天津医药

天津医药 ›› 2021, Vol. 49 ›› Issue (10): 1009-1013.doi: 10.11958/20203592

• 细胞与分子生物学 •    下一篇

OX40Ig修饰大鼠脂肪间充质干细胞的实验研究

黄志伟，刘涛，陈晓波，耿洁，李光，王玉亮   

  1. 1天津医科大学第二医院检验科，天津市泌尿外科研究所（邮编300211）；2天津市第一中心医院器官移植中心；3优赛生命科技 有限公司；4天津医科大学基础医学院遗传学系
  • 收稿日期:2020-12-28 修回日期:2021-06-01 出版日期:2021-10-15 发布日期:2021-10-15
  • 通讯作者: 王玉亮 E-mail:wang_yu_l@163.com
  • 作者简介:基金过期 作者要求今年11期发表
  • 基金资助:
    OX40Ig和ICOSIg基因修饰自体脂肪间充质干细胞诱导大鼠肾移植免疫耐受及机制研究;器官移植诱导免疫耐受及个体化免疫治疗

Experimental study on OX40Ig modified rat adipose derived mesenchymal stromal cells #br#

HUANG Zhi-wei, LIU Tao, CHEN Xiao-bo, GENG Jie, LI Guang, WANG Yu-liang #br#   

  1. 1 Department of Clinical Laboratory Medicine, the Second Hospital of Tianjin Medical University, Tianjin Institute of Urology, Tianjin 300211, China; 2 Organ Transplantation Center, Tianjin First Central Hospital; 3 Unicell Life Science Development Co., Ltd; 4 Department of Genetics, School of Basic Medical Science, Tianjin Medical University
  • Received:2020-12-28 Revised:2021-06-01 Published:2021-10-15 Online:2021-10-15
  • Contact: WANG Yuliang E-mail:wang_yu_l@163.com

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摘要： 目的 构建含OX40融合蛋白（OX40Ig）基因修饰的大鼠脂肪间充质干细胞（ADSCs）即ADSCsOX40Ig，探讨其 在体外对淋巴细胞的免疫调节作用。方法 构建pcDNA3.1（+）/OX40Ig融合基因表达载体，采用核转染技术转染大 鼠ADSCs，Western blot检测ADSCs中OX40Ig表达水平。ADSCs与大鼠异基因淋巴细胞共培养后分为对照组（反应 细胞+刺激细胞）、ADSCs组（反应细胞+刺激细胞+ADSCs）及ADSCsOX40Ig组（反应细胞+刺激细胞+ADSCsOX40Ig），MTT法 检测淋巴细胞增殖抑制率，逆转录聚合酶链反应（RT-PCR）检测淋巴细胞内干扰素（IFN）-γ、白细胞介素（IL）-10及 转化生长因子（TGF）-β mRNA表达。结果 经测序鉴定验证，pcDNA3.1（+）/OX40Ig质粒构建成功，转染ADSCs后 OX40Ig蛋白呈高表达；与ADSCs组比较，ADSCsOX40Ig显著增强异基因淋巴细胞增殖抑制率（P＜0.05）；对照组、ADSCs 组及 ADSCsOX40Ig组的 IFN-γ mRNA 相对表达水平依次降低，而 IL-10、TGF-β mRNA 相对表达水平依次升高（P＜ 0.05）。结论 ADSCsOX40Ig较单纯ADSCs能更好地发挥对异基因淋巴细胞的免疫调节作用。

关键词: 间充质基质细胞, 基因融合, 免疫调节, OX40配体, 脂肪间充质干细胞, 真核表达载体

Abstract: Objective To construct OX40Ig genetically modified rat adipose derived mesenchymal stromal cells (ADSCsOX40Ig), and to investigate its immunomodulatory effect on lymphocytes in vitro. Methods The fusion gene expression vector of pcDNA3.1(+)/OX40Ig was constructed and transfected into rat ADSCs by nuclear transfection. The expression of OX40Ig in ADSCs was measured by Western blot assay. The transfected and untransfected ADSCs were co-cultured with rat allogeneic lymphocytes, and were divided into the control group (responder + stimulator), the ADSCs (responder+stimulator+ ADSCs) group, and the ADSCsOX40Ig (responder+stimulator+ADSCsOX40Ig) group. The proliferatory activity of lymphocytes was detected by MTT method. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect interferon (IFN)-γ, interleukin (IL) -10 and transforming growth factor (TGF) - β mRNA levels in lymphocytes. Results The sequencing confirmed that pcDNA3.1(+)/OX40Ig plasmid was successfully constructed, and OX40Ig was highly expressed in transfected ADSCs. Compared with ADSCs, the proliferation inhibition rate of allogeneic lymphocytes was significantly increased in ADSCsOX40Ig (P＜0.05). The expression levels of IFN-γ mRNA decreased gradually, but the expression levels of IL-10 and TGF-β mRNA increased gradually in the control, ADSCs and ADSCsOX40Ig groups (P＜0.05). Conclusion Compared with ADSCs along, ADSCsOX40Ig has better immunomodulatory effects on allogeneic lymphocytes.

Key words: mesenchymal stromal cells, gene fusion, immunomodulation, OX40 ligand, adipose-derived mesenchymal stem cells, eukaryotic expression vector