發(fā)布時(shí)間：2019-06-11 04:17

【摘要】：一研究背景及目的 粒細(xì)胞集落刺激因子(G-CSF)動(dòng)員的外周血干細(xì)胞采集物作為異基因造血干細(xì)胞移植(allo-HSCT)的移植物現(xiàn)已廣泛應(yīng)用于臨床,此舉措不僅可以滿足臨床對(duì)于采集造血干/祖細(xì)胞的數(shù)量要求,還產(chǎn)生了一種現(xiàn)象：采用G-CSF動(dòng)員后的外周血干細(xì)胞采集物移植其造血與免疫重建快于骨髓采集物移植,盡管外周血干細(xì)胞采集物中T細(xì)胞高于骨髓采集物數(shù)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)發(fā)生率并未增高,當(dāng)中的機(jī)制至今仍未得到完全闡釋。有研究者對(duì)移植采集物中CD4+、CD8+初始及記憶T細(xì)胞亞群、細(xì)胞因子的表達(dá)、CD34+細(xì)胞含量、HLA-G表達(dá)等方面進(jìn)行探討,初步揭示了G-CSF動(dòng)員后外周血干細(xì)胞移植中低GVHD發(fā)生率與改變移植物中的細(xì)胞亞群、細(xì)胞因子表達(dá)等有關(guān)。 髓源性抑制細(xì)胞(MDSCs)來(lái)源于骨髓祖細(xì)胞和未成熟髓細(xì)胞,小鼠中它們都表達(dá)Gr-1和CD11b分子,人類MDSCs的譜系標(biāo)志是CD33陽(yáng)性,CD3、CD14、CD19、CD56及HLA-DR陰性或者CD33+/CD11b+/CD14-細(xì)胞。在健康成年鼠中,僅在骨髓中有大量的Gr-1+/CD11b+的細(xì)胞,而外周血和脾中少于4%。在健康成年人中則未見(jiàn)有相關(guān)報(bào)道。正常情況下,MDSCs可以分化為樹突狀細(xì)胞、巨噬細(xì)胞和(或)粒細(xì)胞。但在某些病理情況下,如自身免疫疾病、腫瘤、炎癥及外傷時(shí),均可檢測(cè)到MDSCs體內(nèi)的擴(kuò)增。尤其在荷瘤小鼠的脾臟、血液及腫瘤組織和腫瘤患者的外周血及腫瘤組織中MDSCs數(shù)量和比例均有大幅度的增加,貫穿腫瘤發(fā)生的整個(gè)過(guò)程,且與腫瘤的大小和惡性程度有一定的相關(guān)性。大量的動(dòng)物模型研究報(bào)道發(fā)現(xiàn),在腫瘤發(fā)生發(fā)展過(guò)程中,MDSCs起到免疫抑制作用。最近有研究者發(fā)現(xiàn)在小鼠模型中G-CSF能從骨髓中動(dòng)員更多的MDSCs到達(dá)外周,但在人體中G-CSF對(duì)MDSCs的影響及MDSCs與aGVHD的關(guān)系卻未見(jiàn)相關(guān)報(bào)道,本文現(xiàn)報(bào)道在人體中G-CSF對(duì)MDSCs的影響及MDSCs與GVHD的關(guān)系。 二方法 對(duì)20例健康造血干細(xì)胞供者,在G-CSF動(dòng)員前和動(dòng)員后第5天分別獲取骨髓(BM)、外周血(PB)及外周血干細(xì)胞采集物,使用流式細(xì)胞術(shù)檢測(cè)其中MDSCs和淋巴細(xì)胞亞群含量,及MDSCs表面粘附分子CD11a、CD44、CD49d、CD49e變化；酶聯(lián)免疫吸附試驗(yàn)(ELISA)檢測(cè)動(dòng)員前后BM及PB中精氨酸酶(ARG)及誘導(dǎo)型一氧化氮合成酶(iNOS)表達(dá)量。以30例接受allo-HSCT患者輸注MDSCs細(xì)胞數(shù)中位數(shù)為界值進(jìn)行分組,比較高M(jìn)DSCs與低MDSCs組移植后造血重建、免疫重建、GVHD發(fā)生率、緩解與復(fù)發(fā)、長(zhǎng)生存等指標(biāo),分析MDSCs移植細(xì)胞數(shù)與aGVHD發(fā)生的相關(guān)關(guān)系。數(shù)據(jù)統(tǒng)計(jì)分析：采用SPSS 13.0統(tǒng)計(jì)軟件包進(jìn)行處理,以均數(shù)±標(biāo)準(zhǔn)差(x±s)表示,動(dòng)員前后比較、組間比較采用兩獨(dú)立t檢驗(yàn)或者配對(duì)樣本t檢驗(yàn)；等級(jí)資料比較采用卡方檢驗(yàn)；采用等級(jí)相關(guān)系數(shù)(Spearman相關(guān)系數(shù))非參數(shù)方法分析非雙變量正態(tài)分布資料。P≤0.05為差異有統(tǒng)計(jì)學(xué)意義。 三結(jié)果 1、G-CSF對(duì)健康供者外周血細(xì)胞的影響：經(jīng)G-CSF動(dòng)員后第5天,健康供者外周血中白細(xì)胞(WBC)數(shù)量明顯提高(P0.001),血紅蛋白(HGB)及血小板(PLT)均無(wú)明顯變化(P=0.205、0.939)。 2、G-CSF動(dòng)員對(duì)PB和BM中MDSCs含量的影響：在正常生理狀態(tài)下,健康供者PB、BM中均能檢測(cè)到MDSCs, MDSCs在PB中占有核細(xì)胞比例為0.35±0.07%,BM中為0.65±0.13%,BM中MDSCs比PB中高(P0.001)；G-CSF動(dòng)員5天后MDSCs在PB中占有核細(xì)胞比例為0.79±0.16%,BM中為0.72±0.13%,動(dòng)員后BM與PB中MDSCs比例相比無(wú)明顯變化(P=0.114)。