NK细胞重建与急性移植物抗宿主病相关性研究

发布时间：2018-04-28 09:04

  本文选题：NK细胞 + 急性移植物抗宿主病　； 参考：《中国人民解放军军事医学科学院》2017年硕士论文

【摘要】：随着医疗技术的飞速发展,异基因造血干细胞移植(allogeneic hematopoietic stem cell transplantation,allo-HSCT)后患者治愈率得到极大的提高。但由于缺乏有效预测指标和治疗措施,急性移植物抗宿主病(acute graft-versus-host disease,aGVHD),特别是激素耐药的aGVHD,仍是制约allo-HSCT治愈率的关键技术性瓶颈[1]。aGVHD对靶细胞的损害可分为三步:(1)治疗相关的损伤导致炎性细胞因子的大量释放,从而启动aGVHD;(2)T细胞激活后开始增殖活化;(3)T细胞攻击患者上皮组织或器官。故aGVHD治疗的关键在于抑制T细胞的增殖活化,但这也带来了肿瘤复发和重症感染的风险[2,3]。自然杀伤(Natural killer,NK)细胞可快速杀伤患者体内变异的细胞,具有抗肿瘤和抗感染的作用,对allo-HSCT具有重要意义。进一步研究发现,NK细胞在抗肿瘤、抗感染的同时,还具有抗aGVHD和促进移植物植入的作用[4],为防治aGVHD带来了新的希望。NK细胞在allo-HSCT后快速实现数量重建[5,6],此时NK细胞抗aGVHD作用是靠杀伤抗原呈递细胞(antigen presenting cells,APCs),阻断T细胞的活化而得以实现[7]。NK细胞同时表达抑制性和活化性受体,其对APCs的杀伤活性是由两种受体与配体的结合情况共同决定的。当APCs表面抑制性配体表达缺失,或活化性配体表达高于抑制性配体时,NK细胞将被激活,特异性的杀伤APCs。近年来的研究发现,allo-HSCT后免疫重建的NK细胞表面的受体谱表达改变,特别是抑制性受体表达上调,导致免疫重建的NK细胞功能受到抑制,杀伤靶细胞的活性低下。NK细胞功能抑制这一情况将持续到allo-HSCT后半年左右[8,9]。免疫重建的NK细胞作为一种功能被抑制的免疫细胞,是否还能杀伤APCs,是否还能有效发挥抗aGVHD作用,目前尚未明确。本研究通过分析NK细胞的重建情况,探讨NK细胞重建与aGVHD的相关性,为进一步研究重建NK细胞的抗aGVHD作用奠定了基础。本研究首先观察了患者allo-HSCT后NK细胞计数和活性与正常人的差异,分析了allo-HSCT后NK细胞计数和活性恢复情况,从而综合评价NK细胞的重建。实验思路简述如下:选取2015年1月至2015年7月,在本院行allo-HSCT的26例患者,其中男性17例,女性9例,患者中位年龄37(12~62)岁。采集患者allo-HSCT后+30d、+60d、+90d时外周血作为实验组,通过流式细胞仪(flowcytometry,FCM)检测外周血中NK细胞计数和活性。将供者作为对照组,界定NK细胞计数和活性的正常值。将外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)分离出外周血后计数。用CD45-APC、CD56-FITC、CD16-FITC和CD3-PerCP标记PBMCs,FCM检测出PBMNs中CD45、CD3、CD56、CD16的表达情况,计算CD3-CD56+CD16+的NK细胞计数。用CSFE荧光标记K562细胞。将K562细胞单独孵育作为对照组,观察K562细胞自然凋亡率。将NK细胞与K562细胞按1:1比例共同培养作为实验组,观察NK细胞对K562细胞杀伤活性。对照组和实验组细胞在培养箱中孵育2.5小时后用碘化丙啶(propidium Iodide,PI)标记,FCM检测CSFE+PI+的细胞(即凋亡的K562),最后计算出NK细胞杀伤活性。根据不同时间点NK细胞计数和活性的检测结果,探索allo-HSCT后NK细胞数量和功能的重建情况。研究结果显示:正常人NK细胞计数和活性为(1198±514)个/μl和(14.0±6.8)%。患者allo-HSCT后+30d、+60d、+90d时NK细胞计数分别为(951±403)个/μl、(1358±418)个/μl和(1255±353)个/μl,与正常人无统计学差异。患者NK细胞活性分别为(7.3±3.7)%、(9.4±3.6)%和(9.3±2.4)%,较正常人显著降低。以上结果说明,allo-HSCT后+30d时,NK细胞计数能恢复到正常人水平。但allo-HSCT后+90d内,NK细胞活性低下。NK细胞计数和活性在allo-HSCT后+30d时较+60d、+90d时低下。Allo-HSCT后NK细胞计数和活性的降低是否会影响其抗aGVHD作用,从而增加aGVHD发病的风险则有待于进一步研究。因此,本研究第二部分对26例患者进行了跟踪随访,以探索NK细胞重建对aGVHD发病的影响。实验思路简述如下:根据患者的临床表现和相关病理指标,统计26例患者aGVHD发病、总体生存(overall survival,OS)和无进展生存(progression-free survival,PFS)等临床资料,分析NK细胞计数和活性与aGVHD发病和预后的相关性。研究结果显示:根据患者是否发生aGVHD,将26例患者分为aGVHD组(11例)和无aGVHD组(15例)。与无aGVHD组相比,aGVHD组患者在allo-HSCT后+30d时NK细胞计数和活性显著降低,即(655±216)个/μlvs(1169±372)个/μl(p=0.002)和(7.3±3.6)%vs(9.0±3.6)%(p=0.008)。根据患者是否发生Ⅱ~Ⅳ度aGVHD,进一步将26例患者分为Ⅱ~Ⅳ度aGVHD组(7例)和0~Ⅰ度aGVHD组(19例)。与0~Ⅰ度aGVHD组相比,Ⅱ~Ⅳ度aGVHD组患者NK细胞计数和活性同样显著降低,且差异更为显著,即(617±220)个/μl vs(1081±399)个/μl(p=0.001)和(4.2±1.7)%vs(8.3±3.5)%(p=0.001)。NK细胞计数(γ=—0.628,p=0.001)及活性(γ=—0.535,p=0.005)与aGVHD发病呈中度负相关。生存结果显示,Ⅱ~Ⅳ度aGVHD组复发率显著升高(57%vs 5%,p=0.010),且1年PFS率明显下降(43%vs 84%,p=0.010)。Ⅱ~Ⅳ度aGVHD组患者的1年OS率同样低于0~Ⅰ度aGVHD组(71%vs 90%),但无统计学差异(p=0.374)。以上结果说明,allo-HSCT后发生aGVHD的患者+30d时NK细胞计数和活性较未发生aGVHD的患者显著降低,且发生Ⅱ~Ⅳ度aGVHD患者的NK细胞计数和活性降低更为显著。Allo-HSCT后+30d时NK细胞计数和活性与aGVHD呈中度负相关性。+30d时NK细胞计数和活性低下的aGVHD患者,其复发率显著高,PFS率显著降低。综上,本研究结果显示,allo-HSCT后+30d时,NK细胞完成数量重建,但+90d内NK细胞活性显著低于正常人水平。Allo-HSCT后+30d时,重建NK细胞的计数和活性处于最低水平,且与aGVHD的发生和预后存在一定联系,通过早期监测NK细胞计数和活性可识别aGVHD高危患者,这将为防治aGVHD提供新的思路。
