不同抗凝剂保存液对脐血培养的NK细胞增殖及杀伤效应的影响

doi:10.3877/cma.j.issn.2095-3232.2023.05.019

目的

探讨不同抗凝剂保存液对脐血培养的NK细胞增殖及杀伤效应的影响。

方法

无菌采集健康孕妇20 ml脐血共10例，分别放入肝素钠和血液保存液的抗凝管中。用淋巴细胞液分离脐血单个核细胞（CBMC），在体外扩增培养NK细胞，分别于培养0、3、6、9、12、15、18、21 d进行细胞计数。在第15天和第21天通过流式细胞仪检测CD3^-CD56⁺、CD16⁺NK细胞比例以及细胞凋亡率。在第15天用CCK8法按照效靶比5∶1、10∶1、20∶1检测两组抗凝剂收集CBMC，在体外扩增获得的NK细胞对人肝癌细胞株（SMMC7721、BEL7402、Sk-hep1、HepG2、Hep3B、Huh7）的细胞毒作用。两组细胞数目等比较采用t检验；多组比较采用单因素方差分析，两两比较采用LSD-t检验。

结果

体外培养第9天开始NK细胞均呈对数生长，培养至第15天后细胞增殖能力下降，细胞数量减少。细胞生长曲线显示，第9、15、21天肝素钠组NK细胞数目分为（1.8±0.8）×10⁸、（6.2±1.9）×10⁸、（4.9±1.3）×10⁸个，明显高于血液保存液组的（1.0±0.5）×10⁸、（4.3±2.0）×10⁸、（3.4±1.9）×10⁸个（t=2.797，2.198，2.177；P<0.05）。NK细胞对肝癌细胞株的细胞毒作用显示，NK细胞对6株人肝癌细胞的细胞毒作用随着效靶比增加而增强，在效靶比20∶1时杀伤效率最高，其中对SMMC7721杀伤作用最强，而两组杀伤率无明显差异（P>0.05）。

结论

肝素钠和血液保存液均可作为脐血NK细胞培养的抗凝剂，肝素钠可增强NK细胞增殖能力，但不增加对肝癌细胞的杀伤效应。

关键词: NK细胞, 细胞杀伤效应, 细胞毒作用, 肝癌细胞, 抗凝剂, 肝素钠, 血液保存液

Objective

To evaluate the effects of different anticoagulant preservation solutions on the proliferation of natural killer (NK) cells cultured in umbilical cord blood and their killing effects on liver cancer cells.

Methods

A portion of 20 ml umbilical cord blood was aseptically collected from 10 healthy pregnant women and transferred into anticoagulation tubes containing heparin sodium and blood preservation solution, respectively. Cord blood mononuclear cells (CBMCs) were isolated by using lymphocyte solutions. NK cells were proliferated and cultured in vitro, and cell count was carried out at 0, 3, 6, 9, 12, 15, 18 and 21 d after culture, respectively. The percentage of CD3^-CD56⁺, CD16⁺NK cells and the apoptosis rate were detected by flow cytometry at 15 and 21 d. At 15 d, CCK8 assay was utilized to detect the cytotoxic effects of CBMCs collected from two anticoagulant solutions and NK cells obtained from in vitro proliferation on human liver cancer cell lines (SMMC7721, BEL7402, Sk-hep1, HepG2, Hep3B, and Huh7) according to the effect-target ratio of 5∶1, 10∶1 and 20∶1, respectively. Cell count between two groups was compared by t test. Multi-group comparison was performed by one-way ANOVA. Two-group comparison was conducted by LSD-t test.

Results

NK cells grew logarithmically from 9 d of in vitro culture, and the proliferation capability was declined and the number of cells was decreased at 15 d. Cell growth curve showed that the number of NK cells at 9, 15 and 21 d in the heparin sodium group was (1.8±0.8)×10⁸, (6.2±1.9)×10⁸ and (4.9±1.3)×10⁸, significantly higher than (1.0±0.5)×10⁸, (4.3±2.0)×10⁸ and (3.4±1.9)×10⁸ in the blood preservation solution group (t=2.797, 2.198, 2.177; P<0.05). The cytotoxic effects of NK cells on 6 human liver cancer cells were strengthened with the increase of effect-target ratio, and the killing efficiency was the highest at an effect-target ratio of 20∶1, especially for SMMC7721. However, no significant difference was observed in the killing efficiency between two groups (P>0.05).

Conclusions

Both heparin sodium and blood preservation solution can be used as anticoagulants for NK cells cultured in umbilical cord blood. Heparin sodium can enhance the proliferation of NK cells, whereas it does not increase the killing effect on liver cancer cells.

