HSP70-TKD诱导NK细胞对胰腺癌细胞杀伤作用及其机制的探讨

Cytolytic activity of NK cell stimulated with HSP70 peptide TKD against pancreatic carcinoma cell

目的:探讨从人外周血单核细胞(PBMC)由干细胞培养基扩增得到的NK细胞,在热休克蛋白70短肽(HSP70-TKD)刺激下杀伤细胞膜HSP70表达阳性胰腺癌细胞的机制。方法:用干细胞培养基从健康人PBMC中高效扩增得到CD3-CD56+NK细胞,ELISA方法测定HSP70-TKD诱导的NK细胞分泌IFN-γ的水平;ELISPOT方法分析其颗粒酶-B的表达情况;用MTT法测定HSP70-TKD诱导的NK细胞,对细胞膜HSP70表达阳性的胰腺癌细胞Colo+和表达阴性的胰腺癌细胞Colo-杀伤活性;从而分析其杀伤活性与IFN-γ分泌和颗粒酶-B表达的相关性。结果:由外周血扩增NK细胞在2.0μg/mL的HSP70-TKD刺激下,其IFN-γ分泌从刺激前的(8.9±0.7)ng/mL增加到(30.2±1.3)ng/mL,差异有统计意义,P<0.05;而颗粒酶-B的表达从(3.2±0.3)%增加到(21.2±0.6)%,差异有统计意义,P<0.05。HSP70-TKD诱导的NK细胞对Colo-杀伤活性低,与未经诱导的NK细胞比较差异无统计学意义;而对Colo+杀伤活性高,诱导组与未诱导组之间差异有统计学意义;当TKD浓度为2.0μg/mL时,杀伤活性最高为(60.1±2.8)%,而未诱导的NK细胞杀伤活性为(27.8±2.6)%。结论:NK细胞在2.0μg/mL的HSP70-TKD诱导下,其IFN-γ分泌、颗粒酶-B的表达和对Colo+杀伤活性达到最高,推断HSP70-TKD诱导NK对细胞膜HSP70表达阳性细胞的杀伤可能与其IFN-γ分泌和颗粒酶-B的表达有关。

OBJECTIVE： To study the cytolytic mechanism of NK cell expanded in stem growth medium SCGM and stimulated with HSP70 peptide TKD against HSP70-positived pancreatic carcinomacells. METHODS： CD3- CD56＋ NK cells were obtained from human peripheral blood mononuclear （PBMC）in stem cell growth medium SCGM. The amount of IFN-γ produced by NK cells activated with HSP70 peptide TKD was quantified by ELISA technique. The expression of Granzyme B was quantified by ELISPOT technique. The cytolytic activity of TKD-activated NK cells against human pan- creatic carcinoma sublines with and without HSP70 expression on their cell surface was analyzed by MTT assay. The rela- tion between cytolytic activity and production of IFN-γ and Granzyme B produced by NK cells was analyzed. RESULTS： When NK cells were expanded from PBMC and stimulated with 2.0μg/mL HSP70 peptide TKD, the amount of IFN-γ produced by NK TKD-activated cells increased up to （30.2±1.3） ng/mL compared to （8.9±0.7） ng/mL produced by unactivated NK cells （P 〈0. 05）. There was significant statistical difference between them. And the expression of Granzyme B increased up to （21. 2±0.6）% compared to （3.2±0.3）% expressed by unactivated NK cells,there was sig- nificant statistically different between them （P 〈0. 05）. The cytolytic activity of TKD-activated NK cells against HSP70-negative pancreatic carcinoma sublines colo was low and there was not statistical difference to unactivated NK cells. The cytolytic activity against HSP70 membrane-positive pancreatic carcinoma sublines colo＋ was high and there was significantly statistical difference between TKD-activated NK cells and unactivated NK cells. The cytolytic activity was（60.1±2.8）% when NK cells were stimulated with 2.0μg/mL HSP70 peptide TKD compared to （27.8±2.6）% by un- activated NK ceils. CONCLUSIONS： The production of IFN-γ, the expression of Granzyme B and The cytolytic activity HSP70 membrane-positive pancreatic carcinoma sublines colo＋ is the highest when NK cells are stimulated with 2.0 μg/mL HSP70 peptide TKD. Our results indicate that the cytotytic activity against HSP70- positive tumor ceils by NK cells is associated with the production of IFN-γ and Granzyme B by NK cells.