细胞自噬对犬骨髓间充质干细胞干性的影响

doi:10.11843/j.issn.0366-6964.2020.02.018

摘要/Abstract

摘要： 本研究旨在探讨细胞自噬对犬骨髓间充质干细胞（cBMSCs）干性的影响。分离培养cBMSCs，将其分为对照组、使用雷帕霉素促进细胞自噬的雷帕霉素组、使用3-MA抑制细胞自噬的3-MA组，于药物处理12、24、48 h后收集细胞，利用间接细胞免疫荧光法检测自噬微管相关蛋白1轻链3 Ⅱ（LC 3Ⅱ）的蛋白表达水平；荧光定量PCR检测自噬相关基因LC 3、Beclin 1、自噬相关基因7（Atg 7）以及干性相关基因性别决定基因相关转录因子2（Sox 2）、特异AT序列结合蛋白2（Satb 2）mRNA转录水平；通过茜素红染色检测经成骨诱导的cBMSCs的成骨分化能力，通过油红O染色检测经成脂诱导的cBMSCs的成脂分化能力。结果显示：雷帕霉素组的LC 3Ⅱ蛋白表达量上调，3-MA组则为下调。雷帕霉素组干性相关基因与自噬相关基因在不同时间点的表达水平均有不同程度的上调，且随感染时间的延长呈上调趋势；3-MA组干性相关基因与自噬相关基因的mRNA转录水平在不同时间点不同程度降低，且随感染时间延长呈下调趋势。成骨诱导试验中雷帕霉素组cBMSCs形态变化最明显，且矿化面积较大，3-MA组细胞矿化面积小；成脂诱导试验中雷帕霉素组脂滴数量少，3-MA组脂滴数量多且聚集程度高。在一定程度上促进cBMSCs自噬水平可以更好地维护其干性并提高成骨分化能力，为优化治疗中cBMSCs的质量提供理论基础和技术支持。

关键词: 犬骨髓间充质干细胞, 自噬基因, 干性基因, 成骨诱导, 成脂诱导

Abstract: This study aimed to investigate the effect of autophagy on the stemness of canine bone marrow mesenchymal stem cells (cBMSCs). The isolated cBMSCs were cultured with ordinary medium, rapamycin-containing medium, 3-MA-containing medium. Cells were collected after 12, 24 and 48 hours' treatment, then autophagy microtubule-associated protein 1 light chain 3 Ⅱ (LC 3Ⅱ) were detected by indirect immunofluorescence; autophagy and stemness related genes, e.g. LC 3, Beclin 1, autophagy-related 7 (Atg 7), SRY-related high-monility-group (HMG)-box protein-2 (Sox 2), special AT-rich sequence-binding protein 2 (Satb 2), were detected by fluorescence quantitative PCR. The cBMSCs differentiation abilities were detected by the dyeing experiment after adipogenic (Oil red O staining) and osteoplastic (alizarin red staining) differentiation. Results were as follows:The LC 3Ⅱ protein expression and gene expression level at the rapamycin group was increased, while which was decreased in the 3-MA group. The results showed that there was a significant increase at the stemness and autophagy related mRNA expression levels of the rapamycin group, while which was decreased in the 3-MA group. The cBMSCs in the rapamycin group showed morphological alterations obviously and the larger mineralized area, while the cBMSCs in the 3-MA group showed the smaller mineralized area. There were fewer lipid droplets at the rapamycin group, while the 3-MA group was more. These results indicated that autophagy could enhance the stemness and osteogenic differentiation of cBMSCs, it was providing theoretical and technical evidence for cBMSCs in the treatment of diseases in dogs.

Key words: canine bone marrow mesenchymal stem cells, autophagy-related genes, stemness-related genes, osteogenesis induction, lipogenesis induction

中图分类号:

S852

郭娟, 张迪, 李龙, 储宗秀, 田仰清, 汪雅媛, 杨蕊, 高洪, 严玉霖. 细胞自噬对犬骨髓间充质干细胞干性的影响[J]. 畜牧兽医学报, 2020, 51(2): 365-373.

GUO Juan, ZHANG Di, LI Long, CHU Zongxiu, TIAN Yangqing, WANG Yayuan, YANG Rui, GAO Hong, YAN Yulin. Effect of Autophagy on the Stemness of Canine Bone Marrow Mesenchymal Stem Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(2): 365-373.

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