用于人类脂肪干细胞的纯化和体外长期培养的聚合物涂层

doi:10.7503/cjcu20170121

摘要/Abstract

摘要：

采用紫外固化法制备了基于丙烯酸酯类水凝胶的聚合物涂层(PC), 并用X射线光电子能谱(XPS)、水接触角(WCA)和原子力显微镜(AFM)分别对PC进行了化学组成和表面性能的表征. 在PC表面进行了人类脂肪干细胞(hASC)的体外长期培养扩增, 得到的第3代细胞的生物学表征结果表明, 干细胞在PC表面能正常黏附生长, 流式细胞仪检测发现干细胞对特征标记物CD49d, CD73, CD105的阳性显性比例较高, 对HLA-DR和CD31几乎不显性, 说明扩增的干细胞具有hASC特征. 对PC上扩增的干细胞进行诱导分化, 并用油红O、茜素红和阿利新蓝分别进行染色分析, 结果表明, 该干细胞保留了hASC的多能特性: 能分化为成脂、成骨和成软骨细胞. 含有单体甲基丙烯酰氧乙基三甲基氯化铵(DMC)、甲基丙烯酸环己酯(CHMA)和甲基丙烯酸-2-(二乙氨基)乙酯(DEAEMA)的PC2(质量比为3:1:2)在用于hASC体外长期培养时, 比其它PC和TCP更有利于细胞的黏附和增殖, 纯化细胞, 保持其多能性. 实时荧光定量PCR(RT-qPCR)的分析表明PC2上得到的细胞更容易向成骨和成软骨细胞分化.

关键词: 聚合物涂层, 人类脂肪干细胞, 体外长期培养, 纯化, 分化, 丙烯酸酯类水凝胶

Abstract:

Polymer coating(PC) based on polyacrylate hydrogels was prepared through UV initiated polymerization and characterized with X-ray photoelectron spectroscopy(XPS), water contact angles(WCA) and atomic force microscopy(AFM) respectively to understand their chemistry and surface properties. The biological characteristics of human adipose-derived stem cell(hASC) were evaluated after long-term cultured on PC in vitro for 3 passages. The results indicated that the hASC proliferated successfully on the PC, expressed high percentage of specific membrane markers CD49d, CD73, CD105, and very low percentage of HLA-DR and CD31 by using flow cytometry. The results suggested that the proliferated stem cells had the characteristics of hASC. The hASC were differentiated to adipocytes, osteoblasts and chondrocytes and stained with Oil Red O, Alizarin Red S and Alcian Blue respectively to demonstrate the maintaining of pluripotency of hASC. The results showed that the PC2 based on methacrylatoethyltrimethyl ammonium chloride, cyclohexyl methacrylate and diethyl amino ethyl methacrylate(mass ratio of 3:1:2) was the best in hASC binding and proliferation, improved the purification, maintained the pluripotency among the three coatings and TCP. Relative gene expression of the differentiated cells was analyzed by quantitative real-time polymerase chain reaction(RT-qPCR). The results show that the harvest hASC from PC2 are apt to differentiate to osteoblasts and chondrocytes.

Key words: Polymer coating, Human adipose-derived stem cell, Long-term culture in vitro, Purification, Differentiation, Polyacrylate hydrogel

中图分类号:

O632

TrendMD:

邱林子, 王桂容, 潘艳, 王溢文, 李新庆, 邓林红, 张嵘. 用于人类脂肪干细胞的纯化和体外长期培养的聚合物涂层. 高等学校化学学报, 2017, 38(10): 1897.

QIU Linzi, WANG Guirong, PAN Yan, WANG Yiwen, LI Xinqing, DENG Linhong, Mark Bradley, ZHANG Rong. Polymer Coatings for Purification and Long-term Culture of Human Adipose-derived Stem Cells^†. Chem. J. Chinese Universities, 2017, 38(10): 1897.

图/表 14

Table 1 Mass ratio of monomers used for the preparation of PC

Polymer coating	Component	m(DMC):m(CHMA):m(DEAEMA)
PC1	Poly(DMC-co-CHMA-co-DEAEMA)	1:1:1
PC2	Poly(DMC-co-CHMA-co-DEAEMA)	3:1:2
PC3	Poly(DMC-co-CHMA-co-DMAEMA)	3:1:2

Table 2 Surface free energy of PC

Polymer coating	Γ/(mN·m^-1)	γ^d/(mN·m^-1)	γ^p/(mN·m^-1)
PC1	33.5	12.9	20.6
PC2	34.7	11.3	23.4
PC3	36.1	11.2	24.9

Fig.1 Water contact angles of PC

Table 3 Elemental compositions(%) of PC based on XPS analysis

Element	PC1		PC2		PC3
Element	Experimental	Theoretical	Experimental	Theoretical	Experimental	Theoretical
C₁_s	68.2	63.8	70.6	63.6	50.0	59.9
N₁_s	4.5	4.8	5.0	4.6	6.9	4.8
O₁_s	18.3	17.5	15.6	16.9	33.5	17.4
C $l 2 p$	0.8	5.7	1.1	8.5	1.8	8.5

Table 3 Elemental compositions(%) of PC based on XPS analysis

Element	PC1		PC2		PC3
Element	Experimental	Theoretical	Experimental	Theoretical	Experimental	Theoretical
C₁_s	68.2	63.8	70.6	63.6	50.0	59.9
N₁_s	4.5	4.8	5.0	4.6	6.9	4.8
O₁_s	18.3	17.5	15.6	16.9	33.5	17.4
C $l 2 p$	0.8	5.7	1.1	8.5	1.8	8.5

Fig.2 XPS spectra of PC(A) Survey spectra; (B—D) high resolution of C1s spectra[PC1(B), PC2(C), PC3(D)].

