双峰驼诱导多能干细胞体外分化为成骨细胞条件及炎症模型构建的初探

doi:10.3969/j.issn.1674-7968.2025.10.009

Abstract
Figure/Table
References
Related Citation (3)

Download: PDF (23057 KB) HTML (1 KB)
Export: BibTeX | EndNote (RIS)

Abstract Stem cells have the ability of self-replication and multidirectional differentiation, which is of great significance in the field of regenerative medicine. As a unique species adapted to extreme environments, the induced pluripotent stem cells (iPSCs) of the bactrian camel (Camelus bactrianus) contain a special differentiation and control mechanism. This study centered on the optimal culture protocol for the osteogenic induced differentiation of bactrian camel induced pluripotent stem cells (bciPSCs). The optimal culture protocol for osteoblast induced differentiation were screened, and then explored the optimal drugs and concentrations for constructing inflammation models using osteoblast (OB) cells obtained from this protocol. The pluripotency and differentiation ability of bciPSCs kept in the laboratory were firstly determined by qRT-PCR, immunofluorescence and karyotyping; then bciPSCs were induced and differentiated by 2 direct and indirect induction methods and 2 different culture mediums. The OB obtained were tested by qRT-PCR, immunofluorescence, Western blot, etc. to determine the effect of osteogenic differentiation and compare the purity of the OB obtained to determine the optimal induction method; The cells obtained by the optimal method of osteogenic induction were selected to construct the inflammation model, and the OB were treated with lipopolysaccharide (LPS) and Interleukin-1β (IL-1β), and then screened the optimal drug and concentration by CCK-8, qRT-PCR, and Western blot. The results showed that compared with bactrian camel embryonic fibroblasts (CEF), the expression of pluripotency genes in laboratory-preserved bciPSCs was extremely significantly up-regulated (P<0.01). During the passaging process, the cells did not undergo morphological and pluripotency changes, indicating their ability to differentiate into three blastomeres and their suitability for subsequent studies. BciPSCs were directionally induced to differentiate via 2 methods: Direct induction and indirect induction. After induction, the expression of osteogenic marker genes—including osteocalcin bone gamma-carboxyglutamate protein (BGLAP), collagen typeⅠ α2 (COL1A2), runt-related transcription factor 2 (Runx2), and secreted phosphoprotein 1 (SPP1)—was significantly up-regulated (P<0.05). Additionally, the expression levels of osteopontin (OPN) and Runx2 were also significantly increased (P<0.05), and both were positively expressed in immunofluorescence staining and osteogenesis-specific staining. Through intergroup comparisons, the group of cells exhibiting the most pronounced osteogenic characteristics was selected for subsequent studies; The cell viability was significantly decreased after 24 h of LPS and IL-1β treatment (P<0.01), and the expression levels of the inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-6 (IL6) and IL-1β were significantly increased (P<0.05), with 70 ng/mL of IL-1β up-regulated inflammatory factors more significantly, which could be used as a condition for constructing an inflammation model. This study provides biological materials and reference data for the study of osteoarthritis disease pathogenesis and drug screening.

Key words： Bactrian camel induced pluripotent stem cells (bciPSCs) In vitro differentiation Osteoblasts(OB) Osteoarthritis (OA)

Received: 06 March 2025

ZTFLH:

S824

Corresponding Authors: *zhychy@163.com

	Service

	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	JIANG Di-Di
	LI Zong-Shuai
	GAO Kun
	MA Tian
	LIU Wan-Ruo
	ZHANG Yong

Cite this article:

JIANG Di-Di,LI Zong-Shuai,GAO Kun, et al. Preliminary Exploration of the Conditions and Inflammatory Model Construction for Bactrian Camels Induced Pluripotent Stem Cells into Osteoblasts in vitro[J]. 农业生物技术学报, 2025, 33(10): 2195-2208.

URL:

https://journal05.magtech.org.cn/Jwk_ny/EN/10.3969/j.issn.1674-7968.2025.10.009 OR https://journal05.magtech.org.cn/Jwk_ny/EN/Y2025/V33/I10/2195