[Abstract]:With the rapid development of medical technology, the cure rate of allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been greatly improved. However, due to the lack of effective predictors and treatment measures, acute graft-versus-host disease (acute graft-versus-host disease, aGVHD), especially hormone Drug-resistant aGVHD is still a key technical bottleneck that restricts the cure rate of allo-HSCT, [1].aGVHD can be divided into three steps: (1) the treatment related injury leads to the massive release of inflammatory cytokines, thus initiating aGVHD; (2) T cells begin to proliferate and activate; (3) T cells attack the epithelial tissue or organs of the patients. So aGVHD treatment The key is to inhibit the proliferation and activation of T cells, but this also brings the risk of tumor recurrence and severe infection. The [2,3]. natural killer (Natural killer, NK) cells can quickly kill the mutant cells in the patient's body. It has the role of anti-tumor and anti infection, and is of great significance to allo-HSCT. Further studies have found that NK cells are anti tumor and anti infection. At the same time, it also has the effect of resisting aGVHD and promoting graft implantation, which brings new hope that.NK cells can quickly reconstruct [5,6] after allo-HSCT for the prevention and control of aGVHD. At this time, the anti aGVHD effect of NK cells is by killing antigen presenting cells (antigen presenting cells, APCs) and blocking the activation of.NK cells. Expression of inhibitory and activated receptors, whose killing activity against APCs is determined by the combination of two receptors and ligands. When the expression of APCs surface inhibitive ligand is missing, or the expression of active ligand is higher than the inhibitory ligand, the NK cells will be activated. The specific killing of APCs. in recent years has found that the immune weight after allo-HSCT is heavy. The changes in the expression of the receptor spectrum on the surface of the NK cells, especially the up regulation of the inhibitory receptor expression, resulted in the inhibition of the function of the immune rebuilt NK cells and the inhibition of the function inhibition of the low active.NK cells of the target cells. This situation will continue to the NK cells reconstructed by [8,9]. immunized as a function that is suppressed in the second half of allo-HSCT. Cell, whether it can kill APCs, and whether it can play an effective anti aGVHD effect, is not clear. This study is to explore the correlation between NK cell reconstruction and aGVHD by analyzing the reconstruction of NK cells. This study has laid the foundation for further study of the anti aGVHD effect of reconstruction of NK cells. The difference between sex and normal people, analysis of the NK cell count and activity recovery after allo-HSCT, and thus comprehensive evaluation of the reconstruction of NK cells. The experimental ideas are summarized as follows: 26 cases of allo-HSCT in our hospital from January 2015 to July 2015 were selected, including 17 men, 9 women and 37 (12~62) years of middle age. After allo-HSCT +30d The peripheral blood was used as the experimental group at +60d and +90d as the experimental group. The count and activity of NK cells in peripheral blood were detected by flow cytometry (flowcytometry, FCM). The donors were used as the control group to define the normal values of NK cell count and activity. The peripheral blood mononuclear cells (peripheral blood mononuclear cells, PBMCs) were separated out of the peripheral blood count. 6-FITC, CD16-FITC and CD3-PerCP were labeled PBMCs, FCM was used to detect the expression of CD45, CD3, CD56, CD16 in PBMNs, and the CD3-CD56+CD16+ NK cell count was calculated. The cytotoxic activity of NK cells to K562 cells was observed. The cells in the control group and the experimental group were incubated in the incubator for 2.5 hours and were labeled with propidium Iodide (PI), and FCM was used to detect the CSFE+PI+ cells (the apoptotic K562). Finally, the NK cell killing activity was calculated. According to the detection results of the count and activity of the non simultaneous NK