地西他滨联合沙利度胺治疗高危骨髓增生异常综合征外周血PD-1、CTLA-4、细胞因子的表达变化及相关性研究

发布时间：2018-08-16 12:18

【摘要】：研究目的:探讨高危骨髓增生异常综合症(myelodysptastic syndrome,MDS)患者经地西他滨联合沙利度胺治疗前后外周血CD3+T、CD4+T、CD8+T、Treg细胞上程序性死亡分子-1(programmed death-1,PD-1)、细胞毒性T淋巴细胞相关抗原4(cytotoxic T lymphocyte-associated antigen 4,CTLA-4)表达,细胞因子干扰素γ(interferon-γ,IFN-γ)、白介素4(interleukin-4,IL-4)、白介素10(interleukin-10,IL-10)、肿瘤坏死因子-α(tumor necrosis factor-alpha,TNF-α)和转化生长因子β(trans-forming growth factorβ,TGF-β)水平的变化和分析T细胞亚群上表达PD-1、CTLA-4与细胞因子水平之间的相关性。研究方法:收集18例不同亚型高度危险分层的初治MDS患者和12例健康对照者,患者均使用地西他滨联合沙利度胺治疗3个疗程以上(包含3个疗程);采用流式细胞技术检测患者治疗前后组及健康对照组外周血CD3+T、CD4+T、CD8+T、Treg细胞上PD-1、CTLA-4表达。同时,应用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测MDS患者及健康对照组外周血细胞因子IFN-γ、IL-4、IL-10、TNF-α和TGF-β水平。并进一步分析T细胞亚群上表达PD-1、CTLA-4与细胞因子水平之间的相关性。研究结果:1.高危MDS治疗前组与健康对照组相比,CD3+T细胞上PD-1的表达升高(19.74±2.08%vs 5.28±1.60%,P0.001);CD4+T细胞上PD-1的表达显著升高(35.35±2.49%vs 2.64±0.79%,P0.001);CD8+T细胞上PD-1的表达升高(24.50±1.47%vs 3.46±0.31%,P0.001)及Treg细胞上PD-1表达明显升高(41.31±4.50%vs 5.01±1.79%,P0.001),差异均有统计学意义。高危MDS治疗后组与治疗前组相比,CD3+T细胞上PD-1表达明显下降(8.21±2.20%vs 19.74±2.08%,P0.001);CD4+T细胞上PD-1的表达明显下降(6.64±1.52%vs 35.35±2.49%,P0.001);CD8+T细胞上PD-1的表达下降(5.61±1.12%vs 24.50±1.47%,P0.001)及Treg细胞上PD-1表达显著降低(7.68±1.73%vs 41.31±4.50%,P0.001),差异均有统计学意义。2.高危MDS治疗前组与健康对照组相比,CD3+T细胞上CTLA-4的表达明显升高(9.71±1.45%vs0.46±0.57%,P0.001);CD4+T细胞上CTLA-4表达升高(3.68±1.17%vs 0.70±0.58%,P0.01);CD8+T细胞上CTLA-4表达升高(7.64±1.69%vs 1.51±0.61,P0.001)及Treg细胞上CTLA-4表达显著升高(14.57±1.87%vs 2.69±1.44%,P0.001),差异均有统计学意义。高危MDS治疗后组与治疗前相比,CD3+T细胞上CTLA-4表达明显下降(2.42±1.09%vs 9.71±1.45%,P0.001);CD4+T细胞上CTLA-4的表达下降(1.96±0.23%vs 3.68±1.17%,P0.001);CD8+T细胞上CTLA-4的表达下降(3.57±0.96%vs 7.64±1.69%,P0.001)及Treg细胞上CTLA-4表达明显下降(6.43±1.98%vs 14.57±1.87%,P0.001),差异均有统计学意义。3.高危MDS治疗前组与健康对照组相比,IFN-γ水平增高(10.61±1.47 vs 5.59±0.36,P0.001),IL-4水平明显增高(11.58±1.86 vs7.66±1.05,P0.001),TGF-β水平增高(29.53±1.60 vs 16.74±1.16,P0.001),TNF-α水平明显升高(24.27±0.88 vs 7.05±0.78,P0.001),IL-10水平显著增高(78.39±2.38 vs 9.63±0.38,P0.001),差异均有统计学意义。高危MDS治疗后组与治疗前组相比,IFN-γ水平降低(7.63±1.28 vs 10.61±1.47,P0.001),IL-4水平降低(9.35±1.09 vs 11.58±1.86,P0.001),TGF-β水平明显降低(19.48±1.50 vs 29.53±1.60,P0.001),TNF-α水平显著降低(10.45±0.49 vs 24.27±0.88,P0.001),IL-10水平明显下降(15.65±1.00 vs 78.39±2.38,P0.001),差异均有统计学意义。4.MDS患者治疗前CD8+T细胞表达PD-1与血清IFN-γ水平呈正相关(r=0.876,P0.01);MDS患者治疗前CD4+T细胞表达PD-1与血清IFN-γ水平呈正相关(r=0.538,P0.05);MDS患者治疗前CD3+T细胞表达PD-1与血清IFN-γ水平呈正相关(r=0.499,P0.05);MDS患者治疗前Treg细胞表达PD-1与血清IFN-γ水平呈正相关(r=0.921,P0.001);MDS患者治疗前CD3+T细胞表达CTLA-4与血清IL-10水平呈正相关(r=0.885,P0.001);MDS患者治疗前CD4+T细胞表达CTLA-4与血清IL-10水平呈正相关(r=0.781,P0.001);MDS患者治疗前CD8+T细胞表达CTLA-4与血清IL-10水平呈正相关(r=0.803,P0.001);MDS患者治疗前Treg细胞表达CTLA-4与血清IL-10水平呈正相关(r=0.726,P0.001),差异均有统计学意义。结论:1.初治高危MDS患者治疗前外周血PD-1及CTLA-4在CD3+T、CD4+T、CD8+T和Treg细胞上表达显著升高,T细胞免疫功能紊乱可能参与MDS发病;2.初治高危MDS患者治疗前外周血细胞因子IFN-γ、IL-4、IL-10、TNF-α和TGF-β水平增高,免疫微环境中细胞因子水平的改变可能参与MDS发病。3.初治高危MDS患者治疗前T细胞亚群上表达的PD-1与外周血细胞因子IFN-γ水平呈正相关及初治高危MDS患者治疗前T细胞亚群上表达的CTLA-4与外周血细胞因子IL-10水平呈正相关,提示IFN-γ和IL-10与T细胞亚群上表达的PD-1及CTLA-4在MDS发病中相互作用,协同致病。
