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

摘要/Abstract

摘要：

通过体外培养获得高质量的肌腱来源间充质干细胞，为运动性损伤治疗、竞赛马运动性损伤治疗、诸多疾病的治疗、单细胞测序、多组学研究、基因组文库构建、cDNA文库构建、线粒体DNA文库构建和基因功能等诸多方面的研究提供种子细胞，具有重要的深入研究价值。本研究选取63只26日龄，平均体重40.3 g，雄性健康北京鸭胎鸭骨骼肌肌腱为研究对象，利用Ⅳ型胶原酶消化法和组织块贴壁培养法在体外进行肌腱来源间充质干细胞培养，同时按照国际细胞质量检测内容和标准对所培养的体外培养细胞进行了全面的质量检测。通过肌腱来源间充质干细胞的形态学、冻存前活率、微生物污染(细菌、真菌、原虫、支原体和病菌)、阳性率、克隆能力、迁移能力、表面标志物反转录PCR、冻存复苏后形态、冻存后活率、生长曲线绘制和群体倍增时间、免疫组织化学、核型、多向诱导分化潜能(向成脂细胞诱导分化能力、向成骨细胞诱导分化能力和向成软骨细胞诱导分化能力)等20项质量检测结果表明，所建细胞系均达到国际优质细胞标准。通过大量的重复试验证明，已建立了用于治疗运动性损伤肌腱来源间充质干细胞体外培养和鉴定的科学体系，并获得了大量的高质量供体细胞，为成功构建治疗运动性损伤医学模型，以及动物学、畜牧学和医学等生命科学领域的相关研究奠定了理论、方法、技术和实物基础。

关键词: 间充质干细胞, 培养与鉴定, 运动性损伤治疗

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

中图分类号:

S813.3

张洋, 王仲发, 李旻娟, 何玉楠, 关伟军. 治疗运动性损伤肌腱来源间充质干细胞体外培养与鉴定[J]. 畜牧兽医学报, 2025, 56(8): 3813-3825.

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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参考文献 28

