G-CSF对骨髓和外周血中MDSCs影响的初步研究
发布时间：2019-06-11 04:17
【摘要】：一研究背景及目的 粒细胞集落刺激因子(G-CSF)动员的外周血干细胞采集物作为异基因造血干细胞移植(allo-HSCT)的移植物现已广泛应用于临床,此举措不仅可以满足临床对于采集造血干/祖细胞的数量要求,还产生了一种现象：采用G-CSF动员后的外周血干细胞采集物移植其造血与免疫重建快于骨髓采集物移植,尽管外周血干细胞采集物中T细胞高于骨髓采集物数倍,但是其移植物抗宿主病(GVHD)、尤其是急性移植物抗宿主病(aGVHD)发生率并未增高,当中的机制至今仍未得到完全阐释。有研究者对移植采集物中CD4+、CD8+初始及记忆T细胞亚群、细胞因子的表达、CD34+细胞含量、HLA-G表达等方面进行探讨,初步揭示了G-CSF动员后外周血干细胞移植中低GVHD发生率与改变移植物中的细胞亚群、细胞因子表达等有关。 髓源性抑制细胞(MDSCs)来源于骨髓祖细胞和未成熟髓细胞,小鼠中它们都表达Gr-1和CD11b分子,人类MDSCs的谱系标志是CD33阳性,CD3、CD14、CD19、CD56及HLA-DR阴性或者CD33+/CD11b+/CD14-细胞。在健康成年鼠中,仅在骨髓中有大量的Gr-1+/CD11b+的细胞,而外周血和脾中少于4%。在健康成年人中则未见有相关报道。正常情况下,MDSCs可以分化为树突状细胞、巨噬细胞和(或)粒细胞。但在某些病理情况下,如自身免疫疾病、肿瘤、炎症及外伤时,均可检测到MDSCs体内的扩增。尤其在荷瘤小鼠的脾脏、血液及肿瘤组织和肿瘤患者的外周血及肿瘤组织中MDSCs数量和比例均有大幅度的增加,贯穿肿瘤发生的整个过程,且与肿瘤的大小和恶性程度有一定的相关性。大量的动物模型研究报道发现,在肿瘤发生发展过程中,MDSCs起到免疫抑制作用。最近有研究者发现在小鼠模型中G-CSF能从骨髓中动员更多的MDSCs到达外周,但在人体中G-CSF对MDSCs的影响及MDSCs与aGVHD的关系却未见相关报道,本文现报道在人体中G-CSF对MDSCs的影响及MDSCs与GVHD的关系。 二方法 对20例健康造血干细胞供者,在G-CSF动员前和动员后第5天分别获取骨髓(BM)、外周血(PB)及外周血干细胞采集物,使用流式细胞术检测其中MDSCs和淋巴细胞亚群含量,及MDSCs表面粘附分子CD11a、CD44、CD49d、CD49e变化；酶联免疫吸附试验(ELISA)检测动员前后BM及PB中精氨酸酶(ARG)及诱导型一氧化氮合成酶(iNOS)表达量。以30例接受allo-HSCT患者输注MDSCs细胞数中位数为界值进行分组,比较高MDSCs与低MDSCs组移植后造血重建、免疫重建、GVHD发生率、缓解与复发、长生存等指标,分析MDSCs移植细胞数与aGVHD发生的相关关系。数据统计分析：采用SPSS 13.0统计软件包进行处理,以均数±标准差(x±s)表示,动员前后比较、组间比较采用两独立t检验或者配对样本t检验；等级资料比较采用卡方检验；采用等级相关系数(Spearman相关系数)非参数方法分析非双变量正态分布资料。P≤0.05为差异有统计学意义。 三结果 1、G-CSF对健康供者外周血细胞的影响：经G-CSF动员后第5天,健康供者外周血中白细胞(WBC)数量明显提高(P0.001),血红蛋白(HGB)及血小板(PLT)均无明显变化(P=0.205、0.939)。 2、G-CSF动员对PB和BM中MDSCs含量的影响：在正常生理状态下,健康供者PB、BM中均能检测到MDSCs, MDSCs在PB中占有核细胞比例为0.35±0.07%,BM中为0.65±0.13%,BM中MDSCs比PB中高(P0.001)；G-CSF动员5天后MDSCs在PB中占有核细胞比例为0.79±0.16%,BM中为0.72±0.13%,动员后BM与PB中MDSCs比例相比无明显变化(P=0.114)。动员后外周血中MDSCs含量明显高于动员前(P0.001),而动员前后BM中MDSCs细胞比例变化无统计学意义(P=0.136)。 3、G-CSF动员对PB和BM中淋巴细胞亚群的影响：动员后PB中CD4+CD25+ Treg细胞较动员前明显上升(P=0.048),其余细胞亚群均未见有显著差别(P均0.05), G-CSF动员前后MDSCs表达量与CD4+CD25+Treg细胞表达量无显著相关关系(Pearson相关系数rs=0.627,P=0.096)。 4、MDSCs表面粘附因子：动员前PB中MDSCs表面粘附因子CD11a、CD44、CD49d和CD49e表达量分别为0.72±0.23%、0.63±0.17%、0.34±0.11%和0.59±0.09%,BM中MDSCs表面粘附因子表达量分别为1.06±0.16%、1.08±0.13%、0.87±0.10%和1.18±0.19%；动员后PB中MDSCs表面粘附因子CD11a、CD44、CD49d和CD49e表达量分别为0.44±0.21%、0.34±0.10%、0.44±0.11%和0.41±0.12%,BM中MDSCs表面粘附因子表达量分别为0.75±0.13%、0.52±0.14%、0.50±0.12%和0.69±0.15%。动员前PB和BM中MDSCs表面粘附因子表达量明显高于动员后(P值均小于0.05)。 5、Arg和iNOS表达量：动员前PB和BM中Arg表达量分别为0.66±0.15mIU/ml和2.20±0.40mIU/ml,动员后分别为1.89±0.19mIU/ml和2.16±0.40mIU/ml;动员前PB和BM中iNOS表达量分别为0.92±0.13mIU/ml和2.68±0.36mIU/ml,动员后分别为2.10±0.13mIU/ml和2.79±0.23mIU/ml。动员后PB中Arg和iNOS表达量均较动员前明显提高,具统计学意义(P值均小于0.01)；动员前后BM中Arg和iNOS表达量均无明显变化(P值均大于0.05)。 6、GVHD发生情况：中位随访时间为227天(48—363天),30例接受allo-HSCT患者,18例患者存活、12例死亡。高MDSCs组aGVHD发生率20.00%(3/15)、Ⅲ-Ⅳ°aGVHD 0.00%(0/15)、cGVHD 30.77%(4/13);低MDSCs组aGVHD发生率66.67%(10/15)、Ⅲ-Ⅳ°aGVHD 20.00%(3/15)、cGVHD 45.45%(5/11)。高MDSCs组aGVHD发生率显著低于低MDSCs组(P=0.011),而两组间Ⅲ-Ⅳ°aGVHD和cGVHD发生率则无显著差异(P=0.073、P=0.469)。 7、MDSCs移植数与aGVHD发生关系：使用相关分析(Bivariate Correlations)分析移植MDSCs数量与aGVHD发生关系,结果显示：移植后aGVHD的发生程度随着移植时回输供者MDSCs细胞数量的增多而降低,两者之间呈显著负相关(Spearman相关系数rs=-0.445,P=0.014)。提示MDSCs细胞数量与aGVHD发生率有显著负相关关系。 8、移植后复发与长生存：高MDSCs组复发率为26.67%(4/15),低MDSCs组复发率为20.00%(3/15),复发率比较无显著差别(P=0.671)。高MDSCs组与低MDSCs组无病生存率分别是46.7±14.2%和38.9±15.8%,总生存率分别是70.9±12.4%和50.6±15.0%,比较两组无病生存率、总生存率均无显著性差异(P值分别为0.833、0.442)。 四结论 在人体G-CSF能动员BM中MDSCs到达外周血,使PB中MDSCs增高,PB中ARG与iNOS表达量增高。G-CSF动员MDSCs到达外周的机制与MDSCs表面粘附因子表达量变化有关。G-CSF动员的外周血干细胞采集物中MDSCs增加与移植后低aGVHD发生率有关,两者有明显的负相关关系。MDSCs细胞输注不会影响GVL和免疫重建功能。
