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.
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