大耳白兔骨髓间充质干细胞的分离培养与生物学鉴定

doi:10.13304/j.nykjdb.2023.0411

摘要/Abstract

摘要：

骨髓间充质干细胞（bone marrow mesenchymal stem cells， BMSCs）来源于发育早期的中胚层和外胚层，作为干细胞中的种子细胞，具有强大的自我复制和更新能力，并对治疗、修复、补充受损或缺损的肝、肌肉等组织有修复和治疗作用。为了研究BMSCs的生物学特性和多化潜能，对日本大耳白兔BMSCs进行体外分离培养及生物学鉴定研究。取日本大耳白胎兔骨髓组织，采用全骨髓贴壁法分离纯化、体外培养，鉴定日本大耳白兔BMSCs生物学特性，并进行细胞形态学观察、生长动力学检测及其成骨、成软骨、成脂肪能力鉴定。结果表明，采用全骨髓贴壁法成功从日本大耳白胎兔骨髓中分离得到生长状态良好的大耳白兔BMSCs，细胞形态为长梭形，生长曲线呈典型“S”形；免疫荧光和RT-PCR结果显示，大耳白兔BMSCs表达CD29、CD44、CD73、CD105间充质干细胞表面标记物基因，但不表达原始造血祖细胞标志物CD45；经特异性染色和RT-PCR分析表明，分离的细胞是间充质干细胞；体外诱导大耳白兔BMSCs能够分化为骨骼细胞、软骨细胞、脂肪细胞。建立了大耳白兔BMSCs体外分离培养体系，为利用骨髓间充质干细胞进行组织与工程学研究和动物遗传资源保存研究奠定了一定的理论基础。

关键词: 日本大耳白兔, 兔骨髓间充质干细胞, 分离培养, 生物学特性, 诱导分化

Abstract:

Bone marrow mesenchymal stem cells （BMSCs） derived from the early development of the mesoderm and ectoderm， as seed cells in stem cells， with strong self-replication and renewal ability， and have repair and therapeutic effects for the damaged or defective liver， muscle and other tissues. In order to study BMSCs， this article conducted in vitro isolation and culture and biological identification of Japanese large ear white rabbit bone marrow mesenchymal stem cells. The bone marrow tissue of Japanese large ear white rabbit was isolated， purified and cultured in vitro by whole bone marrow adhesion method to identify the biological characteristics of Japanese large ear white rabbit BMSCs， observe cell morphology， detect growth kinetics， and identify osteogenesis， cartilage and adipogenic ability. The results showed that BMSCs with good growth status were successfully isolated from the bone marrow of Japanese large ear white rabbits by whole bone marrow adhesion， with long spindle shape and a typical “S” shape in the growth curve. The results of immunofluorescence and RT-PCR showed that BMSCs of Japanese large ear white rabbits expressed CD29， CD44， CD73， CD105 mesenchymal stem cell surface marker genes， but didn’t express the original hematopoietic progenitor cell marker CD45. Specific staining and RT-PCR showed that the isolated cells were mesenchymal stem cells； in vitro induced BMSCs of Japanese large ear white rabbits to differentiate into bone， cartilage and fat cells. In this study， an in vitro isolation and culture system for BMSCs in Japanese large ear white rabbits was established， which laid a theoretical foundation for the use of bone marrow mesenchymal stem cells as tissue and engineering research and the conservation of animal genetic resources.animal genetic resources.

Key words: Japanese large eared white rabbit, rabbit bone marrow mesenchymal stem cells, isolation and cultivation, biological characteristics, induced differentiation

中图分类号:

S831

周世莹, 刘晏辰, 张洋, 杨雪松, 关伟军, 高扬. 大耳白兔骨髓间充质干细胞的分离培养与生物学鉴定[J]. 中国农业科技导报, 2023, 25(8): 96-105.

Shiying ZHOU, Yanchen LIU, Yang ZHANG, Xuesong YANG, Weijun GUAN, Yang GAO. Isolational Culture and Biological Identification of Japanese Large Ear White Rabbit Bone Marrow Mesenchymal Stem Cells[J]. Journal of Agricultural Science and Technology, 2023, 25(8): 96-105.

