脐血单核细胞体外高效扩增NK细胞方法的研究  

Study on a highly efficient ex vivo expansion method for NK cells using umbilical cord blood mononuclear cells

作　　者：汪德海 郭昌龙 周越 高建恩 马旭 WANG Dehai;GUO Changlong;ZHOU Yue;GAO Jianen;MA Xu(National Research Institute for Health Commission,Na-tional Human Genetic Resources Center,Beijing 100051,China)

机构地区：[1]国家卫生健康委员会科学技术研究所国家人类遗传资源中心

出　　处：《中国输血杂志》2018年第7期722-726,共5页Chinese Journal of Blood Transfusion

基　　金：国家重点研发计划(2017YFC1002004和2016YFC1000307-11)

摘　　要：目的建立1种使用脐血单核细胞（UCBMC）制备较高纯度自然杀伤（NK）细胞的体外高效扩增方法,并初步鉴定扩增后的NK细胞杀伤肿瘤细胞（K562）的能力。方法 6人份脐血标本来自北京妇产医院,取20 mL/份、采用密度梯度离心法分离脐血中的UCBMC,提取后的UCBMC以1×10~6个/mL细胞在4 mL含有白细胞介素（IL）-2、IL-12、IL-15和IL-21的X-VIVO15中培养5 d,期间于d2和d3添加等体积含上述细胞因子的新鲜培养基; 800 g离心将细胞后转入含1 000 IU/ml IL-2和50 ug/mL L-抗坏血酸-2-磷酸三钠盐的X-VIVO15培养基中继续培养至14 d,800 g离心收获细胞。无血清RPMI1640重悬后通过台盼蓝染色法做细胞计数测定细胞的扩增倍数;流式细胞仪检测NK细胞重要表面标志物（CD56和CD16等）及与杀伤功能相关的重要分子[CD158a、CD158b、CD158e/k、CD69、CD314（NKG2D）、CD94、CD335（NKp46）、CD337（NKp30）、CD62L、CD226（DNAM-1）、CD57和CD336（NKp44）]的表达情况;胞内染色结合流式细胞仪测定细胞杀伤重要效应分子Perforin和Granzyme B的表达情况,并以乳酸脱氢酶（LDH）释放法检测扩增后的细胞对K562细胞的杀伤能力。结果 6份UCBMC培养14 d：活细胞总数从培养前的4×106个扩增至（3. 6—5. 5）×10~8个,扩增倍数91—137（116±13. 1）; CD3-CD56＋细胞（NK细胞）占比85. 6%—95. 5%（90. 2±3. 4）%;培养得到的NK细胞表面除表达CD56分子外,还表达CD16（91. 2±2. 6）%、CD158a（71. 4±4）%、CD158b（52. 1±2. 2）%、CD158e/k（50. 3±6. 9）%、CD69（95. 6±2. 8）%、CD314（NKG2D）（94. 2±1. 7）%、CD94（94. 1±2. 1）%、CD335（NKp46）（26. 6±4. 1）%、CD337（NKp30）（94. 6±1. 5）%、CD62L（12. 8±3. 4）%、和CD226（DNAM-1）（97. 6±1. 3）%,以及CD57（4. 2±0. 9）%和CD336（NKp44）（7. 1±1）%。胞内染色结果显示：〉90%扩增产物（细胞）内表达与NK细胞杀伤功能相关的Perforin（86. 9±2. 7）%和Granzyme Objective To establish a method for preparing of natural killer（ NK） cells in vitro with high purity by the induction of umbilical cord blood mononuclear cells（ UCBMC） with cytokine cocktail and evaluate the potential anti-tumor capability of the expanded cells.Methods 6 UCB samples were obtained from Beijing Obstetrics and Gynecology Hospital under the term of scientific use. UCBMCs were isolated from the UCB samples by differential density centrifugation using Ficoll-Paque. The isolated UCBMCs were cultured in a serum free medium of X-VIVO 15 containing IL-2（ 1 000 μ/mL）,IL-12（ 20 ng/mL）,IL-15（ 20 ng/mL）,IL-21（ 5 ng/mL） and 10% auto-plasma at a cell density of 1×106/mL for 5 days with equal volume fresh medium addition at day 2 and day 3. Cells were harvested by centrifugation at 800 g,room temperature and re-plated in 15 mL X-VIVO 15 medium containing IL-2（ 1 000 IU/mL） and phospho-ascorbic acid（ 50 μg/mL） for 9 days,with equal volume fresh medium addition every 3 days. After 14 days of culture,cells were harvested and the expression of NK cell immunophenotypic markers,such as CD56 and CD16,and functional molecules,such as CD158 a,CD158 b,CD158 e/k,CD69,CD314（ NKG2 D）,CD94,CD335（ NKp46）,CD337（ NKp30）,CD62 L,CD226（ DNAM-1）,CD57,CD336（ NKp44）,Perforin and Granzyme B on or in expanded cells were analyzed by flow cytometry. In vitro cytotoxic function against tumor cell line K562 was evaluated using LDH release cytotoxic assay. Results Freshly isolated 6 UCBMCs were cultured for 14 days in the expansion medium supplemented with 10% auto-plasma from each UCB unit respectively. The average percentage of CD3-CD56＋subset in expanded cells was85. 6%—95. 5%（ 90. 2±3. 4） % with the average of（ 116± 4. 7）fold expansion. In addition of NK phenotypic markers（ CD56 and CD16）,the expanded cells expressed most of the functional molecules of NK cells,such as CD158 a（ 71. 4 ± 4） %,CD158 b（ 52. 1±2. 2） %,CD158 e/k（ 50. 3±6. 9） %,CD69（ 95. 6�

关 键 词：脐血 单核细胞 自然杀伤细胞 体外扩增 肿瘤细胞 K562 细胞毒 

分 类 号：Q813.11[生物学—生物工程] R392.12[医药卫生—免疫学]