摘要
目的探讨低剂量照射(LDR)对NK细胞体内及体外增殖和杀伤活性的影响,并探讨其相关的信号传导机制,同时将体外LDR后的NK细胞回输小鼠体内,观察LDR后的NK细胞对肿瘤的形成和生长的抑制作用。方法从小鼠脾脏提出的单个核细胞,应用免疫磁珠的方法分离CD3-/CD16+/CD56+细胞,并应用流式细胞仪鉴定后以总剂量为20Gy60Coγ射线照射后的小鼠脾细胞为饲养细胞,以重组人白介素-2(rhIL-2)为诱导因子,在体外进行NK细胞的扩增培养。培养后的细胞经流式细胞仪进一步鉴定其表型。体外实验将NK细胞分成阴性对照组,和25mGy、75mGy、200mGy和500mGy组,每组又分成照射后4h、24h、48h、72h组;体内实验将小鼠分成阴性对照组和75mGy照射后4h、24h、48h、72h组。体内体外实验均应用流式细胞仪检测各组细胞NK细胞的增殖指数,并应用3H-TdR掺入法检测其杀伤活性;体外实验我们同时验证LDR刺激NK细胞生长和活性的信号传导的机制,将细胞分成阴性对照组、75mGy照射组、照射+SB203580组、照射+P79350组,每组细胞检测细胞的增殖强度、活性变化及相关蛋白磷酸化P38MAPK、IFN-γ、FasL、perforin的变化,并统计它们之间的关系;体内实验应用K562尾静脉输入和皮下移植制作K562成瘤模型,观察LDR后NK细胞回输到小鼠体内对K562成瘤的抑制作用及已经成瘤的K562小鼠肿瘤的生长的抑制作用。结果应用上述方法可以成功培养出CD3-/CD16+/CD56+NK细胞,体内、体外低剂量照射后可以增加NK细胞的增殖指数,并增强其对K562细胞的杀伤活性。体外实验75mGy照射后24h最为明显。体内照射48h最明显;LDR后NK细胞表达磷酸化的P38MAPK、IFN-γ、FasL、perforin均增加,且与PI及杀伤活性呈正相关,与未照射的NK细胞比较,差异具有显著性(P<0.05),抑制P38MAPK表达后,PI、杀伤活性及相关蛋白IFN-γ、FasL、perforin均下降;LDR后NK细胞回输到尾静脉注射成瘤模型小鼠中肝、脾CD13+、S期细胞及外周血异常细胞所占的百分率都明显下降,与未照射NK细胞回输组比较,差异具有显著性(P<0.05),回输到经皮下抑制成瘤的小鼠模型中肿瘤体积、重量明显下降,小鼠的生存时间和生存率明显延长,与未照射NK细胞回输组比较,差异具有显著性(P<0.05)。结论低剂量辐射可以提高体内体外NK细胞的增殖和杀伤活性,机制是LDR可以增强P38MAPK信号传导的途径,LDR后NK细胞回输小鼠体内不但可以抑制K562细胞成瘤作用,还可以抑制已经成瘤的小鼠的肿瘤生长并提高生存时间和生存率。
Objective To study the in vitro and in rive effect of LDR on the proliferation and killing activity of mouse NK cells with exploitation of the related mechanism of signal transduetion. The effect of infused NK cells on inhibiton of oncogenesis and tumor burden regression was also studied. Methods Mononuclear cells extracted from mouse spleen were treated with immunomagnetic bead for the isolation of CD3^＋/CD16 ^＋ , CD56 ^＋ cells. After verified with flowcytomctry, these NK cells were cultured with mice splenic cells （irracdiated with 20Gy ^60Co gamma ray） as feeder cells and rhIL-2 as induction factor for 3 rounds （5 days each round）. Specimens of cultured NK cells were reated with differeut doses of radiation （25raCy, 75mGy, 20dinGy, 500raCy）, the proliferation index （PI） with tumor- cidal activity on 1,7562 cells （with ^3H-TdR） ineorporalion was examined at 4h, 24h, 48h, 72h after irradiation respectively. The tole of P38MAPK signal pathw;ty in the LDR effect was examined with adding either inhibitor （SB203580） or activator （P79350） of P38MAIPK into the culture and measuring the PI, Killing activity （as expression of the related factors IFN -gamma, FasL, perforin ） of NK cells thereafter. The in rive test involved exposing mice to whole body 25raCy irradiation, harvesting splenic NK cells at 4h, 24h, 48h, 72h later respectively and performing the above - described in vitro procedures. Inhibition of oncogenesis was examined in rive with infusion of coltured NK cells （ LDR treated vs LDR non - treated） 10 days after infusion of K562 cells into mice and examinatiou of hepatic/splen;CD13^＋ , S - stage cells and peripheral blood tumor cells in the sacrificed animal another 10 days later. Also, K562 cells were innoculated subcutaneously into mice. After tumor nodule formation （2.0 × 2.0mm）, NK cells （LDR treated vs non - treated） wcre infused and regression of the tumor nodule with the weight of hepatic tumor mass was noticed in saeririced animals on d 8 and the survival rate on d 40 recorded. Results With the above methods, CD3^＋/CD16 ^＋ , CD56 ^＋ NK cell were successfully cultured. The PI and killing activity were highest at 24h after 75mGy LDR in vitro, but at 48h in rive. Both PI and killing activity demonstrated as expression of IFN -gamma, FasL, perforin were sigoifieantly increased after LDR （vs without LDR, P 〈 0. 0.5）, with significant decrease after inhibition of P38MAPK pathway with SB203580. infusion of LDR treated NK cells into tumor - forming mouse models （ with K562 cell infusion） resulted in significant decrease of hepatic/splenic CD13^＋ , S - stage cells and periph- eral blood abnormal （tumor） cell percentages （ vs without LDR, P 〈 0.05）. The same was true for tumor bearing mouse models （with K562 cells subeutanenus iunoculation and tumor nnodule formation） i.e. more tumor nodule regression and less hepatic tumor weight with prolonged survival （vs without LDR,P〈 0.05）. Conclusion Both in vitro and in rive, LDR could promote the PI and killing activity of NK cells the through P38MAPK signal pathway augmentation. Better inhibition of oncogenesis and decrease of tumor burden were clearly demonstrated with infusion of LDR treated NK cells into mouse models （prepared with K562 cells）.
出处
《放射免疫学杂志》
CAS
2006年第6期529-535,共7页
Journal of Radioimmanology
