猪软骨脱细胞基质对人脂肪干细胞增殖及分化的影响

doi:10.7518/hxkq.2020.02.002

华西口腔医学杂志 ›› 2020, Vol. 38 ›› Issue (2): 122-127.doi: 10.7518/hxkq.2020.02.002

猪软骨脱细胞基质对人脂肪干细胞增殖及分化的影响

刘茜, 李雪健, 王忠山()

军事口腔医学国家重点实验室,口腔疾病国家临床医学研究中心,陕西省口腔医学重点实验室,空军军医大学口腔医院修复科,西安 710032

收稿日期:2019-09-11 修回日期:2019-12-16 出版日期:2020-04-01 发布日期:2020-04-15
通讯作者: 王忠山 E-mail:wzs_fmmu@163.com
作者简介:刘茜,硕士,E-mail：fmmuliuqian@foxmail.com
基金资助:
国家自然科学基金(81700930)

Effects of porcine acellular cartilaginous matrix on the proliferation and differentiation of human adipose-derived stromal cells

Liu Qian, Li Xuejian, Wang Zhongshan()

State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shanxi Key Laboratory of Stomatology, Dept. of Prosthodontics, School of Stomatology, Air Force Medical University, Xi’an 710032, China

Received:2019-09-11 Revised:2019-12-16 Online:2020-04-01 Published:2020-04-15
Contact: Zhongshan Wang E-mail:wzs_fmmu@163.com
Supported by:
The National Natural Science Foundation of China(81700930)

摘要/Abstract

摘要：

目的研究猪软骨脱细胞基质（pACM）对人脂肪干细胞（hADSCs）增殖及分化的影响。方法将猪关节软骨进行脱细胞处理制备pACM并进行鉴定。分离培养hADSCs并分化鉴定。将不同浓度pACM与hADSCs共培养,以不添加pACM为对照组,通过CCK-8法检测pACM对hADSCs增殖能力的影响,通过荧光定量聚合酶链反应、蛋白质印迹法检测pACM对hADSCs成软骨分化的影响。结果 0.5、1.0、2.0 mg·mL ^-1 pACM组hADSCs细胞增殖速率与对照组无统计学差异,而4.0、8.0 mg·mL ^-1pACM组hADSCs细胞增殖速率低于对照组（P<0.05）。0.5、1.0、2.0 mg·mL ^-1 pACM组成软骨相关基因SOX-9、抗Ⅱ型胶原纤维α1（COL2A1）、抗蛋白聚糖（ACAN）及细胞黏附相关基因层黏连蛋白（LAMININ）的表达均高于对照组（P<0.05）,细胞干性相关基因Notch-1的表达低于对照组（P<0.05）,成脂相关基因过氧化物酶体增殖物激活受体-γ（PPAr-γ）的表达各组间均无统计学差异（P>0.05）。成软骨相关蛋白SOX-9、COL2A1及ACAN的表达高于对照组（P<0.05）。结论适宜浓度的pACM不会影响hADSCs的增殖,并且可以在基因水平上诱导其向成软骨方向分化。

关键词: 脱细胞基质, 软骨, 人脂肪干细胞

Abstract:

Objective This study aimed to evaluate the effects of porcine acellular cartilaginous matrix (pACM) on the proliferation and differentiation of human adipose-derived stromal cells (hADSCs). Methods pACM was prepared from porcine articular cartilage through decellularization treatment. hADSCs were isolated from human adipose tissues and cultured with different pACM concentrations. No pACM was used as the control group. The effect of pACM on hADSCs proliferation was detected by CCK-8 method. Moreover, the effect of pACM on hADSCs chondrogenic differentiation was analyzed through fluorescence quantitative polymerase chain reaction and Western blot. Results hADSCs proliferation rate in 0.5, 1.0, and2.0 mg·mL ^-1 pACM groups was not significantly different from that in the control group, whereas that in 4.0 and 8.0 mg·mL ^-1 pACM group was lower than that in the control group (P<0.05). The expression levels of pACM chondrogenic genes, including SOX-9, collagen type Ⅱ alpha 1 chain (COL2A1), and aggrecan (ACAN) and cell adhesion-related gene LAMININ in 0.5, 1.0, and 2.0 mg·mL ^-1 pACM group were higher than those of the control group (P<0.05), but that of a stemness-related gene Notch-1 was lower than that of the control group (P<0.05). No statistical difference was found in the expression of a lipogenesis-related gene peroxisome proliferator-activated receptor-γ (PPAr-γ) (P>0.05). The expression levels of chondrogenic proteins (SOX-9, COL2A1, and ACAN) were higher than those of the control group (P<0.05). Conclusion Appropriate pACM concentrations do not affect hADSCs proliferation but can induce hADSCs chondrogenic differentiation.

Key words: acellular matrix, cartilage, human adipose-derived stromal cells

中图分类号:

刘茜, 李雪健, 王忠山. 猪软骨脱细胞基质对人脂肪干细胞增殖及分化的影响[J]. 华西口腔医学杂志, 2020, 38(2): 122-127.

Liu Qian, Li Xuejian, Wang Zhongshan. Effects of porcine acellular cartilaginous matrix on the proliferation and differentiation of human adipose-derived stromal cells[J]. West China Journal of Stomatology, 2020, 38(2): 122-127.

图/表 6

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参考文献 16

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目的基因	引物序列（5’-3’）
GAPDH	F：TCAGCAATGCCTCCTGCAC
	R：TCTGGGTGGCAGTGATGGC
SOX-9	F：AGCGAACGCACATCAAGAC
	R：CTGTAGGCGATCTGTTGGGG
COL2A1	F：TGGACGATCAGGCGAAACC
	R：GCTGCGGATGCTCTCAATCT
ACAN	F：ACTCTGGGTTTTCGTGACTCT
	R：ACACTCAGCGAGTTGTCATGG
LAMININ	F：ACTGGACTCACCTACGCCAAC
	R：GGGAGCACACTGGTCACATTT
Notch-1	F：CGAACCAATACAACCCTCTGC
	R：CTGGTAGCTCATCATCTGGGACA
PPAr-γ	F：GGTGACTTTATGGAGCCCAAGT
	R：GACTCATGTCTGTCTCCGTCTTC

猪软骨脱细胞基质对人脂肪干细胞增殖及分化的影响

Effects of porcine acellular cartilaginous matrix on the proliferation and differentiation of human adipose-derived stromal cells

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