绵羊外周血间充质干细胞的生物学特性

doi:10.19723/j.issn.1671-167X.2022.06.015

摘要/Abstract

摘要：

目的: 用粒细胞集落刺激因子(granulocyte colony-stimulating factor，G-CSF)连续动员法获取绵羊与兔外周血间充质干细胞(peripheral blood mesenchymal stem cells，PBMSCs)，通过比较两种来源PBMSCs的获取成功率、细胞产量及生物学特性的差异，为PBMSCs移植修复关节软骨损伤及软骨组织工程的临床前研究提供实验依据。方法: 经G-CSF连续动员获取绵羊与兔外周血单核细胞，通过形态学特征、流式法分析其表面标记、体外定向诱导两种细胞三系分化(即：成脂分化、成骨分化、成软骨分化)，确证获取的细胞为PBMSCs。统计并比较两种PBMSCs的集落形成单位(colony-forming units, CFUs)、获取成功率，用血细胞计数板统计并比较两种第2代PBMSCs的产量，用细胞增殖-毒性检测试剂盒检测两种PBMSCs倍增时间，用图像处理法定量分析三系分化结果。结果: 镜下见梭形绵羊和兔PBMSCs呈鱼群状排列，第2代绵羊与兔PBMSCs表达CD44、CD90，不表达CD34、CD45，三系分化结果良好。原代绵羊与兔PBMSCs的CFUs(个)分别为7.27±1.56、5.73±1.62，绵羊与兔PBMSCs的获取成功率分别为78.57%、36.67%，每毫升外周血可获取的第2代绵羊与兔PBMSCs数(个)分别为29 582±2 138、26 732±2 286，第3代绵羊与兔PBMSCs的细胞倍增时间(h)分别为22.32±0.28、33.21±0.64，第4代绵羊与兔PBMSCs的细胞倍增时间(h)分别为23.62±0.56、35.30±0.38，绵羊与兔PBMSCs量化成脂比分别为7.77%±3.81%、17.05%±1.52%，绵羊与兔PBMSCs软骨球酸性粘多糖阳性比分别为11.67%±0.53%、8.14%±0.57%，以上各组间比较差异均有统计学意义(P < 0.05)。结论: 经G-CSF连续动员获取绵羊PBMSCs更高效，绵羊PBMSCs产量更丰富、增殖能力更强，在适当环境下能产生更多酸性粘多糖，且成脂能力更低，在自体干细胞移植修复关节软骨损伤及软骨组织工程的临床前动物在体实验中具有良好的研究前景，并为该类研究进一步提供实验依据。

关键词: 外周血间充质干细胞, 动员, 生物学特性

Abstract:

Objective: To obtain eripheral blood mesenchymal stem cells (PBMSCs) from sheep and rabbits by continuous mobilization of granulocyte colony-stimulating factor (G-CSF), and by comparing the success rates, cell yields and biological characteristics of the two sources of PBMSCs, and to provide the experimental basis for the preclinical study of PBMSCs transplantation to repair articular cartilage injury and cartilage tissue engineering. Methods: Through morphological characteristics, flow cytometry analysis of its surface markers, and induction of trilineage differentiation of the two cells in vitro (ie: adipogenic differentiation, osteogenic differentiation, chondrogenic differentiation), the obtained cells were finally confirmed to be PBMSCs. The colony-forming units (CFUs) and the acquisition success rates of the two PBMSCs were counted and compared, and the production of the second generation of the two PBMSCs was counted and compared by hemocytometer, and the cell counting kit-8 was used to detect the doubling time of the two PBMSCs, and the results of trilineage differentiation were quantitatively analyzed by image processing. Results: Microscopically, the PBMSCs of fusiform sheep and rabbits were arranged in fish group, and the second generation of sheep and rabbit PBMSCs expressed CD44 and CD90, but not CD34 and CD45. The induction of trilineage differentiation of the two cells in vitro were successful. The CFUs of primary sheep and rabbits PBMSCs were: 7.27±1.56, 5.73±1.62, and the success rate of acquisition of sheep and rabbits PBMSCs were 78.57% and 36.67%. The number of the second-generation sheep and rabbits PBMSCs that obtained per milliliter of peripheral blood were: 29 582±2 138, 26 732±2 286, and the cell doubling times (h) of the third-generation sheep and rabbits PBMSCs were: 22.32±0.28, 33.21±0.64, the cell doubling time (h) of the fourth generation sheep and rabbits PBMSCs were: 23.62±0.56, 35.30±0.38, and the quantitative lipid ratio of sheep and rabbit PBMSCs were: 7.77%±3.81%, 17.05%±1.52%, sheep and rabbit PBMSCs chondroglobus acid mucopolysaccharide positive ratios were: 11.67%±0.53%, 8.14%±0.57%. There were statistical differences among the above groups (P < 0.05). Conclusion: The continuous mobilization of G-CSF to obtain sheep PBMSCs is more efficient. Sheep PBMSCs have more abundant yield and stronger proliferation ability.Sheep PBMSCs can produce more acidic mucopolysaccharides and have lower adipogenic abi-lity under appropriate conditions. Sheep PBMSCs have good research prospects in repair of articular cartilage injury with autologous stem cell transplantation and preclinical animal in vivo experiment of cartilage tissue engineering.This experiment provides further experimental basis for this kind of research.

Key words: Peripheral blood mesenchymal stem cells, Mobilization, Biological characteristics

中图分类号:

韩超,周祝兴,陈有荣,董子慧,余家阔. 绵羊外周血间充质干细胞的生物学特性[J]. 北京大学学报（医学版）, 2022, 54(6): 1151-1157.

Chao HAN,Zhu-xing ZHOU,You-rong CHEN,Zi-hui DONG,Jia-kuo YU. Biological characteristics of sheep peripheral blood mesenchymal stem cell[J]. Journal of Peking University (Health Sciences), 2022, 54(6): 1151-1157.

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