治疗运动性损伤肌腱来源间充质干细胞体外培养与鉴定

doi:10.11843/j.issn.0366-6964.2025.08.022

Abstract

Abstract:

Obtaining high-quality tenogenic mesenchymal stem cells(MSCs) through in vitro culture, providing seed cells for the treatment of sports injuries (including equine athletic injuries), various diseases, single-cell sequencing, multi-omics research, genomic library construction, cDNA library construction, mitochondrial DNA library construction, gene function research and other investigations, had important in-depth research value. In this study, tendon tissues were collected from the skeletal muscles of 63 healthy male Peking duck embryos at 26 days of age (average weight: 40.3 g). Tenogenic MSCs were isolated and cultured in vitro using the type Ⅳ collagenase digestion method and the tissue block adhesion culture methods. Meanwhile, comprehensive quality assessment of the cultured cells in vitro were conducted according to international cell quality inspection standards and parameters. The results of 20 quality tests, including cellular morphology, pre-cryopreservation viability, microbial contamination (bacteria, fungi, protozoa, mycoplasma, viruses), positive marker expression, clonogenic capacity, migration ability, post-thaw morphology, post-thaw viability, growth curve analysis, population doubling time, reverse transcription PCR (RT-PCR) for surface markers, immunohistochemistry, karyotype analysis, and multidirectional differentiation potential (adirogenic, osteogenic, and chondrogenic induction), indicated that all established cell lines met international standarda for high-quality cells. Through a large number of repeated experiments, a scientific system for the culture of tenogenic MSCs in vitro for sports injuries therapy has been established, and a substantial quantity of high-quality donor cells has been acquired. This achievement lays a solid theoretical, methodological, technical, and material foundation for successfully constructing medical models for sports injury treatment, as well as for related research in zoology, animal science, veterinary medicine, and the broader life sciences.

Key words: mesenchymal stem cells, culture and identification, treatment of sports injuries

CLC Number:

S813.3

ZHANG Yang, WANG Zhongfa, LI Minjuan, HE Yunan, GUAN Weijun. Cultivation and Identification of Tenogenic Mesenchymal Stem Cells for Sports-Related Injury Therapy in vitro[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(8): 3813-3825.

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仪器与设备名称 Instrument/Equipment	品牌型号 Manufacturer & Model	产地 Origin	主要试剂 Main reagents
脉冲场电泳系统 Pulsed field electrophoresis system	Biometra	德国	DMEM/F12	Invitrogen
Cell-porator电击仪 Cell-porator electroporator	BTX-1800	德国	FBS	Gibco
电热恒温鼓风干燥箱 Electric thermostatic air drying oven	DHG-9203A	上海	N2 supplement	Gibco
超高速冷冻离心机 Ultra-high-speed	BECKMAN	德国	ITS	Gibco
台式高速低温离心机 High-speed low-temperature desktop centrifuge	Sigma	德国	RNA PCR Kit	TaKaRa
超纯水仪Ultrapure water system	Millipore	美国	胰蛋白酶	BBI
-80 ℃低温冰箱 -80 ℃ ultra low freezer	Forma	美国	马血清	Gibco
制冰机Ice maker	SANYO	日本	胰岛素	Coolaber
自动灭菌锅Automatic autoclave	TOMY	日本	DAPI染色液	Solarbio
酸度计Acidometer	Mettler Toledo	德国	IBMX	Sigma
凝胶成像系统Gel image system	Alpha Innotech	德国	FITC	Bioss
流式细胞仪Flow cytometer	FC-500	美国	desmin抗体	Bioss
倒置相差显微镜 Inverted phase contrast microscope	Olympus IX-71	日本	LIF	Bioss
程序降温仪Programmable Freezer	CBS 2100	美国	SCF	Bioss
液氮储存系统Liquid nitrogen storage system	RCB 500 Liquide	法国	bFGF	Bioss
高精度电子天平 High-precision electronic balance	Mettler Toledo AL104	美国	FGF-4	Bioss
电动移液器Electronic pipette	Eppendorf	德国	HGF	Bioss

基因 Gene	引物序列(5′→3′) Primer sequence	退火温度/℃ Temperature	产物长度/bp Products length
COLLAGEN Ⅰ	F: TCGGCTACTGCGGATTTCTT R: AAGACTGCCCAAGGGTCTTT	58	212
COLLAGEN Ⅲ	F: CTTAATGGCAGTTGTCACCGT R: TGCCAATGACAATCTTTGACAGG	58	247
NUCLEOSTEMIN	F: TCGGCTACTGCGGATTTCTT R: AAGACTGCCCAAGGGTCTTT	58	220
CD44	F: AAAAGACTTGCTGCATGCCC R: CAGCCAGGAAGAGCAGTAGG	57	255
TENASCIN-C	F: TCAGCTTGGGAGTCAGCCTA R: CCACACGCAATTGAGGAGTC	56	270
GAPDH	F: CCTCTACTGGGGCTGCTAAG R: AAGTCGCAGGAGACAACCTG	59	232
PPAR-γ	F: AGGTTTCATGACACGGGAGT R: AACAGCTGTGACGACTCTGG	60	261
LPL	F: GCCCACCTTTGAGTACGCT R: CCATGCTGGGCTTTTCTTCG	59	305
OPN	F: CGGTAAAACCGCACTTTCCTG R: CTTCCTCTGTGGCATCGTCT	59	297
RUNX2	F: TGATTTAGGGCGCATTCCTCA R: ATCTGGTGTGCAAGGCTCTG	59	304
SOX9	F: AAGTGCTCAAGGGCTACGAC R: TAGTCGGGGTGGTCCTTCTT	59	280
ACAN	F: CGACAGGCTAAAGGGTGAGG R: GGGAAATCCCGTCTGGTTGT	59	271
GAPDH	F: TTCGTGATGGGTGTGAACCA R: CTTAGCAGCCCCAGTAGAGG	60	255

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Cultivation and Identification of Tenogenic Mesenchymal Stem Cells for Sports-Related Injury Therapy in vitro

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