图/表 10

表1 引物信息

Table 1 Primer information

基因 Gene	引物序列（3’-5’） Primer sequence（3’-5’）	退火温度 Anneal temperature/℃	产物长度 Product length/bp
GAPDH	F：ACTATGGACGTAGAGCTGGTCCCTGA	61.00	492
GAPDH	R：TCCAGGCAGGCCCCAATTGTGGGTG	61.00	492
CD29	F： CGATGTGATGACTTAGAAGCCTTG	60.85	471
CD29	R：CAGAGACATAAAGAAAAATAAGA	60.85	471
CD44	F：GGAGGATATCACTCAGATCCAGCCA	60.60	373
CD44	R： GTTGAAGTTCAAGCGTGCTGAAGAC	60.60	373
CD73	F：AAGATGATCTGGAGAACGTCAAGA	61.65	623
CD73	R： ACTTCCGAATTGGGTTTGGCTCCTT	61.65	623
CD105	F： GCTCAAGAACCCTTGCACGAGTGAG	60.35	550
CD105	R： ACAGACAGAGGGGAGGAGTTCAACG	60.35	550
Collage typeⅠ	F： CCAACGATGGACAGTGCCACCTGG	59.30	425
Collage typeⅠ	R： GCCCACCTCGTCCAGAAACTCAG	59.30	425
osteopontin	F： CCTGTTTACAAGGAACTGAAAAAC	59.00	512
osteopontin	R： AATGTCATCCAGCTGATCATTGATG	59.00	512
LPL	F：GCCAGAAGGAGTAACGATAAATA	54.50	486
LPL	R：GAAGGAAGTGCTCCAATATCTCCAT	54.50	486
PPAR-γ	F： GCAAACTAACTGGCTATGGTCCT	60.10	473
PPAR-γ	R： ATTAAGTGTCACCCAGGATGTGC	60.10	473
ACAN	F：CACGTTTGAGTGTGGGGCTTGCAG	60.50	464
ACAN	R：GGGAGTGCGTCTGTGGGCAGTGTT	60.50	464
SOX9	F： CCTATTAATAATTTCAAGAATCCAA	60.70	507
SOX9	R： ACTATGGACGTAGAGCTGGTCCCTGA	60.70	507
CD45	F： ATGGTGAAGGTCGGAGTGAACGG	60.40	304
CD45	R： ATTTGGCCGCATTGGGCGCCTGGT	60.40	304

图1 日本大耳白兔骨髓间充质干细胞形态

Fig. 1 BMSCs morphology of Japanese large ear white rabbits

图2 日本大耳白兔骨髓间充质干细胞复苏前后形态

Fig. 2 Resuscitation morphology of Japanese large ear white rabbits BMSCs before and after recovery

图3 日本大耳白兔骨髓间充质干细胞生长曲线及群体倍增时间

Fig. 3 Growth curve， population doubling time of BMSCs in Japanese large ear white rabbits

图4 日本大耳白兔骨髓间充质干细胞划痕试验及不同代次克隆形成能力A： 6 h划痕试验；B： P3、P9、P15克隆团及克隆形成率，*表示差异在P<0.05水平显著

Fig. 4 Scratching experiment and clonogenic ability of Japanese large ear white rabbits BMSCsA： 6 h scratch test；B： P3， P9， P15 clone groups and the cloning rate， * indicates significant difference at P<0.05 level.

图5 日本大耳白兔骨髓间充质干细胞免疫荧光注：DAPI—核染；FITC—免疫荧光；Merge—DAPI和FITC图叠加。

Fig. 5 Immunofluorescence of Japanese large ear white rabbits BMSCsNote： DAPI—Nuclear staining；FITC—Immunofluorescence；Merge—Overlay of DAPI and FITC images.

图6 表面标记物RT-PCR检测注：M为DNA Marker；1~6分别为GAPDH、CD29、CD45、CD44、CD73和CD105。

Fig. 6 RT-PCR result of marker genesNote： M shows DNA Marker；1~6 represent GAPDH，CD29，CD45，CD44，CD73 and CD105，respectively.

图7 日本大耳白兔骨髓间充质干细胞成骨分化钙沉积茜素红染色和RT-PCR检测A：成骨诱导形态；B：成骨诱导后的RT-PCR检测；M为DNA Marker，1为GAPDH，2、4为诱导组，Ⅰ型胶原蛋白和骨桥蛋白基因为阳性；3为阴性对照组，Ⅰ型胶原蛋白基因为阴性

Fig. 7 Detection of calcium deposition in osteoblastic differentiation of Japanese large ear white rabbits BMSCs using alizarin red and RT-PCRA： Osteogenic induction morphology； B： RT-PCR after osteogenic induction；M shows DNA Marker； 1 shows GAPDH； 2 and 4 show the induction group， collagen type I and osteopontin gene are positive；3 show negative control group， collagen type I is negative

图8 日本大耳白兔骨髓间充质干细胞成软骨分化阿利新蓝染色和RT-PCR检测A：成软骨诱导形态；B：成软骨诱导后的RT-PCR检测；M为1 000 DNA Marker，1为GAPDH，2和3为诱导组，SOX9、ACAN为阳性

Fig. 8 Detection of chondrogenic differentiation of Japanese large ear white rabbits BMSCs using Alixin blue and RT-PCRA： Chondrogenic induction morphology； B： RT-PCR after chondrogenic induction；M shows 1 000 DNA Marker，1 shows GAPDH，2 and 3 show the induction group， SOX9 and ACAN are positive

图9 日本大耳白兔骨髓间充质干细胞成脂分化油红O染色和RT-PCR检测A：成脂诱导后形态； B：成脂诱导后RT-PCR检测；M为1 000 DNA Marker，1为GAPDH，2和3为诱导组，LPL、PPAR-γ为阳性

Fig.9 Oil red O staining and RT-PCR detection of adipogenic differentiation of Japanese large ear white rabbits BMSCsA： Lipogenic induction morphology； B： RT-PCR after induction of adipogenesis；M shows 1 000 DNA Marker，1 shows GAPDH，2 and 3 show the induction group，LPL and PPAR-γ are positive

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