天津医药

天津医药 ›› 2015, Vol. 43 ›› Issue (3): 225-228.doi: 10.11958/j.issn.0253-9896.2015.03.001

• 细胞与分子生物学 •    下一篇

人脐带血造血干细胞体外分化为 NK 细胞的效率及功能检测

罗琦 1， 尹洁 2， 李杨 2， 黄珊 2， 王玺 2， 何景华 1△   

  1. 1 天津医科大学基础医学院药理学教研室 （邮编 300070）； 2 天津医科大学细胞生物学系
  • 收稿日期:2014-09-30 修回日期:2014-10-29 出版日期:2015-03-15 发布日期:2015-03-15
  • 通讯作者: 何景华 E-mail:hejinghuacn@163.com
  • 作者简介:罗琦 （1990）， 女， 硕士在读， 主要从事肿瘤与免疫药理学方面研究
  • 基金资助:
    973 国家重大科学研究计划资助项目 （2014CB910104）； 国家自然科学基金资助项目 （81171899）

The efficiency and function detection of NK cell differentiation from human umbilical cord  hematopoietic stem cells in vitro#br#

LUO Qi1, YIN Jie2, LI Yang2, HUANG Shan2, WANG Xi2, HE Jinghua1△   

  1. 1 Department of Pharmacology, College of Basic Medicine, Tianjin Medical University, Tianjin 300070, China; 2 Department of Cell Biology, College of Basic Medicine, Tianjin Medical University

  • Received:2014-09-30 Revised:2014-10-29 Published:2015-03-15 Online:2015-03-15
  • Contact: HE Jinghua E-mail:hejinghuacn@163.com

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摘要： 目的 检测人脐带血造血干细胞 （HSCs） 体外分化为自然杀伤 （NK） 细胞的效率及功能。方法 从人脐带血中分离 CD34+ HSCs， 接种于含 20 μg/L FMS 样酪氨酸激酶 3 配体（Flt3L）、 干细胞生长因子（SCF）、 白细胞介素（IL） -7、 IL-15 及 IL-21 的 SCGM 培养基中， 定向诱导 CD34+ HSCs 分化为 NK 细胞。观察细胞生长状态， 在分化的第 7、 14、 21 及 28 天， 采用流式细胞术检测各阶段 CD56、 NKG2D、 NKp46、 CD3、 CD19 及 CD34 等细胞免疫表型的表达变化； 分化的第 21、 28 天， 以分化得到细胞为效应细胞， K562 细胞作为靶细胞， 设置 8∶1、 4∶1、 2∶1 和 1∶1 4 组效靶细胞比， 分别采用乳酸脱氢酶（LDH）细胞毒性检测法和 7AAD/CFSE 标记法， 检测分化得到的细胞杀伤功能。结果 脐带血来源的 CD34+ HSCs 在体外适宜的条件下可大量增殖； 整个分化进程的不同阶段中， CD3 和 CD19 表达量差异无统计学意义， CD56、 NKG2D 及 NKp46 的表达量逐渐增加， 最高达（72.57±1.60） %、（32.83±1.29） %和（29.53±
2.40） %， CD34 的表达量逐渐降低， 最低为（12.13±2.01） %。LDH 毒性检测法和 7AAD/CFSE 标记法测得最大杀伤效率可达（49.91±2.76） %和（40.87±1.12） %， 8∶1、 4∶1、 2∶1 和 1∶1 组 NK 细胞杀伤能力均依次降低（P < 0.05）， 诱导分化28 d 的 NK 细胞杀伤能力与 21 d 差异无统计学意义。结论 人脐带血 HSCs 在体外适宜的培养条件下， 可定向分化为NK 细胞， 诱导分化得到的 NK 细胞具有杀伤功能。

关键词: 胎血, 造血干细胞, 杀伤细胞, 天然, 免疫表型分型, 细胞毒性试验, 免疫, CD34+ HSCs, 体外分化, 细胞杀伤

Abstract: Objective To detect the efficiency and function of NK cell differentiation from human umbilical cord hematopoietic stem cells (HSCs) in vitro. Methods CD34+ hematopoietic stem cells were isolated from human umbilical cord blood, and inoculated into SCGM medium containing 20 μg/L FMS like tyrosine kinase 3 ligand (Flt- 3L), stem cell factor (SCF), interleukin (IL) -7, IL-15 and IL-21. And CD34+ HSCs were differentiated into NK cells in directional inducing. The growth state of cells was observed. The expressions of CD56, NKG2D, NKp46, CD3, CD19 and CD34 were detected by flow cytometry in the differentiation of 7, 14, 21 and 28 d. In the differentiation of 21 d and 28 d, the differentiation cells were used as effector cells, and K562 cells as target cells. The ratios of effector cells and target cells were 8∶1, 4∶1, 2∶1 and 1∶1. The killing activity of the differentiated cells was detected by lactate dehydrogenase (LDH) cell toxicity assay and 7AAD/CFSE labeling method. Results CD34+ HSCs derived from human umbilical cord blood can proliferate in vitro under appropriate condition. There were no significant differences in the expression of CD3 and CD19 between different differentiation stages (7, 14, 21 and 28 d, P > 0.05). The expressions of CD56, NKG2D and NKp46 were significantly different (P < 0.05), and the ultimate expression amount was (72.57±1.60)%, (32.83±1.29)% and (29.53±2.40)%. The expression of CD34 decreased gradually, and the lowest was (12.13 ± 2.01)%. The maximum killing activity detected by LDH cell toxicity assay and 7AAD/CFSE labeling method reached(49.91±2.76)% and (40.87±1.12)%.The killing activity of NK cells was decreased in the order of 8∶1, 4∶1, 2∶1 and 1∶1 groups (P < 0.05). There was no significant difference in the killing activity between NK cells of 28 d and 21 d. Conclusion Human umbilical cord hematopoietic stem cells can differentiate into NK cells under appropriate conditions in vitro, and the NK cells induced from differentiation are with killing activity.

Key words: fetal blood, hematopoietic stem cells, killer cells, natural, immunophenotyping, cytotoxicity tests, immunologic, CD34+ hematopoietic stem cells, in vitro differentiation, cytotoxicity