白细胞介素11联合粒细胞集落刺激因子对小鼠异基因骨髓移植后移植物抗宿主病影响的研究

目的探讨重组白细胞介素11(rhIL-11)联合重组粒细胞集落刺激因子(rhG-CSF)用于供者对小鼠异基因骨髓移植后移植物抗宿主病(GVHD)的作用及其机制。方法建立同种异基因骨髓移植急性GVHD模型,以C57BL/6H-2b为供鼠,rhIL-11、rhG-CSF或二者联合应用作为动员剂,BALB/CH-2d受鼠经致死剂量(8·0Gy)照射后随机分为5组,A组为单纯照射组(10只),B组为GVHD对照组(18只),C组为G-CSF移植组(18只),D组为IL-11移植组(18只),E组为联合移植组(18只),在照射后4h移植供鼠骨髓细胞和脾细胞。观察移植后小鼠的生存期和病死率,应用流式细胞仪测定嵌合体形成情况,用四甲基偶氮唑盐(MTT)法检测混合淋巴细胞反应,酶联免疫吸附实验(ELISA)检测白细胞介素4(IL-4)、γ干扰素(IFN-γ)等细胞因子的分泌情况,并进行组间比较。结果E组小鼠生存时间明显长于其他各组(B、C、D),差异有统计学意义(P<0·01),30d存活率B、C、D、E组分别是:0、27·3%(3/11)、36·4%(4/11)、63·6%(7/11)。B组小鼠均出现GVHD病理改变,其程度与发生GVHD的时间密切相关,GVHD发生越早,病理改变越重;E组有3只小鼠在移植后20d内发生GVHD,病理程度改变较其他各组轻。与B、C、D组相比,E组移植后14d脾细胞培养上清中IFN-γ水平较低(92pg/ml±14pg/mlvs172pg/ml±31pg/ml、132pg/ml±23pg/ml、117pg/ml±28pg/ml,均P<0·01),IL-4的水平较高(36pg/ml±7pg/mlvs16pg/ml±4pg/ml、26pg/ml±4pg/ml、24pg/ml±4pg/ml,均P<0·05),D、E组肿瘤坏死因子α(TNF-α)水平相近,但明显低于B、C组(均P<0·01),并且E组对宿主抗原的淋巴细胞增殖均低于B、C、D组(0·22±0·08vs0·87±0·14、0·54±0·21、0·49±0·18,均P<0·01)。结论rhIL-11联合rhG-CSF用于供者能产生协同作用,减轻GVHD,其机制可能与受者体内T细胞向Th2细胞因子极化、炎症介质如TNF-α降低有关。

Significance of donors treatment with combination of recombinant human interleukin-11 and recombinant human granulocyte-colony stimulating factor in reduction of incidence of lethal grafts versus host disease after allogeneic bone marrow transplantation

OBJECTIVE: To investigate the effects of donor treatment with combination of recombinant human interleukin-11 (rhIL-11) and recombinant human granulocyte-colony stimulating factor (rhG-CSF) on incidence of lethal graft versus host disease (GVHD) after allogeneic bone marrow transplantation (BMT). METHODS: Forty C57BL/6 mice, as donors, were treated with phosphate buffered saline (PBS), or optimal doses of rhG-CSF, rhIL-11, or rhG-CSF + rhIL-11 for five consecutive days. Fifty BALB/C mice, as recipients, received a lethal dose of 8.0 Gy total body irradiation (TBI) 4-6 h before the transplantation. Ten C57BL/6 mice without transplantation were used as pure radiation control group (group A). The other 40 C57BL/6 mice received the allogeneic bone marrow from the donors treated with PBS (group B), rhG-CSF (group C), rhIL-11 (group D), or rhG-CSF + rgIL-11 (group E) 14 and 30 days after the transplantation, suspensions of bone marrow cells were prepared from the 4 groups. Monoclone antibody of PE-H-2D(b) was added. The percentage of the H-2D(b) positive cells was observed by flow cytometry to detect the chimerism to determine the proportion of the cells of donor origin. The liver, spleen, intestine, and skin tissues of the mice presenting symptoms of GVHD were taken out before the diseased mice died. The long-surviving mice were killed 60 days after the transplantation and the above-mentioned tissues were taken out. Light microscopy was used to observe the pathological changes. 14 days after transplantation, the spleen cells of the different groups were obtained to undergo mixed leucocyte reaction (MLR), and 14 days after transplantation another spleen cells of groups B, C, D, and E were obtained to prepare suspension of mononuclear cells to test the levels of IL-4, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha by ELISA. RESULT: The survival time of group E was 38 d +/- 19 d, significantly longer than the other groups (all P < 0.01). The survival times of groups C and D were 23 d +/- 8 d and 25 d +/- 15 d respectively, both significantly longer than that of group B (14 d +/- 4 d, both P < 0.01). The mice of group A all died within 8 days with a survival time of 6.2 d +/- 1.3 d. The GVHD pathological changes were milder in group E than in the other groups. 14 and 30 days after transplantation, the percentage of H-2D(b) antibody positive cells was over 95% in all surviving transplanted mice. All the mice of group B developed GVHD, and the earlier GVHD appeared the severer the pathological changes. In group E 3 mice developed GVHD more than 20 days after transplantation, 7 mice survived more than 30 days, and 4 survived 60 days without any sign of GVHD. The pathological changes in the tissues of the mice that died within 20 days were more remarkable than those of the mice that died 20 days or more after transplantation. The levels of IFN-gamma of group C, D, and E were significantly lower than that of group B, and the levels of IL-4 of group C, D, and E were significantly higher than that of group B (all P < 0.01). The level of IFN-gamma of group E was significantly lower than those of groups C and D, and the level of IL-4 of group E was significantly higher than those of groups C and D (all P < 0.05). The levels of group D and E were significantly lower than those of group B and C (all P < 0.01). The MLR 14 days after transplantation showed that the proliferation to host alloantigen of group E was 0.22 +/- 0.08, significantly lower than those of groups B, C, and D (0.87 +/- 0.14, 0.54 +/- 0.21, and 0.49 +/- 0.18 respectively, all P < 0.01). CONCLUSION: Donor treatment with the combination of rhIL-11 and rhG-CSF shows a synergic function in inducing immune tolerance, and significantly reduces the incidence of lethal GVHD after BMT.