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  

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

作　　者：罗琦[1] 尹洁[2] 李杨[2] 黄珊[2] 王玺[2] 何景华[1] 

机构地区：[1]天津医科大学基础医学院药理学教研室,300070 [2]天津医科大学细胞生物学系

出　　处：《天津医药》2015年第3期225-228,I0001,共5页Tianjin Medical Journal

基　　金：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)

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20μg/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。Objective To detect the efficiency and function of NK cell differentiation from human umbilical cord he- matopoietic 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 fac- tor （SCF）, interleukin （IL） -7, IL-15 and IL-21. And CD34^＋ HSCs were differentiated into NK ceils 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 ceils 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 CI）19 between different differentia- tion 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 ceils of 28 d and 21 d. Conclusion Human umbilical cord hematopoietic stem ceils can differentiate into NK cells un-der appropriate

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 

分 类 号：R392[医药卫生—免疫学]