[Abstract]:Background and purpose of the study The peripheral blood stem cell collected from the granulocyte colony-stimulating factor (G-CSF) is widely used as a graft for allogeneic hematopoietic stem cell transplantation (allo-HSCT). Bed, this measure not only can meet the clinical value of the collection of hematopoietic stem/ progenitor cells, but also a phenomenon: the peripheral blood stem cell collection after the mobilization of G-CSF is used to transplant the hematopoietic and immune reconstitution faster than the bone marrow collection In spite of the fact that the T cells in the peripheral blood stem cell collection are several times higher than the bone marrow collection, the incidence of graft versus host disease (GVHD), especially the acute graft versus host disease (aGVHD), has not increased, and the mechanism has not yet been fully developed. The changes of CD4 +, CD8 + initial and memory T-cell subpopulations, the expression of cytokines, the content of CD34 + cells, and the expression of HLA-G were discussed. Group, cytokine expression, etc. The myeloid-derived suppressor cells (MDSCs) are derived from bone marrow progenitor cells and immature myeloid cells, both of which express Gr-1 and CD11b molecules, and the lineage marker for human MDSCs is CD33-positive, CD3, CD14, CD19, CD56, and HLA-DR-negative or CD33 +/ CD11b +/ CD14 -Cells. In healthy adult rats, only a large number of cells of Gr-1 +/ CD11b + are present in the bone marrow, while less in the peripheral blood and in the spleen at 4%. No phase was found in healthy adults Off-site reports. In normal cases, MDSCs can be differentiated into dendritic cells, macrophages, and/ or granulocytes. However, in certain pathological conditions, such as autoimmune diseases, tumors, inflammation, and trauma, MDSCs can be detected The number and proportion of MDSCs in the peripheral blood and tumor tissues of the spleen, the blood and the tumor tissues and the tumor tissues of the tumor-bearing mice are greatly increased, Correlations. A large number of animal model studies have found that MDSCs play an immunosuppression role in the development of tumorigenesis. The effect of G-CSF on MDSCs and the relationship between MDSCs and aGVHD in the human body are not reported in this paper. The effect of G-CSF on MDSCs and the relationship between MDSCs and GVHD are reported in this paper. the relationship Methods: In 20 healthy hematopoietic stem cell donors, bone marrow (BM), peripheral blood (PB) and peripheral blood stem cell collection were obtained before and after the mobilization of G-CSF, and the content of MDSCs and lymphocyte subpopulations and the surface adhesion of MDSCs were detected by flow cytometry. Molecular CD11a, CD44, CD49d, and CD49e changes; Enzyme-linked immunosorbent assay (ELISA) for the detection of arginase (ARG) and inducible nitric oxide synthase (i) in BM and PB before and after mobilization The expression of NOS was measured in 30 patients with allo-HSCT. The median of the number of MDSCs in the patients with allo-HSCT was divided into the boundary value, and the number of MDSCs and the aGVHD were analyzed by comparing the indexes