動(dòng)員后外周血中MDSCs含量明顯高于動(dòng)員前(P0.001),而動(dòng)員前后BM中MDSCs細(xì)胞比例變化無(wú)統(tǒng)計(jì)學(xué)意義(P=0.136)。 3、G-CSF動(dòng)員對(duì)PB和BM中淋巴細(xì)胞亞群的影響：動(dòng)員后PB中CD4+CD25+ Treg細(xì)胞較動(dòng)員前明顯上升(P=0.048),其余細(xì)胞亞群均未見(jiàn)有顯著差別(P均0.05), G-CSF動(dòng)員前后MDSCs表達(dá)量與CD4+CD25+Treg細(xì)胞表達(dá)量無(wú)顯著相關(guān)關(guān)系(Pearson相關(guān)系數(shù)rs=0.627,P=0.096)。 4、MDSCs表面粘附因子：動(dòng)員前PB中MDSCs表面粘附因子CD11a、CD44、CD49d和CD49e表達(dá)量分別為0.72±0.23%、0.63±0.17%、0.34±0.11%和0.59±0.09%,BM中MDSCs表面粘附因子表達(dá)量分別為1.06±0.16%、1.08±0.13%、0.87±0.10%和1.18±0.19%；動(dòng)員后PB中MDSCs表面粘附因子CD11a、CD44、CD49d和CD49e表達(dá)量分別為0.44±0.21%、0.34±0.10%、0.44±0.11%和0.41±0.12%,BM中MDSCs表面粘附因子表達(dá)量分別為0.75±0.13%、0.52±0.14%、0.50±0.12%和0.69±0.15%。動(dòng)員前PB和BM中MDSCs表面粘附因子表達(dá)量明顯高于動(dòng)員后(P值均小于0.05)。 5、Arg和iNOS表達(dá)量：動(dòng)員前PB和BM中Arg表達(dá)量分別為0.66±0.15mIU/ml和2.20±0.40mIU/ml,動(dòng)員后分別為1.89±0.19mIU/ml和2.16±0.40mIU/ml;動(dòng)員前PB和BM中iNOS表達(dá)量分別為0.92±0.13mIU/ml和2.68±0.36mIU/ml,動(dòng)員后分別為2.10±0.13mIU/ml和2.79±0.23mIU/ml。動(dòng)員后PB中Arg和iNOS表達(dá)量均較動(dòng)員前明顯提高,具統(tǒng)計(jì)學(xué)意義(P值均小于0.01)；動(dòng)員前后BM中Arg和iNOS表達(dá)量均無(wú)明顯變化(P值均大于0.05)。 6、GVHD發(fā)生情況：中位隨訪時(shí)間為227天(48—363天),30例接受allo-HSCT患者,18例患者存活、12例死亡。高M(jìn)DSCs組aGVHD發(fā)生率20.00%(3/15)、Ⅲ-Ⅳ°aGVHD 0.00%(0/15)、cGVHD 30.77%(4/13);低MDSCs組aGVHD發(fā)生率66.67%(10/15)、Ⅲ-Ⅳ°aGVHD 20.00%(3/15)、cGVHD 45.45%(5/11)。高M(jìn)DSCs組aGVHD發(fā)生率顯著低于低MDSCs組(P=0.011),而兩組間Ⅲ-Ⅳ°aGVHD和cGVHD發(fā)生率則無(wú)顯著差異(P=0.073、P=0.469)。 7、MDSCs移植數(shù)與aGVHD發(fā)生關(guān)系：使用相關(guān)分析(Bivariate Correlations)分析移植MDSCs數(shù)量與aGVHD發(fā)生關(guān)系,結(jié)果顯示：移植后aGVHD的發(fā)生程度隨著移植時(shí)回輸供者M(jìn)DSCs細(xì)胞數(shù)量的增多而降低,兩者之間呈顯著負(fù)相關(guān)(Spearman相關(guān)系數(shù)rs=-0.445,P=0.014)。提示MDSCs細(xì)胞數(shù)量與aGVHD發(fā)生率有顯著負(fù)相關(guān)關(guān)系。 8、移植后復(fù)發(fā)與長(zhǎng)生存：高M(jìn)DSCs組復(fù)發(fā)率為26.67%(4/15),低MDSCs組復(fù)發(fā)率為20.00%(3/15),復(fù)發(fā)率比較無(wú)顯著差別(P=0.671)。高M(jìn)DSCs組與低MDSCs組無(wú)病生存率分別是46.7±14.2%和38.9±15.8%,總生存率分別是70.9±12.4%和50.6±15.0%,比較兩組無(wú)病生存率、總生存率均無(wú)顯著性差異(P值分別為0.833、0.442)。 四結(jié)論 在人體G-CSF能動(dòng)員BM中MDSCs到達(dá)外周血,使PB中MDSCs增高,PB中ARG與iNOS表達(dá)量增高。G-CSF動(dòng)員MDSCs到達(dá)外周的機(jī)制與MDSCs表面粘附因子表達(dá)量變化有關(guān)。G-CSF動(dòng)員的外周血干細(xì)胞采集物中MDSCs增加與移植后低aGVHD發(fā)生率有關(guān),兩者有明顯的負(fù)相關(guān)關(guān)系。MDSCs細(xì)胞輸注不會(huì)影響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，

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