[1] 李宗帅. 2022. 双峰驼诱导多能干细胞的建立、鉴定及特有诱导基因的筛选[D]. 博士学位论文, 甘肃农业大学, 导师: 张勇, pp. 46-54.
(Li Z S.2022. Establishment and identification of bactrian camel induced pluripotent stem cells and screening of unique induced[D]. Thesis for Ph. D., Gansu Agricultural University, Supervisor: Zhang Y, pp. 46-54.)
[2] Abramoff B, Caldera F E.2020. Osteoarthritis: Pathology, diagnosis, and treatment options[J]. Medical Clinics of North America, 104(2): 293-311.
[3] Aboul-Soud M A M, Alzahrani A J, Mahmoud A.2021. Induced pluripotent stem cells (iPSCs)-roles in regenerative therapies, disease modelling and drug screening[J]. Cells, 10(9): 2319.
[4] An J, Yang H, Zhang Q, et al.2016. Natural products for treatment of osteoporosis: The effects and mechanisms on promoting osteoblast-mediated bone formation[J]. Life Sciences, 147: 46-58.
[5] Ahmadi A, Mazloomnejad R, Kasravi M, et al.2022. Recent advances on small molecules in osteogenic differentiation of stem cells and the underlying signaling pathways[J]. Stem Cell Research & Therapy, 13(1): 518.
[6] Barruet E, Hsiao E C.2016. Using human induced pluripotent stem cells to model skeletal diseases[J]. Methods in Molecular Biology, 1353: 101-118.
[7] Jirimutu, Wang Z, Ding G, et al.2012. Genome sequences of wild and domestic bactrian camels[J]. Nature Communications, 3: 1202.
[8] Chang Y H, Wu K C, Ding D C.2020. Induced pluripotent stem cell-differentiated chondrocytes repair cartilage defect in a rabbit osteoarthritis model[J]. Stem Cells International, 2020: 8867349.
[9] Chuluunbat B, Charruau P, Silbermayr K, et al.2014. Genetic diversity and population structure of mongolian domestic bactrian camels (Camelus bactrianus)[J]. Animal Genetics, 45(4): 550-558.
[10] Chen G, Liu T, Yu B, et al.2020. CircRNA-UBE2G1 regulates LPS-induced osteoarthritis through miR-373/HIF-1a axis[J]. Cell Cycle, 19(13): 1696-1705.
[11] Davatchi F, Abdollahi B S, Mohyeddin M, et al.2011. Mesenchymal stem cell therapy for knee osteoarthritis. Preliminary report of four patients[J]. International Journal of Rheumatic Diseases, 14(2): 211-215.
[12] Glyn-Jones S, Palmer A J, Agricola R, et al.2015. Osteoarthritis[J]. Lancet, 386(9991): 376-87.
[13] Heinola T, Grauw D J, Virkki L, et al.2013. Bovine chronic osteoarthritis causes minimal change in synovial fluid[J]. Journal of Comparative Pathology, 148(4): 335-344.
[14] Huang Y, Zhang X, Zhan J, et al.2021. Bone marrow mesenchymal stem cell-derived exosomal mir-206 promotes osteoblast proliferation and differentiation in osteoarthritis by reducing Elf3[J]. Journal of Cellular and Molecular Medicine, 25(16): 7734-7745.
[15] Koh Y G, Choi Y J.2012. Infrapatellar fat pad-derived mesenchymal stem cell therapy for knee osteoarthritis[J]. Knee, 19(6): 902-907.
[16] Kong X, Ning C, Liang Z, et al.2024. Koumine inhibits IL-1β-induced chondrocyte inflammation and ameliorates extracellular matrix degradation in osteoarthritic cartilage through activation of PINK1/Parkin-mediated mitochondrial autophagy[J]. Biomed Pharmacother, 173: 116273.
[17] Liu Y, Lin L, Zou R, et al.2018. MSC-derived exosomes promote proliferation and inhibit apoptosis of chondrocytes via lncRNA-KLF3-AS1/miR-206/GIT1 axis in osteoarthritis[J]. Cell Cycle, 17(21-22): 2411-2422.
[18] Lou X.2015. Induced pluripotent stem cells as a new strategy for osteogenesis and bone regeneration[J]. Stem Cell Reviews and Reports, 11(4): 645-651.
[19] Ma M S, Kannan V, De Vries, et al.2017. Characterization and comparison of osteoblasts derived from mouse embryonic stem cells and induced pluripotent stem cells[J]. Journal of Bone and Mineral Metabolism, 35(1): 21-30.
[20] Mcilwraith C W, Kawcak C E, Frisbie D D, et al.2018. Biomarkers for equine joint injury and osteoarthritis[J]. Journal of Orthopaedic Research, 36(3): 823-831.
[21] Nganvongpanit K, Soponteerakul R, Kaewkumpai P, et al.2017. Osteoarthritis in two marine mammals and 22 land mammals: Learning from skeletal remains[J]. Journal of Anatomy, 231(1): 140-155.
[22] Ossendorff R, Kurth S, Wang S, et al.2024. Comparison of concentration-and homology-dependent effects of the proinflammatory cytokine interleukin-1β (IL-1β) in a Bovine chondrocyte inflammation model[J]. Cells, 14(1): 30.
[23] Schroeder I S.2021. Pluripotent stem cells for cell therapy[J]. Methods in Molecular Biology, 2269: 25-33.
[24] Sobhani A, Khanlarkhani N, Baazm M,et al.2017. Multipotent stem cell and current application[J]. Acta Medica Iranica, 55(1): 6-23.
[25] Son Y B, Jeong Y I, Jeong Y W, et al.2021. Comparative study of biological characteristics, and osteoblast differentiation of mesenchymal stem cell established from Camelus dromedarius skeletal muscle, dermal skin, and adipose tissues[J]. Animals, 11(4): 1017.
[26] Tian Z, Yu T, Liu J, et al.2023. Introduction to stem cells[J]. Progress in Molecular Biology and Translational Science, 199: 3-32.
[27] Uto S, Nishizawa S, Hikita A, et al.2018. Application of induced pluripotent stem cells for cartilage regeneration in CLAWN miniature pig osteochondral replacement model[J]. Regenerative Therapy, 9: 58-70.
[28] Van Weeren P R, De Grauw J C.2010. Pain in osteoarthritis[J]. Veterinary Clinics of North America: Equine Practice, 26(3): 619-642.
[29] Wu W, Ye Z, Zhou Y, et al.2011. AICAR, a small chemical molecule, primes osteogenic differentiation of adult mesenchymal stem cells[J]. International Journal of Artificial Organs, 34(12): 1128-1136.
[30] Zhang F, Citra F, Wang D A.2011. Prospects of induced pluripotent stem cell technology in regenerative medicine[J]. Tissue Engineering Part B-Reviews, 17(2): 115-124.
[31] Zhan J, Yan Z, Kong X, et al.2021. Lycopene inhibits IL-1β-induced inflammation in mouse chondrocytes and mediates murine osteoarthritis[J]. Journal of Cellular and Molecular Medicine, 25(7): 3573-3584.