of hematopoietic reconstruction, immune reconstruction, GVHD incidence, response and recurrence, and long-survival after transplantation in the patients with high MDSCs and low MDSCs. Correlation between data and data: SPSS 13.0 statistical software package was used for processing, and the mean square standard deviation (x% s) was used to represent, and before and after mobilization, two independent t-test or paired sample t-test were used for comparison between groups; and the grade data ratio The chi-square test was used, and the non-parametric method was used to analyze the non-parametric method using the Spearman correlation coefficient. Normal distribution data. P-0.05 is poor heterophyton The results showed that the number of white blood cells (WBC) in the peripheral blood of healthy donors increased significantly after the mobilization of G-CSF (P = 0.001), and the hemoglobin (HGB) and the platelet (PLT) had no significant change (P = 0). (.205, 0.939).2, The effect of G-CSF mobilization on the content of MDSCs in PB and BM: In normal physiological state, MDSCs were detected in PB and BM of healthy donors, and the proportion of MDSCs in PB was 0.35-0.07%, in BM, 0.65-0.13%, and MDSCs in BM. The proportion of MDSCs in PB was 0.79-0.16%, 0.72-0.13% in BM and 0.72-0.13% in BM after 5-day mobilization of G-CSF. The content of MDSCs in peripheral blood after mobilization was significantly higher than that before mobilization (P <0.001), while the proportion of MDSCs in the BM before and after mobilization was significantly higher than that before mobilization (P = 0.114). The study significance (P = 0.136).3. The effect of G-CSF mobilization on the lymphocyte subpopulation in PB and BM: the CD4 + CD25 + Treg cells in the post-mobilization PB were significantly increased before mobilization (P = 0.048) and the remaining cell subpopulations There was no significant difference between the expression of MDSCs and the expression of CD4 + CD25 + Treg cells before and after the mobilization of G-CSF (Pearson correlation coefficient rs). The surface adhesion factor of MDSCs was 0.72-0.23%, 0.63-0.17%, 0.34-0.11% and 0.59-0.09%, respectively. The expression of CD11a, CD44, CD49d and CD49e was 0.44-0.21%, 0.34-0.10%, 0.44-0.11% and 0.41-0.12%, respectively, and the expression of MDSCs in BM was 0.75-0.13%, 0.52-0.14%, 0.50%, respectively, and the expression of CD11a, CD44, CD49d and CD49e was 0.44-0.21%, 0.34-0.10%, 0.44-0.11% and 0.41-0.12%, respectively. 0.12% and 0.69-0.15% of the surface adhesion factor of MDSCs in the pre-mobilization PB and BM. The expression of Arg and iNOS was 0.66, 0.15mIU/ ml and 2.20-0.40 mIU/ ml, respectively. The expression of iNOS in PB and BM were 0.92-0.13mIU/ ml and 2.68-0.36mIU/ ml, respectively. The expression of Arg and iNOS in the post-mobilization of PB was significantly higher than that in the pre-mobilization group (P <0.01), and the contents of Arg and iNOS in the BM were mobilized before and after mobilization. There was no significant change in the amount of dabigatran (P <0.05).6. The occurrence of GVHD: the median follow-up time was 227 days (48 to 363 days) and 30 received allo. -HSCT,18 patients survived,12 died. The incidence of aGVHD in the high MDSCs group was 20.00% (3/15), the 鈪，
