间充质干细胞产品生物学活性检测方法建立  

Research on biological activity methods of mesenchymal stem cell products

作　　者：王瑶 房吉庆 袁子维 李瑶玲 杨英 饶春明 WANG Yao;FANG Ji-qing;YUAN Zi-wei;LI Yao-ling;YANG Ying;RAO Chun-ming(JOINN Pharmaceutical Quality Research and Testing(Beijing)Co.,Ltd.,Beijing 102605,China)

机构地区：[1]北京昭衍药物检定研究有限公司,北京102605

出　　处：《药物分析杂志》2025年第1期12-19,共8页Chinese Journal of Pharmaceutical Analysis

基　　金：北京市科技计划课题:基因修饰免疫细胞和基因治疗药物质量控制关键技术与服务平台建设(Z221100007922015)。

摘　　要：目的:建立间充质干细胞(MSCs)生物学活性检测方法,应用于MSCs产品的质量控制。方法:采用流式细胞术检测MSCs表面标志物;采用抗坏血酸、β-甘油磷酸钠等诱导MSCs成骨分化,诱导后用茜素红S染色,检测成骨分化能力;采用IBMX、罗格列酮等诱导MSCs成脂分化,诱导后用油红O染色,检测成脂分化能力;采用ITS、TGF-β3等诱导MSCs成软骨分化,诱导后用阿利新蓝染色,检测成软骨分化能力;采用细胞共培养和ELISA方法检测MSCs对巨噬细胞极化的作用,将THP-1细胞诱导为巨噬细胞后与MSCs共培养,ELISA方法检测共培养上清中IL-10、TNFα表达水平;采用CFSE标记细胞法、CD3/CD28刺激PBMC活化法和细胞共培养方法,用流式细胞术检测MSCs对淋巴细胞增殖抑制的能力。结果:间充质干细胞表面标志物CD105、CD73和CD90表达均≥95%,CD45、CD34、CD14、CD19和HLA-DR表达≤2%;MSCs经成骨诱导分化后显微镜下可观察到红色钙结节;MSCs经成脂诱导分化后显微镜下可见红色的脂质空泡,呈典型的脂质分化;MSCs经软骨诱导分化形成软骨细胞球,显微镜下可见典型的软骨陷窝;MSCs与诱导的THP-1巨噬细胞共培养后,MSCs能刺激IL-10表达增多,并下调TNFα分泌;MSCs对CD3/CD28活化后的PBMC的增殖具有抑制作用,抑制率为76.4%。结论:本研究建立了MSCs表面标志物、分化能力、促进巨噬细胞极化和淋巴细胞增殖抑制的检测方法,可用于MSCs产品生物学活性检测。Objective:To explore the biological function methods of mesenchymal stem cells(MSCs)for quality analysis.Methods:The surface markers of MSCs were detected by flow cytometry.MSCs osteogenic differentiation was induced by ascorbic acid andβ-glycerophosphate sodium,etc.,followed by Alizarin Red S staining.MSCs adipogenic differentiation was induced by IBMX,Rosiglitazone,etc.,followed by Oil Red O staining.MSCs could differentiate into chondrocytes with treatment of ITS and TGFβ3,etc.,followed by Alcian Blue staining.Cell co-culture of THP-1-macrophage with MSCs and ELISA assay were applied to detect the effects of MSCs on macrophage polarization.The expression levels of IL~(-1)0 and TNFαin the cell co-culture supernatant were detected by ELISA.To observe the effects of MSCs on lymphocyte proliferation,MSCs cultured with PBMCs,which were labeled with CFSE and activated by CD3/CD28,followed by flow cytometry.Results:The expression of MSCs surface markers,CD105,CD73,and CD90,was more than 95%respectively,while the expression of CD45,CD34,CD14,CD19,and HLA-DR expression was less than 2%.MSCs osteogenic differentiation assay showed red calcium nodules.Red lipid vacuoles were observed in MSCs adipogenic induction differentiation.Furthermore,MSCs have the differentiation potential to chondrocyte spheroids,and typical cartilage pits were observed.Coculture of MSCs with THP-1 macrophages,an increase in IL~(-1)0 expression and downregulate TNFαsecretion were observed.MSCs played inhibitory effects on the proliferation of PBMC activated by CD3/CD28,with an inhibition rate of 76.4%.Conclusions:This study established some of biological activity detection methods for MSCs,including MSCs surface markers,differentiation abilities,promotion of macrophage polarization,and inhibitory effects on lymphocyte proliferation.It provides a potential application for MSCs products quality control.

关 键 词：间充质干细胞 表面标志物 分化 免疫调控 巨噬细胞极化 淋巴细胞增殖抑制 

分 类 号：R917[医药卫生—药物分析学]