.
27	Bonifacio A. ; Beleites C. ; Vittur F. ; Marsich E. ; Semeraro S. ; Paoletti S. ; Sergo V. Analyst 2010, 135, 3193.
28	Pudlas M. ; Brauchle E. ; Klein T. J. ; Hutmacher D. W. ; Schenke-Layland K. J. Biophotonics 2013, 6, 205.
29	Frank C. J. ; McCreery R. L. ; Redd D. C. Anal. Chem. 1995, 67, 777.
30	Lakshmi R. J. ; Kartha V. B. ; Krishna C. M. ; Solomon J. G. R. ; Ullas G. ; Devi P. U. Radiat. Res. 2002, 157, 175.
31	Notingher I. ; Green C. ; Dyer C. ; Perkins E. ; Hopkins N. ; Lindsay C. ; Hench L. L. J. Roy. Soc. Interface 2004, 1, 79.
32	Ruiz-Chica A. J. ; Medina M. A. ; Sánchez-Jiménez F. ; Ramírez F. J. J. Raman Spectrosc. 2004, 35, 93.
33	Fung M. F. K. ; Senterman M. K. ; Mikhael N. Z. ; Lacelle S. ; Wong P. T. T. Biospectroscopy 1996, 2, 155.
34	Tarnowski C. P. ; Ignelzi M. A. ; Morris M. D. J. Bone Miner. Res. 2002, 17, 1118.
35	Verrier S. ; Notingher I. ; Polak J. M. ; Hench L. L. Biopolymers 2004, 74, 157.
36	Vacanti C. A. ; Langer R. ; Schloo B. ; Vacanti J. P. Plast. Reconstr. Surg. 1991, 88, 753.
37	Rosenberg L. J. Bone Joint Surg.-Am. Vol. 1971, 53, 69.
38	Huang Z. W. ; Lui H. ; Chen X. K. ; Alajlan A. ; McLean D. I. ; Zeng H. S. J. Biomed. Opt. 2004, 9, 1198.
39	Potter K. ; Kidder L. H. ; Levin I. W. ; Lewis E. N. ; Spencer R. G. S. Arthritis Rheum. 2001, 44, 846.
40	Anderson D. E. J. ; Athanasiou K. A. Ann. Biomed. Eng. 2008, 36, 1992.
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.
42	Mogilner I. G. ; Ruderman G. ; Grigera J. R. J. Mol. Graph. 2002, 21, 209.
43	Dehring K. A. ; Crane N. J. ; Smukler A. R. ; McHugh J. B. ; Roessler B. J. ; Morris M. D. Appl. Spectrosc. 2006, 60, 1134.
44	Yin J. ; Yang Z. ; Cao Y. P. ; Ge Z. G. Chin. Med. J. 2011, 124, 4245.
45	Takahashi Y. ; Sugano N. ; Takao M. ; Sakai T. ; Nishii T. ; Pezzotti G. J. Mech. Behav. Biomed. Mater. 2014, 31, 77.
46	Dehring K. A. ; Smukler A. R. ; Roessler B. J. ; Morris M. D. Appl. Spectrosc. 2006, 60, 366.
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.
50	Chua K. H. ; Aminuddin B. S. ; Fuzina N. H. ; Ruszymah B. H. Eur. Cells Mater. 2005, 9, 58.
51	Kino-Oka M. ; Yashiki S. ; Ota Y. ; Mushiaki Y. ; Sugawara K. ; Yamamoto T. ; Takezawa T. ; Taya M. Tissue Eng. 2005, 11, 597.
52	Furukawa K. S. ; Imura K. ; Tateishi T. ; Ushida T. J. Biotechnol. 2008, 133, 134.

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软骨细胞体外增殖去分化的拉曼光谱分析

Raman Spectroscopic Analysis of Chondrocyte Dedifferentiation during in vitro Proliferation

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