[Abstract]:Objective: To investigate the programmed death-1 (PD-1) and cytotoxic T lymphocyte-associated antigen-4 (cytotoxic T-lymphocyte-associated antigen-4) in peripheral blood of patients with high-risk myelodysplastic syndrome (MDS) before and after treatment with destabine and thalidomide. Gene 4, CTLA-4 expression, cytokine interferon-gamma (IFN-gamma), interleukin-4 (IL-4), interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-alpha) and trans forming growth factor-beta (TGF-bet) expression in T cell subsets were analyzed. Methods: 18 primary MDS patients with different subtypes of high risk stratification and 12 healthy controls were enrolled in this study. All patients were treated with DXT plus thalidomide for more than 3 courses (including 3 courses); flow cytometry (FCM) was used to detect the levels of cytokines before and after treatment and healthy control group. The expressions of PD-1 and CTLA-4 in peripheral blood CD3+T, CD4+T, CD8+T and Treg cells were detected. The levels of cytokines IFN-gamma, IL-4, IL-10, TNF-alpha and TGF-beta in peripheral blood of MDS patients and healthy controls were detected by enzyme linked immunosorbent assay (ELISA). The expressions of PD-1, CTLA-4 and cytokine water in T cell subsets were further analyzed. Results: 1. Compared with the healthy control group, the expression of PD-1 on CD3 + T cells in high-risk MDS group increased (19.74 65507 The expression of PD-1 on CD3 + T cells in high-risk MDS treatment group was significantly lower than that in pre-treatment group (8.21 + 2.20% vs 19.74 + 2.08%, P 0.001); the expression of PD-1 on CD4 + T cells was significantly lower than that in pre-treatment group (6.64 + 1.52% vs 35.35 + 2.49%, P 0.001). The expression of PD-1 on CD3+T cells was significantly higher than that on healthy control group (9.71+1.45% vs 0.46+0.57%, P 0.001), and that on Treg cells was significantly lower (7.68+1.73% vs 41.31+4.50%, P 0.001). The expression of CTLA-4 on CD8 + T cells was increased (7.64 (+ 1.69%) vs 1.51 (+ 0.61, P 0.001) and that on Treg cells was significantly increased (14.57 (+ 1.87%) vs 2.69 (+ 1.44%, P 0.001). The expression of CTLA-4 on CD4+T cells was decreased (1.96+0.23% vs 3.68+1.17%, P 0.001); the expression of CTLA-4 on CD8+T cells was decreased (3.57+0.96% vs 7.64+1.69%, P 0.001) and the expression of CTLA-4 on Treg cells was