中国组织工程研究

中国组织工程研究 ›› 2021, Vol. 25 ›› Issue (19): 2988-2993.doi: 10.3969/j.issn.2095-4344.3522

• 脂肪干细胞 adipose-derived stem cells • 上一篇    下一篇

两种月龄近交系五指山小型猪脂肪间充质干细胞生物学特性的比较

高元慧1，向  杨2，曹  卉1，王顺兰1，郑琳麟1，何浩伟1，张应爱1，张淑芳1，黄邓高1   

  1. 中南大学湘雅医学院附属海口医院，1中心实验室，2泌尿外科，海南省海口市  570208
  • 收稿日期:2020-05-19 修回日期:2020-05-20 接受日期:2020-06-29 出版日期:2021-07-09 发布日期:2021-01-13
  • 通讯作者: 黄邓高，硕士，助理研究员，中南大学湘雅医学院附属海口医院中心实验室，海南省海口市 570208
  • 作者简介:高元慧，女，1988年生，海南省海口市人，汉族，中南大学在读硕士，主要从事细胞生物学方面的研究。
  • 基金资助:
    海南省自然青年基金(819QN390)，项目负责人：高元慧；海南省自然青年基金(819QN386)，项目负责人：黄邓高；海南省重大科技计划项目(ZDKJ2017007)，项目负责人：张淑芳

Comparison of biological characteristics of adipose derived mesenchymal stem cells in Wuzhishan inbreed miniature pigs aged two different months

Gao Yuanhui1, Xiang Yang2, Cao Hui1, Wang Shunlan1, Zheng Linlin1, He Haowei1, Zhang Yingai1, Zhang Shufang1, Huang Denggao1   

  1. 1Central Laboratory, 2Department of Urology, Affiliated Haikou Hospital of Xiangya School of Medicine, Central South University, Haikou 570208, Hainan Province, China
  • Received:2020-05-19 Revised:2020-05-20 Accepted:2020-06-29 Online:2021-07-09 Published:2021-01-13
  • Contact: Huang Denggao, Master, Assistant researcher, Central Laboratory, Affiliated Haikou Hospital of Xiangya School of Medicine, Central South University, Haikou 570208, Hainan Province, China
  • About author:Gao Yuanhui, Master candidate, Central Laboratory, Affiliated Haikou Hospital of Xiangya School of Medicine, Central South University, Haikou 570208, Hainan Province, China
  • Supported by:
    the Natural Youth Fund of Hainan Province, No. 819QN390 (to GYH); the Natural Youth Fund of Hainan Province, No. 819QN386 (to HDG); the Major Science and Technology Plan Project of Hainan Province, No. ZDKJ2017007 (to ZSF)

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摘要：

文题释义：
脂肪间充质干细胞：是成体间充质干细胞，主要从脂肪组织分离获得，具有侵入性小、分布广泛、容易获取、干细胞数量多等优点，可向脂肪、成骨、软骨及神经元等细胞分化，是再生医学的主要种子细胞来源之一。
β-半乳糖苷酶：是一种水解酶，可催化衰老细胞中β-半乳糖苷水解为单糖，形成蓝色产物，是细胞衰老的生物标志物。

背景：干细胞是再生医学研究的热点，影响干细胞生物学特性的因素很多，其中供体年龄是重要的影响因素之一。
目的：观察两种月龄近交系五指山小型猪脂肪间充质干细胞生物学特性的变化，筛选最佳种子细胞来源。
方法：以4月龄和12月龄近交系五指山小型猪脂肪间充质干细胞为研究对象，分别选取第3代细胞和第10代细胞，观察细胞形态，绘制细胞增殖曲线，检测细胞表面抗原、细胞周期、细胞凋亡情况，然后进行成骨诱导分化茜素红染色和成脂诱导分化油红O染色以及细胞衰老β-半乳糖苷酶染色等。
结果与结论：①4月龄和12月龄细胞均呈漩涡状贴壁生长，表面标志物CD29、CD44和CD90表达均为阳性，CD8a、CD45和HLA-DR表达均为阴性；②4月龄和12月龄第3代细胞增殖能力均较强；第10代细胞，与4月龄相比，12月龄G0/G1期百分比增高(P < 0.001)，早期凋亡增加(P < 0.001)，而S期和G2/M期百分比显著下降(P < 0.001)；③4月龄和12月龄第3代细胞的体外诱导分化能力无显著性差异；12月龄第10代细胞的钙化结节率和脂滴率与4月龄相比显著降低(P < 0.001)；④4月龄和12月龄第3代细胞β-半乳糖苷酶几乎不表达；12月龄第10代细胞β-半乳糖苷酶阳性细胞与4月龄相比显著增加(P < 0.001)，且细胞形态变得更加扁平和肥大、折光性弱；⑤结果表明，4月龄近交系五指山小型猪脂肪间充质干细胞具有更强的增殖和分化能力，且细胞衰老活性更低，提示4月龄更适合用于自体移植的种子细胞。
https://orcid.org/0000-0003-3872-0567(高元慧) 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 干细胞, 脂肪间充质干细胞, 近交系五指山小型猪, 年龄, 增殖, 分化, 衰老

Abstract: BACKGROUND: Stem cells have been the focus of regenerative medicine research. Many factors affect the biological characteristics of stem cells, among which donor age is one of the critical factors.
OBJECTIVE: To observe biological characteristics of two types of inbreed line miniature pigs of Wuzhishan adipose-derived mesenchymal stem cells so as to screen the optimal seed cell source.
METHODS:  Inbreed line miniature pigs of Wuzhishan adipose-derived mesenchymal stem cells aged 4 and 12 months were selected as materials. The passage 3 and passage 10 of cells were selected to observe their cell morphology, draw cell proliferation curve, and detect cell surface antigen, cell cycle and apoptosis. Afterwards, osteogenic differentiation was performed with Alizarin red staining, followed by adipogenic differentiation with Oil Red O staining and cell senescence β-galactosidase staining.
RESULTS AND CONCLUSION: (1) The morphology of cells at 4 and 12 months both showed vortex-shaped adherent growth. The expression of surface markers CD29, CD44 and CD90 was positive, while that of CD8a, CD45 and HLA-DR was negative. (2) The proliferation of cells at 4 and 12 months was stronger in passage 3. In passage 10, the percentage of cells in the G0/G1 phase (P < 0.001) and early apoptosis (P < 0.001) increased, while in the S and G2/M phases, it decreased at 12 months compared with 4 months (P < 0.001). (3) There was no significant difference in the differentiation ability of cells at passage 3 in vitro between 4 and 12 months. The calcification nodule rate and lipid droplet rate of cells at 12 months of passage 10 were significantly lower than at 4 months of passage 10 (P < 0.001). (4) The β-galactosidase of cells was almost not expressed at 4 and 12 months of passage 3. However, in the passage 10, the positive rate of cells at 12 months was significantly higher than that at 4 months (P < 0.001); and the cell morphology became more flattened, hypertrophic and less refractive. (5) It is concluded that adipose-derived mesenchymal stem cells of inbreed line miniature pigs of Wuzhishan at 4 months showed stronger proliferation and differentiation ability, while showed lower senescence on β-galactosidase activity, suggesting that 4-month cells are more suitable for autograft. 

Key words: stem cells, adipose mesenchymal stem cells, inbred Wuzhishan miniature pig, age, proliferation, differentiation, senescence

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