HSP70-TKD诱导的NK细胞对胰腺癌细胞裸鼠移植瘤的抑制作用

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2 μg/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/NKG2 C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.%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 CD 94/NKG2C 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 HSP 70 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 Colo 357 tumors in SCID/beige mice was detected with Immunohis-tochemistry.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 Colo 357 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 Colo 357 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 Colo 357 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 .