significantly decreased (6.43+1.98% vs 14.57+1.87%, P 0.001) in the high-risk MDS group before treatment. Compared with the healthy control group, the levels of IFN-gamma, IL-4, TGF-beta, TNF-alpha, IL-10 and IL-10 were significantly increased (10.61 (+ 1.47 vs 5.59 (+ 0.36, P 0.001), 11.58 (+) 1.86 vs 7.66 (+) 1.05, P 0.001), 29.53 (+) 1.60 vs 16.74 (+) 1.16, P 0.001), and 24.27 (+) 0.88 vs 7.05 (+) 0.78 vs 2.63 (+) and P 0.001), respectively. After treatment, the levels of IFN-gamma, IL-4, TGF-beta, TNF-alpha and TNF-alpha were significantly decreased (10.45 [0.49] vs 24.27 [0.88, P 0.001] and 9.35 [1.09] vs 11.58 [1.86, P 0.001] respectively, compared with those before treatment. The levels of IL-10, IL-10 and CD8+T cell expression PD-1 were positively correlated with serum IFN-gamma (r = 0.876, P 0.01) and CD4+T cell expression PD-1 was positively correlated with serum IFN-gamma (r = 0.538, P 0.05) in MDS patients before treatment. PD-1 was positively correlated with serum IFN-gamma level (r=0.499, P 0.05); Treg cell expression of PD-1 was positively correlated with serum IFN-gamma level before treatment in MDS patients (r=0.921, P 0.001); CD3+T cell expression of CTLA-4 was positively correlated with serum IL-10 level before treatment in MDS patients (r=0.885, P 0.001); CD4+T cell expression of CTLA-4 was positively correlated with serum IL-10 level before treatment in MDS patients. There was a positive correlation (r = 0.781, P 0.001); the expression of CTLA-4 in CD8 + T cells was positively correlated with the level of serum IL-10 in MDS patients before treatment (r = 0.803, P 0.001); the expression of CTLA-4 in Treg cells was positively correlated with the level of serum IL-10 in MDS patients before treatment (r = 0.726, P 0.001). The expression of 3+T, CD4+T, CD8+T and Treg cells increased significantly, T cell immune dysfunction may be involved in the pathogenesis of MDS; 2. The levels of peripheral blood cytokines IFN-gamma, IL-4, IL-10, TNF-alpha and TGF-beta increased before treatment in patients with high-risk MDS, and the changes of cytokines in the immune microenvironment may be involved in the pathogenesis of MDS. There was a positive correlation between the expression of PD-1 and IFN-gamma in peripheral blood. The expression of CTLA-4 in peripheral blood cytokines IL-10 was positively correlated with the level of peripheral blood cytokines IL-10 in high risk MDS patients before treatment. It suggested that IFN-gamma and IL-10 interacted with PD-1 and CTLA-4 in the pathogenesis of MDS.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R551.3

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