Journal of Agricultural Science and Technology ›› 2023, Vol. 25 ›› Issue (8): 96-105.DOI: 10.13304/j.nykjdb.2023.0411
• BIOTECHNOLOGY & LIFE SCIENCE • Previous Articles Next Articles
Shiying ZHOU1,2(), Yanchen LIU2(
), Yang ZHANG1, Xuesong YANG1, Weijun GUAN2, Yang GAO1(
)
Received:
2023-05-30
Accepted:
2023-07-04
Online:
2023-08-20
Published:
2023-09-07
Contact:
Yang GAO
周世莹1,2(), 刘晏辰2(
), 张洋1, 杨雪松1, 关伟军2, 高扬1(
)
通讯作者:
高扬
作者简介:
周世莹 E-mail:643135130@qq.com基金资助:
CLC Number:
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.
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.
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 |
R:TCCAGGCAGGCCCCAATTGTGGGTG | |||
CD29 | F: CGATGTGATGACTTAGAAGCCTTG | 60.85 | 471 |
R:CAGAGACATAAAGAAAAATAAGA | |||
CD44 | F:GGAGGATATCACTCAGATCCAGCCA | 60.60 | 373 |
R: GTTGAAGTTCAAGCGTGCTGAAGAC | |||
CD73 | F:AAGATGATCTGGAGAACGTCAAGA | 61.65 | 623 |
R: ACTTCCGAATTGGGTTTGGCTCCTT | |||
CD105 | F: GCTCAAGAACCCTTGCACGAGTGAG | 60.35 | 550 |
R: ACAGACAGAGGGGAGGAGTTCAACG | |||
Collage typeⅠ | F: CCAACGATGGACAGTGCCACCTGG | 59.30 | 425 |
R: GCCCACCTCGTCCAGAAACTCAG | |||
osteopontin | F: CCTGTTTACAAGGAACTGAAAAAC | 59.00 | 512 |
R: AATGTCATCCAGCTGATCATTGATG | |||
LPL | F:GCCAGAAGGAGTAACGATAAATA | 54.50 | 486 |
R:GAAGGAAGTGCTCCAATATCTCCAT | |||
PPAR-γ | F: GCAAACTAACTGGCTATGGTCCT | 60.10 | 473 |
R: ATTAAGTGTCACCCAGGATGTGC | |||
ACAN | F:CACGTTTGAGTGTGGGGCTTGCAG | 60.50 | 464 |
R:GGGAGTGCGTCTGTGGGCAGTGTT | |||
SOX9 | F: CCTATTAATAATTTCAAGAATCCAA | 60.70 | 507 |
R: ACTATGGACGTAGAGCTGGTCCCTGA | |||
CD45 | F: ATGGTGAAGGTCGGAGTGAACGG | 60.40 | 304 |
R: ATTTGGCCGCATTGGGCGCCTGGT |
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 |
R:TCCAGGCAGGCCCCAATTGTGGGTG | |||
CD29 | F: CGATGTGATGACTTAGAAGCCTTG | 60.85 | 471 |
R:CAGAGACATAAAGAAAAATAAGA | |||
CD44 | F:GGAGGATATCACTCAGATCCAGCCA | 60.60 | 373 |
R: GTTGAAGTTCAAGCGTGCTGAAGAC | |||
CD73 | F:AAGATGATCTGGAGAACGTCAAGA | 61.65 | 623 |
R: ACTTCCGAATTGGGTTTGGCTCCTT | |||
CD105 | F: GCTCAAGAACCCTTGCACGAGTGAG | 60.35 | 550 |
R: ACAGACAGAGGGGAGGAGTTCAACG | |||
Collage typeⅠ | F: CCAACGATGGACAGTGCCACCTGG | 59.30 | 425 |
R: GCCCACCTCGTCCAGAAACTCAG | |||
osteopontin | F: CCTGTTTACAAGGAACTGAAAAAC | 59.00 | 512 |
R: AATGTCATCCAGCTGATCATTGATG | |||
LPL | F:GCCAGAAGGAGTAACGATAAATA | 54.50 | 486 |
R:GAAGGAAGTGCTCCAATATCTCCAT | |||
PPAR-γ | F: GCAAACTAACTGGCTATGGTCCT | 60.10 | 473 |
R: ATTAAGTGTCACCCAGGATGTGC | |||
ACAN | F:CACGTTTGAGTGTGGGGCTTGCAG | 60.50 | 464 |
R:GGGAGTGCGTCTGTGGGCAGTGTT | |||
SOX9 | F: CCTATTAATAATTTCAAGAATCCAA | 60.70 | 507 |
R: ACTATGGACGTAGAGCTGGTCCCTGA | |||
CD45 | F: ATGGTGAAGGTCGGAGTGAACGG | 60.40 | 304 |
R: ATTTGGCCGCATTGGGCGCCTGGT |
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.
Fig. 5 Immunofluorescence of Japanese large ear white rabbits BMSCsNote: DAPI—Nuclear staining;FITC—Immunofluorescence;Merge—Overlay of DAPI and FITC images.
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
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
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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