生长激素对人外周血来源NK细胞杀伤活性的影响及机制探讨

Effect of growth hormone on cytotoxicity of NK cells from PBMC

目的观察生长激素(GH)对人外周血来源自然杀伤(NK)细胞杀伤活性的影响,并探讨其机制。方法选取10名健康志愿者,采用免疫磁珠法分离并培养其外周血NK细胞。将NK细胞分为A、B、C组,分别与终浓度0、5、20μg/mL的重组人GH共培养48 h。以人类髓性白血病K562细胞为靶细胞,NK细胞为效应细胞,按不同的效靶比(25∶1、50∶1、100∶1)混合,采用CCK-8法检测NK细胞对K562细胞的杀伤率;取各组培养48 h的NK细胞,分别采用流式细胞术和Real-time RT-PCR法检测NK细胞表面活化受体NKG2D蛋白及基因。结果三个不同效靶比下,B、C组NK细胞对K562细胞的杀伤率高于A组,且C组高于B组(P均<0.01)。A、B、C组NKG2D阳性表达的NK细胞百分数分别为78.8%±2.8%、87.5%±3.1%、91.8%±3.3%,B、C组NKG2D阳性表达的NK细胞百分数高于A组(P均<0.05)。A、B、C组NKG2D mRNA相对表达量分别为1±0.01、5±2.32、10±3.21,B、C组NKG2D mRNA相对表达量高于A组,且C组高于B组(P均<0.05)。结论 GH可通过上调NK细胞NKG2D的表达,增强其杀伤活性。

Objective To investigate the effect of growth hormone on the cytotoxicity of natural killer （NK） cells from human peripheral blood mononuclear cells （PBMC） and the possible mechanism. Methods NK cells, purified by mag- netic activated cell sorting （MACS） from 10 volunteers, were divided into three groups. Cells in the groups A, B and C were co-cultured with recombinant human growth hormone （rhGH） （A： 0μg/mL, B：5 μg/mL, C：20 μg/mL）. After 48 h, the cytotoxicity of NK cells against K562 cells, with different effector-target ratio （25： 1, 50： 1, 100：1 ） , was analyzed by CCK-8. Real-time RT-PCR and flow cytometry were used to detect the expression of natural killer cell group 2 member D （NKG2D） , in the levels of gene and protein molecules. Results The cytotoxicity of NK cells against K562 cells in the group B and C was significantly higher than that in the group A at different effector-target ratio, and group C was higher than group B （ all P 〈 0. 01 ）. The percentages of NK cells with positive expression of NKG2D in the groups A, B and C were 78.8% ±2.8%, 87.5%±3.1% and 91.8%±3.3%, respectively, and group B and C were higher than group A （ all P 〈 0.05 ）. The relative expression of NKG2D mRNA of NK cells in the groups A, B and C was 1± 0.01, 5±2.32 and 10±3.21 respectively. The expression of NKG2D mRNA in the group B and C was significantly higher than that of group A, and group C was higher than group B （ all P 〈 0.05 ）. Conclusion Growth hormone may enhance the cytotoxici- ty of NK cells from PBMC by up-regulating the expression of NKG2D.