本文编号：2497007
[Abstract]:Background and purpose of the study The peripheral blood stem cell collected from the granulocyte colony-stimulating factor (G-CSF) is widely used as a graft for allogeneic hematopoietic stem cell transplantation (allo-HSCT). Bed, this measure not only can meet the clinical value of the collection of hematopoietic stem/ progenitor cells, but also a phenomenon: the peripheral blood stem cell collection after the mobilization of G-CSF is used to transplant the hematopoietic and immune reconstitution faster than the bone marrow collection In spite of the fact that the T cells in the peripheral blood stem cell collection are several times higher than the bone marrow collection, the incidence of graft versus host disease (GVHD), especially the acute graft versus host disease (aGVHD), has not increased, and the mechanism has not yet been fully developed. The changes of CD4 +, CD8 + initial and memory T-cell subpopulations, the expression of cytokines, the content of CD34 + cells, and the expression of HLA-G were discussed. Group, cytokine expression, etc. The myeloid-derived suppressor cells (MDSCs) are derived from bone marrow progenitor cells and immature myeloid cells, both of which express Gr-1 and CD11b molecules, and the lineage marker for human MDSCs is CD33-positive, CD3, CD14, CD19, CD56, and HLA-DR-negative or CD33 +/ CD11b +/ CD14 -Cells. In healthy adult rats, only a large number of cells of Gr-1 +/ CD11b + are present in the bone marrow, while less in the peripheral blood and in the spleen at 4%. No phase was found in healthy adults Off-site reports. In normal cases, MDSCs can be differentiated into dendritic cells, macrophages, and/ or granulocytes. However, in certain pathological conditions, such as autoimmune diseases, tumors, inflammation, and trauma, MDSCs can be detected The number and proportion of MDSCs in the peripheral blood and tumor tissues of the spleen, the blood and the tumor tissues and the tumor tissues of the tumor-bearing mice are greatly increased, Correlations. A large number of animal model studies have found that MDSCs play an immunosuppression role in the development of tumorigenesis. The effect of G-CSF on MDSCs and the relationship between MDSCs and aGVHD in the human body are not reported in this paper. The effect of G-CSF on MDSCs and the relationship between MDSCs and GVHD are reported in this paper. the relationship Methods: In 20 healthy hematopoietic stem cell donors, bone marrow (BM), peripheral blood (PB) and peripheral blood stem cell collection were obtained before and after the mobilization of G-CSF, and the content of MDSCs and lymphocyte subpopulations and the surface adhesion of MDSCs were detected by flow cytometry. Molecular CD11a, CD44, CD49d, and CD49e changes; Enzyme-linked immunosorbent assay (ELISA) for the detection of arginase (ARG) and inducible nitric oxide synthase (i) in BM and PB before and after mobilization The expression of NOS was measured in 30 patients with allo-HSCT. The median of the number of MDSCs in the patients with allo-HSCT was divided into the boundary value, and the number of MDSCs and the aGVHD were analyzed by comparing the indexes of hematopoietic reconstruction, immune reconstruction, GVHD incidence, response