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

摘要/Abstract

摘要： 【目的】研究骨髓来源的犬造血干细胞（cannie hematopoietic stem cells，cHSCs）的分离、鉴定和培养特性，建立体外培养最优条件，为临床应用奠定理论基础。【方法】取3月龄犬骨髓，采用Ficoll密度梯度离心法分离出单个核细胞（mononuclear cells，MNCs），通过流式细胞仪分选cHSCs并进行体外悬浮培养。用CCK-8法检测cHSCs在不同培养基、不同浓度胎牛血清（FBS）、不同细胞因子、不同cHSCs与cBMSCs接种比例条件下的细胞活性，筛选最优体外培养方案。使用台盼蓝染色计数法统计体外共培养1、7、14、21 d后cHSCs增殖情况。利用流式细胞术和实时荧光定量PCR分别检测P0、P1、P2和P3代cHSCs表面标志物CD34分子阳性百分比以及干性基因性别决定相关基因簇2（SOX2）和八聚体结合蛋白-4（OCT-4）的mRNA表达水平。【结果】成功分离获得cHSCs，不同培养基中细胞活性无显著差异（P>0.05）；不同浓度FBS中，5% FBS组cHSCs活性显著低于10%、15%、20% FBS组（P<0.05），10% FBS组cHSCs活性显著低于20% FBS组（P<0.05）；不同细胞因子条件下，TPO＋FLT-3＋SCF＋IL-6组cHSCs活性显著高于TPO组和TPO+FLT-3组（P<0.05）；不同cHSCs与cBMSCs接种比例条件下，1：10组cHSCs活性极显著高于1：5、5：1和10：1组（P<0.01），5：1接种组cHSCs活性极显著高于1：5和10：1组（P<0.01）。通过筛选的最优培养方案进行体外共培养，第7天时cHSCs进入对数生长期且达到高峰，其中cHSCs＋cBMSCs＋细胞因子组细胞数量显著高于cHSCs＋细胞因子组（P<0.05）。随着cHSCs传代培养，细胞分化潜能发生了变化，P1、P2、P3代细胞CD34分子阳性百分比、SOX2基因mRNA表达水平均极显著低于P0代（P<0.01），P1、P2代OCT-4基因mRNA表达水平极显著高于P0代（P<0.01），P1代OCT-4基因mRNA表达水平极显著高于P2代（P<0.01）。【结论】本研究成功从犬骨髓中分离出cHSCs，筛选的cHSCs体外增殖最优培养条件为含10% FBS的IMDM培养基、cHSCs与cBMSCs接种比例为1：10，同时联合添加TPO＋FLT-3＋SCF＋IL-6细胞因子，P1代cHSCs处于对数生长期且CD34阳性百分比较高，可为今后临床应用提供参考。

关键词: 犬造血干细胞(cHSCs); 分离; 纯化; 体外培养

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

中图分类号:

S858.292

严涵, 田仰清, 昂艳芬, 汪娅媛, 张学峰, 严玉霖. 犬造血干细胞分离、鉴定和培养特性研究[J]. 中国畜牧兽医, 2023, 50(7): 2678-2687.

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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