γ射线照射对脐血淋巴细胞增殖和NK细胞活性的影响

Effect of gamma-ray irradiation on cord blood lymphocyte proliferation and NK cell activity

目的 观察γ射线照射对脐血淋巴细胞增殖和NK细胞活性的影响。方法 取正常分娩的新生儿脐带血，体外肝素抗凝，应用常规密度梯度离心法分离脐血单个核细胞（MNC），计数并调整细胞浓度。Gamma Cell 1000数控细胞照射仪体外照射脐血MNC细胞，吸收剂量率3．72Gy／min。①采用^3H-TdR掺入法测量脐血淋巴细胞增殖：取已分离的单个核细胞，调至细胞浓度1×10^6个／ml，接受0．248、0．496、0．992、1．984、3．968、5．952、7．936和15．872Gy γ射线照射。照射后继续培养56h后加^3H-TdR（3．7×10^7Bq／孔）再培养8h后收集细胞，用Beckman液体闪烁计数器计数放射性核素每分钟闪烁数（计数／min）。②采用^3H-TdR释放法检测脐血NK细胞活性：取传代培养24～48h的K562细胞为靶细胞，调整浓度1×10^6个／ml，加入^3H-TdR（3．7×10^8Bq／m1）孵育6h，洗涤2次去除游离的^3H-TdR，再制备成5×10^5个／ml备用。脐血单个核细胞调整细胞浓度为1×10^3个／ml作为效应细胞（效靶比20：1），给以上述不同剂量γ射线照射。将效应细胞与制备好的靶细胞继续培养18h后收集细胞，用Beckman液体闪烁计数器计数放射性核素每分钟闪烁数。结果 在γ射线对脐血淋巴细胞增殖活性影响的研究中，0．248～15．872Gy γ射线照射显著降低淋巴细胞增殖能力，增殖活性抑制程度与辐射剂量呈正相关（r=0．839，P〈0．05）；3．968Gy γ射线照射组增殖活性仅为对照组的30．86％（P〈0．01），SI=7．8，其中3．968～15．872Gy γ射线照射未发现其明显的增殖活性。而γ射线对脐血NK细胞活性的影响的观察中发现0．248～1．984Gy γ射线照射可引起脐血NK细胞活性显著增高（P〈0．01）；3．968Gy γ射线照射保持NK细胞活性，与对照组相比差异无统计学意义（P〉0．05）；5．952～15．872Gy γ射线可引起脐血淋巴细胞NK细胞活性显著降低（P〈0．01）。结论 3．968Gy γ射线照射完全抑制脐血淋巴细胞增殖的同时仍呈现脐血NK细胞抗肿瘤活性。因此，在进行供者淋巴细胞输注或混合脐血移植时，应用3．968Gy γ射线照射淋巴细胞，再进行过继性细胞免疫治疗可能降低移植物抗宿主病效应，同时不影响移植物抗白血病效应；混合脐血移植时增强移植物抗白血病效应。

Objective To investigate the effects of gamma-ray irradiation on cord blood lymphocyte proliferation and NK cell activity. Methods Freshly isolated mononuclear cells from human cord blood were irradiated with different doses （0.248 ～ 15.872 Gy） of gamma-rays. The lymphocyte proliferation and NK cell activity were measured using ^3H-TdR incorporation assay and ^3H-TdR release assay, respectively. Results In dose range of 0.248 ～ 15.872 Gy, lymphocyte proliferation was inhibited and the inhibition rate was positively correlated with the irradiation dose（ r = 0.839, P 〈 0.05）. Lymphocyte proliferation was not found in dose range of 3.968 ～ 15.872 Gy. Irradiation doses from 0.248 to 1.984 Gy could enhance NK cell activity. The activity of NK cells was reserved after irradiated with 3.968 Gy. Within the dose range of 5.952～ 15.872 Gy, NK cell activity was significantly inhibited. Conclusion Lymphocyte proliferation is inhibited and the activity of NK cell is reserved when irradiated with the dose of 3.968 Gy gamma-rays. So if the lymphocytes are irradiated with such a dose before donor lymphocytes infusion or mix-cord blood transplantation, the effect of graft versus host disease （GVHD） could be decreased whereas the effect of graft versus leukemia （GVL） reaction could be reserved simultaneously during adoptive cellular immunotherapy.