年龄对人脂肪来源间充质干细胞生物学特性的影响

doi:10.11958/20220808

天津医药 ›› 2022, Vol. 50 ›› Issue (9): 912-916.doi: 10.11958/20220808

年龄对人脂肪来源间充质干细胞生物学特性的影响

杜鹏¹(), 王峰², 陈晓波³, 郑燕⁴, 吴倩倩¹, 刘卫军⁵, 王玉亮⁴^,^△()

1 天津市人民医院急诊检验科（邮编300121）
2 天津医科大学基础医学院遗传学系
3 优赛生命科学发展有限公司
4 天津医科大学第二医院,天津市泌尿外科研究所
5 天津市儿童医院麻醉科

收稿日期:2022-05-23 修回日期:2022-07-05 出版日期:2022-09-15 发布日期:2022-09-05
通讯作者: 王玉亮 E-mail:1821262326@qq.com;wang_yu_l@163.com
作者简介:杜鹏（1968）,男,副主任技师,主要从事医学检验方面研究。E-mail： 1821262326@qq.com
基金资助:
国家自然科学基金资助项目(81470982);国家自然科学基金资助项目(32170762)

Impact of age on the biological properties of human adipose tissue-derived mesenchymal stromal cells

DU Peng¹(), WANG Feng², CHEN Xiaobo³, ZHENG Yan⁴, WU Qianqian¹, LIU Weijun⁵, WANG Yuliang⁴^,^△()

1 Clinical Laboratory of Emergency Medicine, Tianjin Union Medical Centre, Tianjin 300121, China
2 Department of Genetics, School of Basic Medical Sciences, Tianjin Medical University
3 Unicell Life Science Development Co., Ltd
4 The Second Hospital of Tianjin Medical University, Tianjin Institute of Urology
5 Department of Anesthesiology, Tianjin Children's Hospital

Received:2022-05-23 Revised:2022-07-05 Published:2022-09-15 Online:2022-09-05
Contact: WANG Yuliang E-mail:1821262326@qq.com;wang_yu_l@163.com

杜鹏, 王峰, 陈晓波, 郑燕, 吴倩倩, 刘卫军, 王玉亮. 年龄对人脂肪来源间充质干细胞生物学特性的影响[J]. 天津医药, 2022, 50(9): 912-916.

DU Peng, WANG Feng, CHEN Xiaobo, ZHENG Yan, WU Qianqian, LIU Weijun, WANG Yuliang. Impact of age on the biological properties of human adipose tissue-derived mesenchymal stromal cells[J]. Tianjin Medical Journal, 2022, 50(9): 912-916.

摘要/Abstract

摘要：

目的探讨供体年龄对脂肪来源间充质干细胞（ADSCs）体外生物学特性的影响。方法收集外科腹部术后留取的皮下脂肪组织和外周血,按年龄分为儿童组、成年组和>50岁组,每组10例,分离培养ADSCs;流式细胞术分析ADSCs免疫表型,实时细胞分析系统检测ADSCs增殖细胞指数（CI）及迁移CI,使用不同的诱导分化培养液检测ADSCs向脂肪细胞和成骨细胞的分化能力,实时逆转录-聚合酶链反应检测骨桥蛋白（OPN）和过氧化物酶体增殖物激活受体-γ（PPAR-γ）mRNA表达水平。将ADSCs与植物血凝素刺激的外周血单个核细胞（PBMCs）共培养,酶联免疫吸附试验检测上清液干扰素（IFN）-γ含量。结果传3代后3组ADSCs形态均为典型的纺锤形,均表达典型间充质干细胞（MSCs）表面标志物CD90、CD105和CD73。与成年组、>50岁组比较,儿童组细胞体外增殖CI、迁移CI及OPN mRNA相对表达水平均增加（P<0.05）。儿童组ADSCs抑制PBMCs的IFN-γ分泌水平优于成年组及>50岁组ADSCs（P<0.05）。结论儿童来源ADSCs具有更强体外增殖、迁移、多向分化以及免疫抑制能力。

