犬造血干细胞分离、鉴定和培养特性研究

doi:10.16431/j.cnki.1671-7236.2023.07.009

Abstract

Abstract: 【Objective】 This research was aimed to study the isolation, identification and culture characteristics of cannie hematopoietic stem cells (cHSCs) derived from bone marrow, establish the optimal conditions for in vitro culture, and lay a theoretical foundation for clinical application.【Method】 Mononuclear cells(MNCs)were isolated from 3-month-old Beagle dogs by Ficoll density gradient centrifugation.cHSCs were separated by flow cytometer and cultured in vitro.CCK-8 method was used to detect the cell activity of cHSCs in different media, different concentrations of fetal bovine serum (FBS), different cytokines, and different proportions of cHSCs to cBMSCs, and to screen the optimal in vitro culture scheme.Trypan blue staining counting method was used to calculate the proliferation of cHSCs after 1, 7, 14 and 21 days of co-culture in vitro.Flow cytometry and Real-time quantitative PCR were used to detect the positive percentage of CD34 molecules as surface markers of cHSCs in P0, P1, P2 and P3, and the mRNA expression of stemness genes sex determining region Y-box 2 (SOX2) and octamer binding factor 4 (OCT-4), respectively.【Result】 cHSCs were isolated and obtained successfully, and there was no significant difference of cell activity in different medium (P>0.05).In different concentrations of FBS, the cHSCs activity in 5% FBS group was significantly lower than that in 10%, 15% and 20% FBS groups (P<0.05), the cHSCs activity in 10% FBS group was significantly lower than that in 20% FBS group(P<0.05).Under different cytokine conditions, the cHSCs activity in TPO+FLT-3+SCF+IL-6 group was significantly higher than that in TPO+FLT-3 and TPO+FLT-3 group (P<0.05).Under different cHSCs and cBMSCs inoculation ratios, the cHSCs activity in 1:10 group was extremely significantly higher than that in 1:5, 5:1 and 10:1 groups (P<0.01), and the cHSCs activity in 5:1 group was extremely significantly higher than that in 1:5 and 10:1 groups (P<0.01).cHSCs entered the logarithmic growth stage and reached the peak on the 7th day after co-culture with the optimal culture scheme, and the number of cells in cHSCs+cBMSCs+cytokine group was significantly higher than that in cHSCs+ cytokine group (P<0.05).With the passage culture of cHSCs, the cell differentiation potential was changed.The positive percentage of CD34 molecule and the mRNA expression of SOX2 gene in P1, P2 and P3 were extremely significantly lower than those in P0 (P<0.01), and the mRNA expression of OCT-4 gene in P1 and P2 was extremely significantly higher than that in P0 group (P<0.01), the mRNA expression of OCT-4 gene in P1 group was significantly higher than that in P2 (P<0.01).【Conclusion】 In this study, cHSCs were successfully isolated from canine bone marrow.The optimal culture conditions for cHSCs were IMDM medium containing 10% FBS, cHSCs and cBMSCs inoculated at a ratio of 1:10, and TPO+FLT-3+SCF+IL-6 cytokines were added.P1 cHSCs were in logarithmic growth stage with a high positive percentage of CD34, which could provide reference for future clinical application.

Key words: canine hematopoietic stem cells(cHSCs); isolation; purification; in vitro culture

CLC Number:

S858.292

YAN Han, TIAN Yangqing, ANG Yanfen, WANG Yayuan, ZHANG Xuefeng, YAN Yulin. Study on the Isolation, Identification and Culture Characteristics of Canine Hematopoietic Stem Cells[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2678-2687.

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Study on the Isolation, Identification and Culture Characteristics of Canine Hematopoietic Stem Cells

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