Fig.3 Representative AFM images of PC1(A), PC2(B) and PC3(C)

Fig.4 Root-mean-square roughness of PC

Fig.5 Average hASC cell numbers per unit area after 1, 4 and 7 d of incubation on PC

Fig.6 Fluorescence images of hASC cultured on the surface of PC2 for 1 d(A, A'), 4 d(B, B') and 7 d(C, C'), respectively(A—C) DAPI(λEx/λEm 358 nm/461 nm)stained nuclei of hASC;(A'—C') calcein-AM(λEx/λEm 490 nm/515 nm)stained hASC.

Fig.7 Expression of surface antigenes before culture and after 3 passages on PC2

Table 4 Expression of CD105 and Cd49d of hASC cultured on PC and TCP

Polymer coating	CD105			CD49d
Polymer coating	p0	p1	p3	p0	p1	p3
TCP	42.04	75.42	87.91	37.91	71.79	77.23
PC1		67.18	93.44		59.09	69.38
PC2		89.90	97.11		61.40	85.09
PC3		54.57	95.51		31.91	80.18

Fig.8 Images of adipogenic(A1—D1), osteogenic(A2—D2) and chondrogenic(A3—D3) differentiation of hASC cultured on PC1(A1—A3), PC2(B1—B3), PC3(C1—C3) and TCP(D1—D3) respectivelyAdipocytes were stained with Oil Red O, Osteocytes with Alizarin Red S and Chondrocytes with Alcian Blue.

Fig.9 Relative gene expression of adipogenic, osteogenic and chondrogenic differentiation of hASC cultured on PC2 and TCP*Denotes a statistical significance of P<0.05, compared to data obtained on TCP.

Table 5 Primers information used in RT-qPCR

Gene name	Primer sequence(5' to 3')	Length(bp)	T_m/℃
PPARγ2	CAGGCTCCACTTTGATTGC	19	57.6
PPARγ2	TCTCCAGCATTTCTACTACACA	22	58.2
ALP	CCACTGACTTCCCTGCCTTC	20	59.8
ALP	GGGCAACTTCCAGACCATT	19	57.6
SOX9	TGGTGGTCGGTGTGATCGTA	20	59.8
SOX9	CGAACGCACATCAAGACG	18	57.3

参考文献 27

[1]	Duffy C. R., Zhang R., How S. E., Biomaterials,2014, 35(23), 5998—6005
[2]	Mauney J. R., Kaplan D. L., Volloch V., Biomaterials,2004, 25(16), 3233—3243
[3]	Mauney J. R., Volloch V., Kaplan D. L., Biomaterials,2005, 26(31), 6167—6175
[4]	Duffy C. R. E., Zhang R., How S. E., Biomater. Sci., 2014, 2(11), 1683—1692
[5]	Kim M. R., Jeong J. H., Park T. G., Biotechnol. Progr., 2002, 18(3), 495—500
[6]	Stile R. A., Healy K. E., Biomacromolecules,2001, 2(1), 185—194
[7]	Haraguchi K. T. T., Ebato M., Biomacromolecules,2006, 7, 3267—3275
[8]	Mant A., Tourniaire G., Diaz-Mochon J. J., Biomaterials,2006, 27(30), 5299—5306
[9]	Anderson D. G., Levenberg S., Langer R., Nat. Biotechnol., 2004, 22(7), 863—866
[10]	Fisher J. P., Dean D., Engel P. S., Annu. Rev. Mater. Res., 2001, 31(1), 171—181
[11]	Burkoth A. K., Burdick J., Anseth K. S., J. Biomd. Mater. Res., 2000, 51(3), 352—359
[12]	Benoit D. S. W., Schwartz M. P., Durney A. R., Nat. Mater., 2008, 7(10), 816—823
[13]	Luo Y., Shoichet M. S., Nat. Mater., 2004, 3(4), 249—253
[14]	Tibbitt M. W., Anseth K. S., Biotechnol. Bioeng., 2009, 103(4), 655—663
[15]	Mann B. K., Biomaterials, 2001, 22(22), 3045—3051
[16]	Zuk P. A., Zhu M., Ashjian P., Mol. Microbiol., 2002, 13(12), 4279—4295
[17]	Zuk P. A., Zhu M., Mizuno H., Tissue Eng., 2001, 7(2), 211—228
[18]	Zhang R., Liberski A., Sanchez-Martin R., Biomaterials,2009, 30(31), 6193—6201
[19]	López-Pérez P. M., Marques A. P., J. Mater. Chem., 2007, 17(38), 4064—4071
[20]	Feng X. J., Shi Y. L., Yang W., Chemistry,2014, 77(5), 418—424
[21]	Kurkuri M. D., Driever C., Thissen H., Biomacromolecules,2009, 10(5), 1163—1172
[22]	Liu X., Feng Q., Bachhuka A., ACS Appl. Mater. Interfaces,2014, 6(12), 9733—9741
[23]	Yao X., Peng R., Ding J., Adv. Mater., 2013, 25(37), 5257—5286
[24]	Balloni S., Calvi E. M., Damiani F., Int. J. Oral Maxillofac. Implants,2009, 24(4)
[25]	De Ugarte D. A., Morizono K., Elbarbary A., Cells Tissues Organs, 2003, 174(3), 101—109
[26]	Moriguchi T., Yano K., Nakagawa S., J. Colloid Interface Sci., 2003, 260(1), 19—25
[27]	Paul H., Reginato A. J., Ralph S. H., Arthritis Rheumatism, 1983, 26(2), 191—200