关键词: 脂肪组织, 间质干细胞移植, 免疫,细胞, 细胞分化, 年龄因素

Abstract:

Objective To investigate the impact of donor age on biological properties of human adipose tissue-derived mesenchymal stromal cells (ADSCs). Methods Subcutaneous adipose tissue and peripheral blood sample were collected after abdominal surgery. They were divided into the child group (n=10), the adult group (n=10) and the >50 years old group (n=10). ADSCs were isolated and cultured. Cell immunophenotypic characteristics were determined by flow cytometry. The proliferation and migration cell index (CI) were monitored with a Real-Time Cell Analyzer. Multilineage differentiation potential was investigated by evaluating the induction response. The mRNA expression of osteopontin (OPN) and peroxisome proliferator-activated receptor-γ (PPAR-γ) from ADSCs were determined by reverse transcription-polymerase chain reaction. ADSCs were co-cultured with peripheral blood mononuclear cells (PBMCs) in presence of concomitant phytohemagglutinin stimulation. Enzyme linked immunosorbent assay was performed to detect the IFN-γ levels in culture supernatants. Results After 3 passages, ADSCs from the three groups showed typical spindle-shaped, and all expressed typical mesenchymal stem cell (MSCs) surface markers CD90, CD105 and CD73. The proliferation, migration CI, and the expression levels of OPN mRNA were significantly increased in ADSCs derived from the child group compared with those in the adult group and the >50 years old group (P<0.05). ADSCs derived from the child group significantly decreased the secretion of IFN-γ in culture supernatants compared with the adult group and the >50 years old group (P<0.05). Conclusion ADSCs derived from children have biological advantages in the proliferation, migration, multilineage differentiation potential and immunosuppressive effects in vitro.

Key words: adipose tissue, mesenchymal stem cell transplantation, immunity,cellular, cell differentiation, age factors

中图分类号:

R392.12

图/表 6

图1 3个年龄组ADSCs形态图（×200）

Fig.1 Morphologic characteristics of ADSCs from three age groups (×200)

图2 姬姆萨染色后的ADSCs的形态图（×400）

Fig.2 Morphologic characteristics of ADSCs after Gemsa staining (×400)

图3 流式细胞术检测ADSCs表面标志物情况 A：儿童组;B：成年组;C：>50岁组。

Fig.3 Detection of surface markers of ADSCs detected by flow cytometry

表1 3组ADSCs增殖及迁移CI比较（n=10,$\bar{x}±s$）

Tab.1 Comparison of cell proliferation and migration cell index of ADSCs derived from three age groups

组别	增殖CI	迁移CI
儿童组	2.65±0.18	2.34±0.17
成年组	2.43±0.15^a	1.92±0.14^a
>50岁组	2.32±0.15^a	1.78±0.20^a
F	11.580^＊＊	29.460^＊＊

图4 3组ADSCs诱导分化成脂及成骨细胞图（×200）脂肪细胞（油红O染色）、成骨细胞（茜素红染色）,箭头所示为脂滴及矿物质沉积。

Fig.4 Adipogenic and osteogenic induction differentiation of isolated ADSCs derived from three age groups (×200)

表2 3组ADSCs中PPAR-γ、OPN mRNA表达及PBMCs中IFN-γ分泌水平比较（n=10,$\bar{x}±s$）

Tab.2 Comparison of PPAR-γ,OPN mRNA expression in ADSCs and IFN-γ secretion in PBMCs from three age groups

组别	PPAR-γ mRNA	OPN mRNA	IFN-γ（ng/L）
儿童组	0.55±0.11	0.58±0.07	638.5±40.4
成年组	0.57±0.08	0.50±0.06^a	687.4±44.8^a
>50岁组	0.63±0.08	0.36±0.07^ab	740.6±36.6^ab
F	2.066	26.539^＊＊	15.701^＊＊

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年龄对人脂肪来源间充质干细胞生物学特性的影响

Impact of age on the biological properties of human adipose tissue-derived mesenchymal stromal cells

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