粒细胞集落刺激因子对急性辐射损伤小鼠中枢及外周血淋巴细胞亚群重建的影响

Effects of granulocyte colony-stimulating factor on central and peripheral lymphocyte subsetreconstitution after sublethal irradiation in mice

目的观察粒细胞集落刺激因子（G—CSF）对急性辐射损伤小鼠中枢及外周血淋巴细胞亚群重建的影响。方法雌性BALB／c小鼠60只经6Gy照射后随机分为照射组、G—CSF＋照射组。G—CSF＋照射组小鼠给与重组人G—CSF100μg·kg^-1·d^-1皮下注射，连续14d，照射组小鼠给与等体积磷酸盐缓冲液（PBS）皮下注射，连续14d，另设空白对照组小鼠20只。照后7、14、21和28d颈部脱臼处死小鼠，取出胸腺制成单个核细胞悬液，使用流式细胞仪检测胸腺CD4＋CD8＋、CD4＋CD8-、CD4-CD8＋、CD4-CD8-细胞亚群的比例。使用全血细胞计数仪进行外周血白细胞计数及淋巴细胞绝对值测定，流式细胞仪检测照后14、28、60d外周血淋巴细胞亚群比例，CCK-8法检测脂多糖（LPS）、刀豆蛋白A（ConA）刺激后脾脏淋巴细胞增殖指数。结果照后7d胸腺CD4＋CD8＋细胞比例降至最低，14d出现反弹，21d再次下降，以后逐渐恢复。照后28dG．CSF＋照射组CD4＋CD8＋细胞比例恢复正常并高于照射组（t=12．22，P〈0．05）。照后21dG—CSF＋照射组CD4-CD8＋细胞比例亦明显高于照射组（t=3．77，P〈0．05）。照后7d外周血白细胞及淋巴细胞绝对值降至最低，照后14和60d，G—CSF＋照射组CD3＋CD8＋T细胞比例明显高于照射组（t=4．31，5．78，P〈0．05），但两组间CD3＋CD4＋T细胞比例在各时间点无明显差异。G—CSF＋照射组B淋巴细胞比例在照后14d明显低于照射组（t=7．3，P〈0．05），但很快恢复，照后28和60d两组B淋巴细胞比例差异无统计学意义。照后14d，G—CSF＋照射组脾脏淋巴细胞对LPS、ConA刺激的增殖指数分别为照射组的4．37和2．98倍。结论G—CSF可促进照后胸腺细胞亚群的恢复，提高外周血淋巴细胞数量，调节外周血淋巴细胞亚群比例，提高淋巴细胞增殖功能，促进急性辐射损伤后中枢及外周免疫重建。

Objective To investigate the effects of recombinant human granulocyte colonystimulating factor（G-CSF） on central and peripheral lymphocyte subset reconstitution after a sublethal dose of irradiation. Methods Sixty female BALB/c mice were given a 6.0 Gy γ-ray total body irradiation （TBI） and randomly divided into 2 equal groups. The mice in G-CSF ＋ TBI group were injected subcutaneously with recombinant human G-CSF 100 μg·kg^-1·d^-1 for 14 d and the mice in TBI group were injected subcutaneously with the same volume of phosphate buffered solution （PBS） once daily for 14 d. 7,14,21, and 28 d later the mice were killed and their thymus were taken out to prepare of the mononuclear cell suspension to analysis the percentage of thymic CD4 ＋ CD8 ＋ double positive, CD4 ＋ CD8 - single positive, CD4 - CD8 ＋ single positive and CD4 - CD8 - double negtive cells by flow cytometry. Peripheral blood samples were collected from the caudal vein twice a week, and the white blood cell（WBC） counts and absolute number of lymphocytcs were assessed by automatic hemocyte analyzer. 14, 28, and 60 d later blood samples were collected from angular vein to examine the peripheral lymphocyte subsets by flow cytometry. Cell counting kit-8 was used to detect lipopolysaccharide （LPS） or concanavalinA （ConA） stimulated splenic lymphocyte proliferation. Results The percentage of thymic CD4 ＋ CD8 ＋ double positive cells decreased 7 d after irradiation, rebounded at 14 d, decreased again at 21 d, and then got a permanent recovery. 28 d after irradiation the percentage of thymic CD4 ＋ CD8 ＋ double positive cells in the G-CSF ＋ TBI group recovered to normal and was significantly higher than that of the TBI group （t = 12.22, P 〈0. 05）. 21d after irradiation the percentage of thymic CD4-CD8 ＋ single positive cells of the G-CSF ＋ TBI group was significantly higher than that of the TBI group （ t = 3.77, P 〈 0. 05）. The peripheral WBCs and lymphocytes decreased to the lowest levels 7 d after irradiation and then gradually increased, however, WBCs and lymphocytes of the G-CSF ＋ TBI group began to recover earlier and faster than the TBI group. The proportion of CD3 ＋ CD8 ＋ T ceils of the G-CSF ＋ TBI group was significantly higher than that of the TBI group 14 and 60 d after irradiation （t =4.31,5.78, P 〈0. 05）. But there was no significant difference in the proportion of CD3 ＋ CD4 ＋ T cells between the two groups. The proportion of B lymphocytes of the G-CSF ＋ TBI group was significantly lower than that of the TBI group 14 d after irradiation（ t = 7.30, P 〈 0.05 ） , but it recovered quickly, and there were no significant differences in the proportion of B lymphocytes between the two groups 28 and 60 d after irradiation. The proliferation indexes of splenic lymphocytes in response to LPS and ConA in the G-CSF ＋ TBI group were 4. 37 and 2.98 times higher than those in the TBI group 14 d after irradiation. Conclusions G-CSF could accelerate the recovery of central and peripheral lymphocyte subsets, raise the absolute number of lymphocytes, and enhance their proliferative function, which contributes to the central and peripheral immune reconstitution after acute irradiation.