cells, the allo-HSCT was explored. The results of the number and function of the post NK cells showed that the count and activity of NK cells in normal people were (1198 + 514) / mu L and (14 + 6.8)%. The count of NK cells was (951 + 403) / u l, (1358 + 418) / L and (1255 + 353) / mu l at +30d, +60d and +90d after allo-HSCT, and there was no statistical difference from normal people. (7.3 + 3.7)%, (9.4 + 3.6)% and (9.3 + 2.4)%, significantly lower than normal people. The results showed that the number of NK cells could be restored to normal level at +30d after allo-HSCT, but in +90d after allo-HSCT, the count and activity of.NK cells in NK cells were lower than +60d after allo-HSCT, and the count and activity of the cells after.Allo-HSCT were low. The second part of this study was followed up to explore the effect of NK cell reconstruction on the incidence of aGVHD. The second part of this study was followed up to explore the effect of NK cell reconstruction on the incidence of aGVHD. The experimental ideas were summarized as follows: according to the patient's clinical manifestation and the related pathological indexes, 26 cases were counted. Patients with aGVHD, overall survival (overall survival, OS) and progression free survival (progression-free survival, PFS) were used to analyze the correlation between the count and activity of NK cells and the morbidity and prognosis of aGVHD. The results showed that 26 patients were divided into aGVHD group (11 cases) and no aGVHD group (15 cases) according to the occurrence of aGVHD. Compared with group aGVHD, the count and activity of NK cells decreased significantly at +30d after allo-HSCT, namely (655 + 216) / mu LVS (1169 + 372) / mu L (p=0.002) and (7.3 + 3.6)%vs (9 + 3.6)% (p=0.008). According to the occurrence of patients with II to IV degree aGVHD, 26 patients were further divided into class II ~ IV aGVHD group (7 cases) and 0~ I degree group (19 cases). Compared with group aGVHD, the count and activity of NK cells in group II ~ IV degree aGVHD were also significantly decreased, and the difference was more significant, that is, (617 + 220) / mu l vs (1081 + 399) / mu L (p=0.001) and (4.2 + 1.7)%vs (8.3 + 3.5)% (p=0.001).NK cell count (gamma = 0.628, p= 0.001) and activity (gamma = 0.535, p=0.005) had a moderate negative correlation with the onset of disease. The results showed that the recurrence rate of group II ~ IV aGVHD group was significantly higher (57%vs 5%, p=0.010), and the 1 year PFS rate decreased significantly (43%vs 84%, p=0.010). The 1 year OS rate in group II ~ IV aGVHD group was also lower than 0~ I aGVHD group (71%vs 90%), but there was no statistical difference (p=0.374). The patients who had no aGVHD significantly decreased, and the number and activity of NK cells in patients with 2 ~ IV degree aGVHD were more significant than those of NK cell count and NK cell count and activity with aGVHD, and the NK cell count and low activity of aGVHD were significantly higher and the PFS rate decreased significantly. The results showed that the number of NK cells was rebuilt at +30d after allo-HSCT, but when the activity of NK cells in +90d was significantly lower than that of normal human.Allo-HSCT, the count and activity of NK cells were at the lowest level, and there was a relationship with the occurrence and prognosis of aGVHD, and the aGVHD high-risk patients could be identified by early monitoring of NK cell count and activity. This will provide a new way of thinking for the prevention and control of aGVHD.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R457.7

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