1	WEIJ P,NAWATAM,WAKITANIS,et al.Human amniotic mesenchymal cells differentiate into chondrocytes[J].Cloning Stem Cells,2009,11(1):19-26. doi: 10.1089/clo.2008.0027
2	OHW,KIMD S,YANGY S,et al.Immunological properties of umbilical cord blood-derived mesenchymal stromal cells[J].Cell Immunol,2008,251(2):116-123. doi: 10.1016/j.cellimm.2008.04.003
3	WAKITANIS,OKABET,HORIBES,et al.Safety of autologous bone marrow-derived mesenchymal stem cell transplantation for cartilage repair in 41 patients with 45 joints followed for up to 11 years and 5 months[J].J Tissue Eng Regen Med,2011,5(2):146-150. doi: 10.1002/term.299
4	MATSUMOTOT,COOPERG M,GHARAIBEHB,et al.Cartilage repair in a rat model of osteoarthritis through intraarticular transplantation of muscle-derived stem cells expressing bone morphogenetic protein 4 and soluble flt-1[J].Arthr Rheumatol,2009,60(5):1390-1405. doi: 10.1002/art.24443
5	TOPOLUKN,HAWKINSR,TOKISHJ,et al.Amniotic mesenchymal stromal cells exhibit preferential osteogenic and chondrogenic differentiation and enhanced matrix production compared with adipose mesenchymal stromal cells[J].Amer J Sports Med,2017,45(11):2637-2646. doi: 10.1177/0363546517706138
6	DOMINICIM,LE BLANCK,MUELLERI,et al.Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement[J].Cytotherapy,2006,8(4):315-317. doi: 10.1080/14653240600855905
7	ZUKP A,ZHUM,MIZUNOH,et al.Multilineage cells from human adipose tissue: implications for cell-based therapies[J].Tissue Engineering,2001,7(2):211-228. doi: 10.1089/107632701300062859
8	李爱国,陈鸿辉,江斌,等.骨膜促进骨-肌腱结点愈合的研究[J].中国矫形外科杂志,2011,19(1):50-53.
	LIA G,CHENH H,JIANGB,et al.Periosteum promotes healing of bone-tendon junction[J].Orthopedic Journal of China,2011,19(1):50-53.
9	KIMT W,LEEM C,BAEH C,et al.Direct coculture of human chondrocytes and synovium-derived stem cells enhances in vitro chondrogenesis[J].Cell J,2018,20(1):53-60.
10	LIL F,BAIC Y,GONGX L,et al.Directed neural differentiation of duck embryonic germ cells[J].J Cell Biochem,2011,112(6):1514-1523. doi: 10.1002/jcb.23060
11	NIUH,SONGH,GUANY,et al.Chicken bone marrow mesenchymal stem cells improve lung and distal organ injury[J].Sci Rep,2021,11(1):17937-17953. doi: 10.1038/s41598-021-97383-4
12	LAIJ,YANGQ,LIANGR,et al.Isolation, culture and identification of bovine skeletal stem cells[J].J Biomater Tissue Engineer,2021,11(12):2337-2345. doi: 10.1166/jbt.2021.2816
13	吴月,王育南,宋哈楠,等.固始鸡脐带间充质干细胞原代培养及其诱导分化潜能研究[J].中国农业科技导报,2022,24(9):79-87.
	WUY,WANGY N,SONGH N,et al.Primary culture and induced differentiation potential of umbilical cord mesenchymal stem cells in Gushi Chickens[J].Journal of Agricultural Science and Technology,2022,24(9):79-87.
14	宗俊霖,王亚芹,刘晏辰,等.荷斯坦牛脐带间充质干细胞分离鉴定及分化研究[J].中国农业科技导报,2023,25(9):105-112.
	ZONGJ L,WANGY Q,LIUY C,et al.Isolation, identification and differentiation of umbilical cord mesenchymal stem cells in Holstein Cows[J].Journal of Agricultural Science and Technology,2023,25(9):105-112.
15	王亚芹,宗俊霖,张涛,等.牛脂肪间充质干细胞的分离培养及分化潜能的研究[J].中国畜牧兽医,2023,50(9):3530-3540.
	WANGY Q,ZONGJ L,ZHANGT,et al.Isolation, culture and differentiation potential of bovine adipose-derived mesenchymal stem cells[J].China Animal Husbandry & Veterinary Medicine,2023,50(9):3530-3540.
16	牛锐利,宋哈楠,吴月,等.西门塔尔牛表皮干细胞的分离培养与生物学特性研究[J].畜牧兽医学报,2023,54(1):168-177. doi: 10.11843/j.issn.0366-6964.2023.01.016
	NIUR L,SONGH N,WUY,et al.Isolation, culture and biological characteristics of epidermal stem cells in Simmental Cattle[J].Acta Veterinaria et Zootechnica Sinica,2023,54(1):168-177. doi: 10.11843/j.issn.0366-6964.2023.01.016
17	LIUX,ZHANGS,GUANW,et al.Isolation and characterization of mesenchymal stem cells from chicken liver[J].J Biomater Tissue Engineer,2020,10(1):8-16. doi: 10.1166/jbt.2020.2225
18	YANB,LIANGR,JIM,et al.Biological characteristics and multilineage differentiation of kidney mesenchymal stem cells derived from the Tibetan Mastiff[J].J Biomater Tissue Engineer,2019,9(7):904-913. doi: 10.1166/jbt.2019.2093
19	LUT,WANGX,ZHENGY,et al.Isolation, culture and biological characteristics of Tibetan Mastiff (Canis lupus Familiaris) muscle satellite cells[J].J Biomater Tissue Engineer,2019,9(5):573-582. doi: 10.1166/jbt.2019.2030
20	WUX,GAOF,WUY,et al.Isolation and biological characteristics of sheep amniotic epithelial cells[J].Cytotechnology,2019,71(2):539-551. doi: 10.1007/s10616-019-00299-1
21	GAOF,WUY,WENH,et al.Multilineage potential research on pancreatic mesenchymal stem cells of bovine[J].Tissue Cell,2019,2(56):60-70.
22	LUT,PEIW,WANGK,et al.In vitro culture and biological properties of broiler adipose-derived stem cells[J].Exp Ther Med,2018,16(3):2399-2407.
23	MAC,GUOY,WENH,et al.Identification and multilineage potential research of a novel type of adipose-derived mesenchymal stem cells from goose inguinal groove[J].DNA Cell Biol,2018,37(9):731-741. doi: 10.1089/dna.2017.4061
24	WANGJ,WUX,ZHENGY,et al.Isolation and biological characterization of mesenchymal stem cells from goose dermis[J].Poult Sci,2018,97(9):3236-3247. doi: 10.3382/ps/pey178
25	MAC,WANGK,JIH,et al.Multilineage potential research of Beijing duck amniotic mesenchymal stem cells[J].Cell Tissue Bank,2018,19(4):519-529. doi: 10.1007/s10561-018-9701-6
26	WANGX,ZHAOS,LAIJ,et al.Anti-inflammatory, antioxidant, and antifibrotic effects of gingival-derived MSCs on bleomycin-induced pulmonary fibrosis in mice[J].I J Mol Sci,2021,23(1):99-115. doi: 10.3390/ijms23010099
27	YUANH,DUH,YUANZ,et al.Correlation analysis of skeletal muscle regeneration and differentiation of satellite cells[J].Acta Medica Mediterranea,2021,37(2):1321-1328.
28	ZHANGS,YINJ,JIH,et al.Functional β-cell differentiation of small-tail han sheep pancreatic mesenchymal stem cells and the therapeutic potential in type 1 diabetic mice[J].Pancreas,2020,49(7):947-954. doi: 10.1097/MPA.0000000000001604

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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