Key words: NK cells, Cell killing effect, Cytotoxicity, Liver cancer cells, Anticoagulant, Heparin sodium, Blood preservation solution

图1 NK细胞生长曲线注：^*为与血液保存液组比较P<0.05

表1 流式细胞术检测NK细胞表型和细胞凋亡率（%）

检测时间	肝素钠组			血液保存液组
检测时间	CD3^-CD56⁺占比	CD16⁺占比	凋亡率	CD3^-CD56⁺占比	CD16⁺占比	凋亡率
第15天	86.1±7.5	91.1±2.5	8.6±5.4	85.5±9.2	92.0±3.2	13.5±6.2
第21天	64.9±10.2	68.3±8.3	26.0±13.9	70.7±8.0	72.5±9.8	27.3±9.1
t值	5.297	8.328	-3.690	3.821	5.974	-3.968
P值	<0.001	<0.001	0.002	0.001	<0.001	<0.001

表1 流式细胞术检测NK细胞表型和细胞凋亡率（%）

检测时间	肝素钠组			血液保存液组
检测时间	CD3^-CD56⁺占比	CD16⁺占比	凋亡率	CD3^-CD56⁺占比	CD16⁺占比	凋亡率
第15天	86.1±7.5	91.1±2.5	8.6±5.4	85.5±9.2	92.0±3.2	13.5±6.2
第21天	64.9±10.2	68.3±8.3	26.0±13.9	70.7±8.0	72.5±9.8	27.3±9.1
t值	5.297	8.328	-3.690	3.821	5.974	-3.968
P值	<0.001	<0.001	0.002	0.001	<0.001	<0.001

表2 不同效靶比NK细胞对肝癌细胞的杀伤率（%）

肝癌细胞株	5∶1		10∶1		20∶1
肝癌细胞株	肝素钠组	血液保存液组	肝素钠组	血液保存液组	肝素钠组	血液保存液组
SMMC7721	56.5±3.4	57.6±4.0	74.6±4.8	74.0±3.3	84.7±3.7^a,b	84.6±3.2^a,b
BEL7402	35.2±3.0^c	35.7±3.7^c	54.7±5.6^a,c	57.6±5.0^a,c	71.3±5.2^a,c	80.7±3.5^a,b
SK-hep1	33.5±2.8^c	37.4±1.7^c	48.9±4.2^a,c	56.6±3.4^a,c	70.1±4.6^a,b,c	78.0±3.8^a,b
HepG2	30.3±3.9^c	36.8±3.2^c	49.1±4.1^a,c	50.9±3.3^a,c	64.7±4.2^a,b,c	67.3±4.1^a,b,c
HEP3B	32.0±3.1^c	31.4±2.7^c	47.8±3.0^a,c	46.0±3.6^a,c	68.2±4.5^a,b,c	64.0±4.1^a,b,c
Huh7	26.4±2.2^c	29.5±2.4^c	41.8±4.2^a,c	43.7±4.8^c	57.4±4.1^a,b，c	59.4±5.2^a,b,c
F值	11.740	10.950	6.877	7.677	4.183	6.317
P值	<0.001	<0.001	<0.001	<0.001	0.003	<0.001

表2 不同效靶比NK细胞对肝癌细胞的杀伤率（%）

肝癌细胞株	5∶1		10∶1		20∶1
肝癌细胞株	肝素钠组	血液保存液组	肝素钠组	血液保存液组	肝素钠组	血液保存液组
SMMC7721	56.5±3.4	57.6±4.0	74.6±4.8	74.0±3.3	84.7±3.7^a,b	84.6±3.2^a,b
BEL7402	35.2±3.0^c	35.7±3.7^c	54.7±5.6^a,c	57.6±5.0^a,c	71.3±5.2^a,c	80.7±3.5^a,b
SK-hep1	33.5±2.8^c	37.4±1.7^c	48.9±4.2^a,c	56.6±3.4^a,c	70.1±4.6^a,b,c	78.0±3.8^a,b
HepG2	30.3±3.9^c	36.8±3.2^c	49.1±4.1^a,c	50.9±3.3^a,c	64.7±4.2^a,b,c	67.3±4.1^a,b,c
HEP3B	32.0±3.1^c	31.4±2.7^c	47.8±3.0^a,c	46.0±3.6^a,c	68.2±4.5^a,b,c	64.0±4.1^a,b,c
Huh7	26.4±2.2^c	29.5±2.4^c	41.8±4.2^a,c	43.7±4.8^c	57.4±4.1^a,b，c	59.4±5.2^a,b,c
F值	11.740	10.950	6.877	7.677	4.183	6.317
P值	<0.001	<0.001	<0.001	<0.001	0.003	<0.001

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Effects of different anticoagulant preservation solutions on the proliferation and killing effects of NK cells cultured in umbilical cord blood

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Effects of different anticoagulant preservation solutions on the proliferation and killing effects of NK cells cultured in umbilical cord blood