and recurrence, and long-survival after transplantation in the patients with high MDSCs and low MDSCs. Correlation between data and data: SPSS 13.0 statistical software package was used for processing, and the mean square standard deviation (x% s) was used to represent, and before and after mobilization, two independent t-test or paired sample t-test were used for comparison between groups; and the grade data ratio The chi-square test was used, and the non-parametric method was used to analyze the non-parametric method using the Spearman correlation coefficient. Normal distribution data. P-0.05 is poor heterophyton The results showed that the number of white blood cells (WBC) in the peripheral blood of healthy donors increased significantly after the mobilization of G-CSF (P = 0.001), and the hemoglobin (HGB) and the platelet (PLT) had no significant change (P = 0). (.205, 0.939).2, The effect of G-CSF mobilization on the content of MDSCs in PB and BM: In normal physiological state, MDSCs were detected in PB and BM of healthy donors, and the proportion of MDSCs in PB was 0.35-0.07%, in BM, 0.65-0.13%, and MDSCs in BM. The proportion of MDSCs in PB was 0.79-0.16%, 0.72-0.13% in BM and 0.72-0.13% in BM after 5-day mobilization of G-CSF. The content of MDSCs in peripheral blood after mobilization was significantly higher than that before mobilization (P <0.001), while the proportion of MDSCs in the BM before and after mobilization was significantly higher than that before mobilization (P = 0.114). The study significance (P = 0.136).3. The effect of G-CSF mobilization on the lymphocyte subpopulation in PB and BM: the CD4 + CD25 + Treg cells in the post-mobilization PB were significantly increased before mobilization (P = 0.048) and the remaining cell subpopulations There was no significant difference between the expression of MDSCs and the expression of CD4 + CD25 + Treg cells before and after the mobilization of G-CSF (Pearson correlation coefficient rs). The surface adhesion factor of MDSCs was 0.72-0.23%, 0.63-0.17%, 0.34-0.11% and 0.59-0.09%, respectively. The expression of CD11a, CD44, CD49d and CD49e was 0.44-0.21%, 0.34-0.10%, 0.44-0.11% and 0.41-0.12%, respectively, and the expression of MDSCs in BM was 0.75-0.13%, 0.52-0.14%, 0.50%, respectively, and the expression of CD11a, CD44, CD49d and CD49e was 0.44-0.21%, 0.34-0.10%, 0.44-0.11% and 0.41-0.12%, respectively. 0.12% and 0.69-0.15% of the surface adhesion factor of MDSCs in the pre-mobilization PB and BM. The expression of Arg and iNOS was 0.66, 0.15mIU/ ml and 2.20-0.40 mIU/ ml, respectively. The expression of iNOS in PB and BM were 0.92-0.13mIU/ ml and 2.68-0.36mIU/ ml, respectively. The expression of Arg and iNOS in the post-mobilization of PB was significantly higher than that in the pre-mobilization group (P <0.01), and the contents of Arg and iNOS in the BM were mobilized before and after mobilization. There was no significant change in the amount of dabigatran (P <0.05).6. The occurrence of GVHD: the median follow-up time was 227 days (48 to 363 days) and 30 received allo. -HSCT,18 patients survived,12 died. The incidence of aGVHD in the high MDSCs group was 20.00% (3/15), the 鈪，
本文编号：2497007
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