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王柔嘉,王紫薇,赵潇溟,唐古生,胡晓霞,王健民*.异基因造血干细胞移植后小鼠共刺激分子表达与急性移植物抗宿主病的关系[J].Academic Journal of Second Military Medical University ,2019,40(6):595-601
异基因造血干细胞移植后小鼠共刺激分子表达与急性移植物抗宿主病的关系    点此下载全文 Fulltext
王柔嘉  王紫薇  赵潇溟  唐古生  胡晓霞  王健民*
海军军医大学(第二军医大学)
长海医院血液内科, 上海 200433
*通信作者
基金项目:国家自然科学基金(81270638,81530047,81871043).
DOI:10.16781/j.0258-879x.2019.06.0595
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摘要:
      目的 探究异基因造血干细胞移植后小鼠共刺激分子表达及其与急性移植物抗宿主病(aGVHD)发病的关系。方法 供体为5只C57BL/6J(H2KD-H2KB+)雄性小鼠,受体为30只CB6F1(H2KD+H2KB+)雌性小鼠。将受体小鼠随机分为单纯照射组、骨髓移植组和aGVHD组,每组10只。单纯照射组:受体小鼠接受γ射线照射后不注射任何细胞;骨髓移植组:受体小鼠接受γ射线照射后注射5×106个供体来源去T淋巴细胞的骨髓有核细胞;aGVHD组:受体小鼠接受γ射线照射后,同时注射5×106个供体来源去T淋巴细胞的骨髓有核细胞和3×107个供体来源脾细胞。采用log-rank生存曲线分析各组小鼠移植后生存情况。分别在移植后7、14、21、28 d采用流式细胞术检测骨髓移植组和aGVHD组小鼠外周血CD4+T淋巴细胞和CD8+T淋巴细胞表面共刺激分子细胞毒性T淋巴细胞相关抗原4(CTLA-4)、程序性死亡蛋白1(PD-1)、可诱导共刺激分子(ICOS)和CD28的表达情况,以及移植后21 d两组小鼠脾组织CD4+T淋巴细胞内白细胞介素(IL)-17、γ干扰素、IL-4的表达情况,并在移植后21 d采用3,3'-二氨基联苯胺(DAB)染色检测两组小鼠结肠组织共刺激分子配体的表达情况。结果 单纯照射组受体小鼠均在γ射线照射后19 d内死亡;骨髓移植组小鼠在γ射线照射后均未出现aGVHD症状,30 d内均存活;aGVHD组小鼠在γ射线照射后出现aGVHD症状的时间为14(11~18)d,生存时间为22(13~30)d。随着时间的推移,aGVHD组小鼠外周血CD4+和CD8+T淋巴细胞表面CTLA-4、PD-1表达在移植后均呈下降趋势,ICOS、CD28表达均呈上升趋势。在移植后28 d时aGVHD组小鼠外周血CD4+和CD8+T淋巴细胞表面CTLA-4、PD-1表达均低于骨髓移植组,ICOS、CD28表达均高于骨髓移植组,差异均有统计学意义(P均<0.05)。DAB染色结果示,骨髓移植组结肠组织CD80、ICOS配体(ICOSL)、PD-1配体(PD-L1)表达均为阴性,aGVHD组结肠组织CD80、ICOSL、PD-L1阳性表达率分别为40%、80%、80%。与骨髓移植组比较,aGVHD组小鼠脾组织CD4+T淋巴细胞内IL-17、γ干扰素表达均增加,IL-4表达减少,差异均有统计学意义(P均<0.05)。结论 异基因造血干细胞移植小鼠发生aGVHD后,共刺激分子CTLA-4表达随时间推移逐渐降低。CTLA-4、ICOS、PD-1均可能通过调节辅助性T细胞(Th)1、Th2、Th17等T淋巴细胞亚群分布参与aGVHD的发生、发展。
关键词:移植物抗宿主病  造血干细胞移植  共刺激分子  细胞毒性T淋巴细胞相关抗原4  细胞因子
Expression of costimulators in mice after allogenetic hematopoietic stem cell transplantation and its relationship with acute graft versus host disease    Fulltext
WANG Rou-jia  WANG Zi-wei  ZHAO Xiao-ming  TANG Gu-sheng  HU Xiao-xia  WANG Jian-min*
Department of Hematology, Changhai Hospital, Naval Medical University(Second Military Medical University)
, Shanghai 200433, China
*Corresponding author
Fund Project:Supported by National Natural Science Foundation of China (81270638, 81530047, 81871043).
Abstract:
      Objective To explore the expression of costimulators in mice after allogenetic hematopoietic stem cell transplantation and its relationship with acute graft versus host disease (aGVHD). Methods Five C57BL/6J (H2KD-H2KB+) male mice were selected as donors. Thirty CB6F1 (H2KD+H2KB+) female mice were selected as recipients and randomized into three groups:total body irradiation (TBI), bone marrow transplantation (BM) and aGVHD groups, with 10 mice in each group. The mice in the TBI group received radiation only without injecting any cells. The mice in the BM group were injected with 5×106 bone marrow cells without T lymphocytes from donors after radiation. The mice in the aGVHD group were simultaneously injected with 5×106 bone marrow cells without T lymphocytes and 3×107 spleen cells from donors after radiation. The survival rates of mice in the three groups were evaluated using log-rank survival curve. The expression levels of costimulators, including cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), programmed death 1 (PD-1), inducible costimulator (ICOS) and CD28, on CD4+ and CD8+ T lymphocytes, were detected using flow cytometry at 7, 14, 21 and 28 d after transplantation. At 21 d after transplantation, the expression levels of interleukin (IL)-17, interferon γ (IFN-γ) and IL-4 in CD4+ T lymphocytes, and the costimulator ligands in the colon tissues, were detected with flow cytometry and 3,3'-diaminobenzidine (DAB) staining, respectively. Results The mice in the TBI group all died within 19 d after irradiation. The mice in the BM group survived 30 d after irradiation without aGVHD. The median onset time of aGVHD symptoms and survival of mice in the aGVHD group were 14 (11-18) d and 22 (13-30) d, respectively. The expression levels of CTLA-4 and PD-1 on CD4+ and CD8+ T lymphocytes were decreased after transplantation, while the expression levels of ICOS and CD28 were on the rise. At 28 d after transplantation, the expression levels of CTLA-4 and PD-1 on CD4+ and CD8+ T lymphocytes were significantly lower in the aGVHD group than those in the BM group (all P<0.05), and the expression levels of ICOS and CD28 were significantly higher (all P<0.05). DAB staining showed that CD80, ICOS ligand (ICOSL), and PD-1 ligand (PD-L1) were negative in the colon tissues of mice in the BM group, and the positive expression rates of CD80, ICOSL, and PD-L1 in the aGVHD group were 40%, 80% and 80%, respectively. Compared with the BM group, the expression levels of IL-17 and IFN-γ in CD4+ T lymphocytes in the aGVHD group were significantly increased, while the expression level of IL-4 was significantly decreased (all P<0.05). Conclusion In aGVHD mice after allogeneic hematopoietic stem cell transplantation, the expression of costimulator CTLA-4 is gradually decreased over time. CTLA-4, ICOS and PD-1 may participate in the development and progression of aGVHD by regulating the distribution of T lymphocyte subgroups such as T-helper cell (Th)1, Th2 and Th17.
Keywords:graft versus host disease  hematopoietic stem cell transplantation  costimulators  cytotoxic T lymphocyte-associated antigen 4  cytokines
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