摘要
目的 探讨自然杀伤（NK）细胞的体外扩增及对卵巢上皮性癌（卵巢癌）细胞的杀伤作用.方法 （1）NK细胞的体外扩增及表型鉴定：收集2015年11月北京大学人民医院1名健康志愿者捐赠的外周血20 ml,分离外周血淋巴细胞,采用转染后过度表达白细胞介素21（IL-21）的慢性髓系白血病细胞系K562细胞（IL-21-K562细胞）作为饲养细胞（经放射线照射）,与外周血中分离的淋巴细胞混合培养,体外诱导、扩增培养14 d,监测扩增过程中淋巴细胞数量和存活细胞比例的变化,14 d后收获扩增的淋巴细胞并计数,采用流式细胞仪鉴定扩增的淋巴细胞中NK细胞比例（其表型为CD3CD56＋）.（2）NK细胞对卵巢癌细胞的杀伤作用：效应细胞（即NK细胞）与靶细胞（即卵巢癌细胞系SKOV3、HOC1A、3AO、CAOV3细胞及其对照K562细胞和正常卵巢上皮细胞系T29细胞）的比值（效靶比）分别为50∶1、20∶1、10∶1、5∶1、1∶1,采用乳酸脱氢酶（LDH）释放实验检测NK细胞对卵巢癌细胞的杀伤作用.结果 （1）NK细胞的体外扩增及表型鉴定结果：体外诱导、扩增培养14d,淋巴细胞数从扩增当天（第0天）的2.0× 107个至第14天时为5.1×109个,扩增后淋巴细胞数增加至扩增前的255倍;存活细胞比例从扩增当天的95％以上降至第8天的80％左右,随后逐渐升高,至第14天时又达95％以上.扩增前、后表型为CD3-CD56＋的NK细胞占CD3-的非T淋巴细胞总数的比例分别为2.33％、85.32％,占总淋巴细胞数的比例分别为1.06％、69.42％,扩增前、后分别比较,差异均有统计学意义（P＜0.01）.（2）NK细胞对卵巢癌细胞的体外杀伤率：LDH释放实验显示,NK细胞对卵巢癌SKOV3、HOC1A、3AO、CAOV3细胞及其对照K562细胞和T29细胞均有很强的杀伤作用,随着效靶比的增加其杀伤率均明显增高（P＜0.01）.当效靶比为1∶1时,NK细胞对K562细胞的杀伤率为37％,而对卵巢癌细胞及T29细胞的杀伤率均在10％左右,分别比较,差异均有统计学意义（P＜0.05）;当效靶比为50∶1和20∶1时,除CAOV3细胞（杀伤率＞70％）以外,NK细胞对其他卵巢癌细胞及其对照K562、T29细胞的杀伤率均大于80％,各细胞间比较,差异均无统计学意义（P＞0.05）.结论 NK细胞能够在体外得到高效扩增,且其对卵巢癌细胞有良好的非特异性杀伤作用.
Objective To amplify natural killer （NK） cells in vitro and explore its killing effect on ovarian cancer cells.Methods （1） The separation of NK cells and identification.A total of 20 ml peripheral blood of one healthy volunteer was collected in Nov.2015,Peking University People＇s Hospital.The peripheral blood mononuclear cells of normal volunteers were isolated,cultured in vitro and amplificated cultivation for 14 days with K562 cells transfected and expressing interleukin 21 （IL-21-K562） as nourish cells.The number and dynamic state of the growth cells were monitored during the cultured process.Cells were harvested and counted after 14 days cultured.The NK cells phenotypes were detected by flow cytometry.（2） The killing effect of NK cells on ovarian cancer cells：the ratio of effector cells （NK cells） and target cells （ovarian cancer cells and its control） was 50∶ 1,20∶ 1,10∶ 1,5∶1 or 1 ∶ 1,NK cells killing effect on ovarian cancer cells was detected by the lactate dehydrogenase （LDH） release experiments.Results （1） The results of NK cells establishment and phenotypic characterization：the cells were induced in vitro for 14 days by amplification culture.With the extension of incubation time,the number of NK cells increased constantly,from 2.0× 107 on day 0 to 5.1 × 109 on day 14.Obvious amplification of the total number of cells were detected for 255 times.Living cells unstained by trypan blue eventually reached 95% above.Before and after the induction and amplification in vitro,the percentage of NK cells （CD3-CD56＋cells） in CD3-cells were 2.33% and 85.32%,respectively （P〈0.01）,which covered the whole lymphocytes 1.06% and 69.42%,respectively （P〈0.01）,which showed that NK was the main cell type in the amplificated lymphocytes.（2） The killing rate of NK cells on ovarian cancer cells in vitro：the results detected by LDH release experiments showed that NK cells could performed strong nonspecific killing effect on ovarian cancer cell lines SKOV3,HOC1A,3AO and CAOV3,as well the normal ovarian cell line T29 and NK sensitive cell line K562,and the killing effect increased significantly along with the increase of effector cells and target cells ratio （P〈0.01）.When the ratio was 1 ∶ 1,the killing rate was 37% for K562,while the rate of killing of other cells was around 10% （P〈0.05）.When the effect-target ratio was 20∶1 and 50∶ 1,in addition to CAOV3 cells （more than 70%）,NK cells had a kill rate of more than 80% for other ovarian cancer cells lines and their control cell K562 and T29 cells （P〉0.05）.Conclusion NK cells could be established in vitro and have a good non-specific killing effect on ovarian cancer cells.
出处
《中华妇产科杂志》
CAS
CSCD
北大核心
2017年第8期545-550,共6页
Chinese Journal of Obstetrics and Gynecology
基金
基金项目：国家自然科学基金（81172454）
关键词
卵巢肿瘤
杀伤细胞
天然
免疫疗法
过继
Ovarian neoplasms
Killer cells,natural
Immunotherapy,adoptive
