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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 investigate the mechanism of NK cell proliferation from human peripheral blood mononuclear cells (PBMCs) derived from stem cell culture medium and HSP70-TKD-stimulated HSP70-positive pancreatic cancer cells. Methods: CD3-CD56 + NK cells were efficiently expanded from healthy human PBMCs with stem cell culture medium and the level of IFN-γ secreted by HSP70-TKD-induced NK cells was measured by ELISA. The expression of Granzyme B was analyzed by ELISPOT ; MTT assay was used to determine the cytotoxicity of NK cells induced by HSP70-TKD to Colo + and pancreatic cancer cells expressing HSP70; and to analyze the cytotoxicity of NK cells induced by HSP70-TKD and IFN- Correlation of expression. Results: NK cell proliferation from peripheral blood increased from (8.9 ± 0.7) ng / mL to (30.2 ± 1.3) ng / mL before stimulated with 2.0 μg / mL HSP70-TKD, (P <0.05). The expression of Granzyme-B increased from (3.2 ± 0.3)% to (21.2 ± 0.6)%, the difference was statistically significant, P <0.05. HSP70-TKD-induced NK cell colony-killing activity was low, compared with the non-induced NK cells was no significant difference; and colo + killing activity, induced group and non-induced group differences were statistically significant; when The highest killing activity was (60.1 ± 2.8)% at TKD concentration of 2.0μg / mL, while the uninhibited NK cell killing activity was (27.8 ± 2.6)%. CONCLUSION: NK cells induced by HSP70-TKD at 2.0μg / mL showed the highest IFN-γ secretion, granzyme-B expression and the highest cytotoxicity against Colo +, suggesting that HSP70-TKD induces NK to kill HSP70 positive cells May be related to its IFN-γ secretion and granzyme-B expression.