S1PR5在NK细胞介导移植物抗宿主病中的作用及其机制

发布时间：2018-06-23 15:37

本文选题：S1PR5 + NK细胞　；参考：《中国人民解放军医学院》2015年博士论文

【摘要】：研究背景：异基因造血干细胞移植(allogeneic hematopoietic stem cell transplantation, allo-HSCT)技术已经成为治疗血液系统疾病和某些实体肿瘤的重要方法之一。急性移植物抗宿主病(acute graft versus host disease, aGVHD)仍是影响移植成功率的重要因素。NK细胞是allo-HSCT后最早恢复免疫重建的细胞,目前的研究已经证实NK细胞可以抑制aGVHD的发生但不影响移植物抗肿瘤作用。鞘氨醇-1-磷酸受体5(sphingosine-1-phosphate receptor 5, S1PR5)是近年来发现的表达于NK细胞表面的一种G蛋白耦联受体,能够介导NK细胞由骨髓和淋巴结向其他组织的迁移,进而发挥免疫学功能。我们前期的临床研究发现allo-HSCT患者中S1PR5表达水平与aGVHD的发生率密切相关。研究目的：主要通过动物试验探讨S1PR5对aGVHD的影响及相关作用机制。研究方法及结果：①应用CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat and cas9 protein)技术构建S1PR5基因敲除(S1PR5-/-)的小鼠；通过qPCR技术及蛋白免疫印迹杂交技术分别在RNA水平及蛋白水平上成功验证了S1PR5-/-纯合子小鼠中S1PR5被敲除。②流式细胞术分别检测S1PR5-/-和WT C57BL/6小鼠体内骨髓、脾脏、淋巴结及外周血中淋巴细胞亚群分布,结果发现S1PR5-/-小鼠体内,骨髓(P=0.002)、淋巴结(P=0.016)中NK细胞比例较WT小鼠明显增高,但是脾脏(P=0.000)、外周血(P=0.010)中NK细胞比例较WT小鼠明显降低。③分别将供者WT(对照组)或者S1PR5-/-(实验组)C57BL/6小鼠移植物(脾细胞+骨髓细胞)经尾静脉注射入致死剂量照射后的受者BALB/c小鼠体内,建立aGVHD模型，，随访小鼠体重、aGVHD评分及生存时间。移植后观察60天,发现实验组小鼠较对照组小鼠生存率明显降低(P=0.019)；aGVHD评分明显升高(P=0.010)；两组体重减轻变化没有统计学差异(P=0.119)。④移植后第4天分别取受者小鼠的骨髓、脾脏、淋巴结及外周血,应用流式细胞术检测不同部位淋巴细胞亚群分布。移植后第4天,实验组小鼠骨髓中NK细胞含量明显高于对照组(P=0.036)；而在脾脏、淋巴结及外周血中,两组间的NK细胞含量没有明显差异。实验组小鼠骨髓T细胞含量明显低于对照组小鼠中T细胞量(P=0.023)；在脾脏、淋巴结及外周血中,两组间T细胞含量均没有明显差异。⑤移植后第30天分别取出受者小鼠的骨髓、脾脏、淋巴结及外周血,应用流式细胞术检测不同组织中淋巴细胞亚群分布。实验组小鼠骨髓中NK细胞含量明显高于对照组骨髓中NK细胞含量(P=0.037)；在脾脏、淋巴结及外周血中,两组NK细胞含量没有明显差异。实验组小鼠外周血中T细胞含量明显高于对照组外周血中T细胞含量(P=0.031)；在骨髓、脾脏及淋巴结中,两组间T细胞含量没有明显差异。研究结论：① CRISPR/Cas9技术是建立基因敲除小鼠模型有效而快捷的方法之一；② S1PR5影响NK细胞的在小鼠体内的分布；③供者小鼠中S1PR5表达缺失能够加重受者小鼠的aGVHD:④ S1PR5表达缺失致使NK细胞由骨髓迁出受阻，外周循环中对aGVHD起保护作用的NK细胞减少,这可能是受者S1PR5表达缺失导致aGVHD加重的重要机制。
[Abstract]:Background: allogeneic hematopoietic stem cell transplantation (allo-HSCT) has become one of the most important methods for the treatment of hematological diseases and some solid tumors. Acute graft versus host disease (acute graft versus host disease, aGVHD) is still an important factor affecting the success rate of transplantation. .NK cells are the earliest cells to restore the immune reconstruction after allo-HSCT. Current studies have shown that NK cells can inhibit the occurrence of aGVHD but do not affect the anti-tumor effect of the grafts. The -1- phosphoric acid receptor 5 (sphingosine-1-phosphate receptor 5, S1PR5) is a G protein coupled receptor expressed on the surface of NK cells in recent years. It can mediate the migration of NK cells from bone marrow and lymph nodes to other tissues, and then play immunological functions. Our previous clinical studies have found that the expression level of S1PR5 in allo-HSCT patients is closely related to the incidence of aGVHD. Objective: To investigate the effect of S1PR5 on aGVHD and the mechanism of its related action through animal experiments. And results: (1) CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat and cas9 protein) was used to construct S1PR5 gene knockout mice. The distribution of lymphocyte subsets in bone marrow, spleen, lymph node and peripheral blood in S1PR5-/- and WT C57BL/6 mice was detected by flow cytometry. The results showed that the proportion of NK cells in S1PR5-/- mice, bone marrow (P=0.002) and lymph node (P=0.016) was significantly higher than that of WT mice, but the proportion of NK cells in the