【摘要】 目的:研究HSP70-TKD诱导的自然杀伤细胞(NK)对胰腺癌原位移植模型的体内抗肿瘤作用。方法:用干细胞培养基富集人外周血单个核细胞(Peripheral blood mononuclear cell,PBMC)至第10天时,加入2μg/ml TKD,4 d后用流式细胞仪检测诱导后NK细胞表面杀伤性受体CD94/NKG2C的表达情况,MTT法测定诱导后NK细胞体外对细胞表面HSP70表达阳性的胰腺癌Colo357细胞的杀伤活性。在BALB/c裸鼠胰腺原位接种胰腺癌Colo357细胞至第15天时,尾静脉注射HSP70-TKD诱导的NK细胞,观察NK细胞对荷瘤鼠的抑瘤作用。免疫组化染色检测NK细胞在胰腺癌组织的浸润情况。结果:NK细胞在干细胞培养基中可大量增殖,14 d时平均细胞纯度为(87.50±1.35)%。HSP70-TKD诱导的NK细胞其细胞表面CD94/NKG2C的表达明显增加,其平均荧光强度为(220.56±6.82),高于未诱导组的(68.72±1.85)。HSP70-TKD诱导的NK细胞对胰腺癌细胞Colo357有更强的杀伤活性,当效靶比为40∶1时杀伤率高达(68.72±2.55)%。体内实验表明,TKD诱导的NK细胞能显著抑制胰腺癌的生长,其抑瘤率可达(61.3±1.5)%,与对照组差异显著(P<0.01)。免疫组化染色表明IL-2和TKD同时诱导的NK细胞更易向胰腺癌组织浸润。结论:HSP70-TKD诱导的NK细胞是一种新型、高效的免疫活性细胞,具有较强的体外抗细胞表面HSP70表达阳性的胰腺癌细胞活性,具有明显的抑制胰腺肿瘤生长的作用。更多还原

【Abstract】 Objective: To investigate the antitumor effect of TKD-activated NK cells on tumor growth inhibition of human pancreatic carcinoma in nude mice. Methods: CD3-CD56+NK cells were obtained from human peripheral blood mononuclear( PBMC) in stem cell growth medium SCGM,2 μg / ml TKD was added to the medium on day 10. The activating receptor CD94 / NKG2 C expression levels on NK cells was detected with FAC after 4 days. The cytolytic activity of TKD-activated NK cells against human pancreatic carcinoma subline with HSP70 expression on their cell surface was analyzed by MTT assay. Established a new model of orthotopic-transplantation tumor of human pancreas. NK cells were injected i. v. into the tail vein of tumor-bearing mice on day 15,the antitumor activity of the NK were evaluated. The capacity to infiltrate Colo357 tumors in SCID / beige mice was detected with Immunohistochemistry. Results:Flow cytometry analysis showed that CD3-CD56+NK cells could expanded in SCGM medium,and the average percentage of NK cell was( 87. 50 ± 1. 35) %. CD94 expression levels on NK cells increased obviously,the mean fluorescence intensity of CD94 was( 220. 56± 1. 82),compared to( 68. 72 ± 1. 85) of control group cell. The cytolytic activity against HSP70 membrane-positive pancreatic carcinoma sublines Colo357 cells was high and there was significantly statistical difference between TKD-activated NK cells and unactivated NK cells. The cytolytic activity was( 68. 72 ± 2. 55) % when ratio of effector cells and target cell was 40∶ 1. TKD-activated NK cells had a stronger suppressive effect on tumor growth in BALB / c nude mice bearing Colo357 cells in vivo,Median inhibitory rates was( 61. 3± 1. 5) %. There was significant statistical difference compare to control group( P < 0. 01). The result of Immunohistochemistry indicated that predominantly NK cells induced with TKD had the capacity to infiltrate Colo357 tumors in SCID / beige mice. Conclusion:TKD-activated NK cells are highly efficient cytolytic effector cells which have a stronger significant suppression against pancreatic carcinoma growth in vivo.更多还原