spleen (P=0.000) and peripheral blood (P=0.010) was larger than that of WT. Mice were significantly reduced. (3) the donor WT (control group) or S1PR5-/- (experimental group) C57BL/6 mice (spleen cells + bone marrow cells) were injected into the BALB/c mice after the tail vein injection into the lethal dose irradiated mice. The aGVHD model was established, and the weight, aGVHD score and survival time of the mice were followed up. After 60 days after transplantation, the mice in the experimental group were found. The survival rate of the control group was significantly lower (P=0.019) and the aGVHD score increased significantly (P=0.010), and there was no significant difference in weight loss between the two groups (P=0.119). (4) the bone marrow, spleen, lymph node and peripheral blood of the recipient mice were taken on the fourth day after transplantation, and the lymphocyte subsets in different parts were detected by flow cytometry. Fourth The content of NK cells in the bone marrow of the mice in the experimental group was significantly higher than that of the control group (P=0.036), but there was no significant difference in the content of NK cells between the two groups in the spleen, lymph nodes and peripheral blood. The content of T cells in the bone marrow of the mice in the experimental group was significantly lower than that of the T cells in the control group (P=0.023); in the spleen, lymph nodes and peripheral blood, the T cells in the spleen, lymph nodes and peripheral blood contained T cells in the experimental group. The bone marrow, spleen, lymph node and peripheral blood of the recipient mice were removed thirtieth days after transplantation. The distribution of lymphocyte subgroups in different tissues was detected by flow cytometry. The content of NK cells in the bone marrow of the experimental group was significantly higher than that in the bone marrow of the control group (P=0.037), and in the spleen, lymph nodes and peripheral blood, the bone marrow of the mice in the experimental group was significantly higher than that of the control group. In the blood, there was no significant difference in the content of NK cells in the two groups. The content of T cells in the peripheral blood of the experimental group was significantly higher than the content of T cells in the peripheral blood of the control group (P=0.031). There was no significant difference in the content of T cells between the two groups in the bone marrow, spleen and lymph nodes. The conclusion: (1) the CRISPR/ Cas9 technique was effective and fast for the establishment of a gene knockout mouse model. One of the methods of Czechoslovakia (S1PR5) affects the distribution of NK cells in mice; (3) the lack of S1PR5 expression in the donor mice can aggravate the deletion of the aGVHD: (S1PR5) expression in the recipient mice, resulting in the removal of the NK cells from the bone marrow, and the decrease in the NK cells that protect the aGVHD in the peripheral circulation, which may result in the S1PR5 expression loss of the recipient leading to aGV. The important mechanism of HD aggravation.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R457.7

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