重型再生障碍性贫血骨髓造血免疫损伤机制及其相关抗原的研究
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摘要
目的
通过研究重型再生障碍性贫血(Severe Aplastic Anemia, SAA)患者CD8+HLA-DR+效应T细胞功能因子表达水平、对骨髓造血细胞的损伤及其作用的靶细胞,证实CD8+HLA-DR+效应T细胞与SAA患者骨髓衰竭之间的“因果效应”及“因果方式”,进一步阐明SAA免疫损伤骨髓造血的机制；同时采用差异蛋白质组学技术,对SAA患者与正常人髓样树突状细胞(myeloid dendritic cells,mDC)的蛋白质成份进行分析,筛选激活SAA免疫反应可能的抗原物质,即导致SAA发病可能的自身抗原,为开发靶向治疗药物提供依据。方法
研究对象为天津医科大学总医院自2010年4月至2012年4月初治SAA患者、治疗后缓解的SAA患者及正常对照者。第一部分研究效应T细胞损伤骨髓造血细胞的途径,采用免疫磁珠分选24例SAA初治患者及23名健康正常人外周血CD8+HLA-DR+效应T细胞,半定量RT-PCR检测分选细胞效应因子穿孔素、颗粒酶B、肿瘤坏死因子-p(tumor necrosis factor beta, TNF-β)、FasL mRNA表达情况。第二部分验证SAA患者效应T细胞对骨髓造血细胞的损伤作用,以免疫磁珠阳性分选12例SAA初治患者、12例SAA缓解患者及12名正常健康人CD8+HLA-DR+T细胞作为效应细胞,阴性分选去除SAA缓解患者、正常健康人骨髓单个核细胞中CD3+T细胞后作为靶细胞,将SAA初治患者效应细胞分别与SAA缓解患者及正常健康人靶细胞混合培养(分别为实验组1、实验组2),同时设SAA缓解患者效应T细胞与SAA缓解患者骨髓靶细胞混合培养组及正常健康人效应T细胞与正常健康人靶细胞混合培养组作为对照(分别为对照组1、对照组2),72h后通过流式细胞术以膜联蛋白V(Annexin V)检测靶细胞凋亡状况,全自动生化分析仪检测培养体系上清液乳酸脱氢酶(lactate dehydrogenase, LDH)水平。第三部分明确SAA患者靶向损伤的骨髓造血细胞,分别将SAA患者、正常人CD8+HLA-DR+效应T细胞与正常人的CD3-细胞(免疫磁珠分选获得)混合培养,72h后采用流式细胞术检测培养系中CD14+、CD33+、CD34+,GlycoA+细胞凋亡状况；以单克隆抗体标记SAA患者和正常人骨髓CD34+、GlycoA+、CD33+、CD14+细胞,比较其Fas蛋白的表达量。第四部分探索导致SAA免疫激活的抗原物质,采用贴壁培养法将SAA患者、缓解患者及正常人单核细胞,体外诱导、分化为成熟的mDC,流式细胞仪分选得到高纯度mDC,提取总蛋白,进行双向电泳分离蛋白质,取差异蛋白质点进行比对,明确SAA患者mDC内与正常人不同的蛋白质成分,即为激活mDC,引起SAA一系列自身免疫反应可能的抗原物质。结果
第一部分SAA初治组CD8+HLA-DR+效应T细胞穿孔素、颗粒酶B mRNA的表达量分别为(0.66±0.25)、(0.56±0.26),正常对照组表达量为(0.53±0.14)、(0.40±0.13),前者明显高于后者(P值分别为0.042、0.012)；SAA初治组CD8+HLA-D时效应T细胞FasL nRNA的表达量为(0.77±0.24),明显高于正常对照组(0.61±0.16)(P=0.011)；SAA初治组CD8+HLA-DR+效应T细胞TNF-β mRNA的表达量为(0.58±0.16),亦明显高于正常对照组(0.46±0.15)(P=0.011)。
第二部分SAA初治患者效应细胞与SAA缓解患者靶细胞混合培养组(实验组1)、SAA初治患者效应细胞与正常健康人靶细胞混合培养组(实验组2)靶细胞凋亡比例分别为(41.12±24.84)%、(45.81±20.47)%,SAA缓解患者效应细胞与SAA缓解靶细胞混合培养组(缓解组)、正常人效应细胞与正常人靶细胞混合培养组(正常组)靶细胞凋亡比例分别为(35.03±22.09)%、(20.95±13.82)%,实验组1、实验组2、缓解组三组间两两比较差异无统计学意义(P>0.05),实验组1、实验组2、缓解组均明显高于正常组(P<0.05)；实验组1、实验组2培养体系上清液LDH水平分别为(74.56±49.13)U/L、(62.61±31.76)U/L,明显高于正常组的LDH水平(28.60±8.91)U/L(P<0.05),与缓解组LDH水平(61.06±28.41)U/L比较差异无统计学意义(P>0.05),缓解组LDH水平明显高于正常组(P<0.05)。
第三部分SAA组骨髓CD34+细胞Fas蛋白的表达量为46.59±27.60%,明显高于对照组(8.89±7.28%,P<0.01)；SAA组CD14+、CD33+、GlycoA+细胞Fas蛋白的表达量分别为29.29±9.23%、46.88±14.30%、15.15±9.26%,明显低于对照组(51.25±38.36%、72.06±39.88%、50.38±39.88%,P<0.05)。SAA初治患者效应细胞与正常健康人靶细胞(实验组)CD34+、CD33+、CD14+细胞凋亡率分别为55.43±20.50%、38.13±20.10%、61.87±21.65%,均明显高于正常人效应细胞与正常人靶细胞混合培养组(对照组)35.02±13.95%、23.44±10.33%、37.04±22.41%(P均<0.05)；实验组CD45-细胞凋亡率为26.43±24.99%,与对照组22.76±7.62%无明显差异(P>0.05)。
第四部分SAA患者mDC细胞内蛋白成分与缓解后患者及正常人之间进行对比,其凋亡相关蛋白(p53、caspase3、Fas),核糖体连接蛋白、泛素化相关蛋白表达增高,与SAA免疫发病机制中mDC细胞激活相关。结论
(1)SAA患者外周血CD8+HLA-DR+效应T细胞穿孔素、颗粒酶表达水平均较正常人明显升高,提示穿孔素、颗粒酶可能在SAA患者细胞毒性T淋巴细胞(cytotoxic lymphocyte, CTL)损伤骨髓造血过程起到了重要的作用；SAA患者CD8+HLA-DR+T效应细胞FasL表达亦明显增高,可能参与了骨髓造血靶细胞的凋亡损伤,同时高表达的FasL也可能参与了辅助性T细胞(helper T cell,Th)亚群的调节,但对CTL本身可能没有作用；此外,TNF-β也是参与CTL损伤骨髓造血靶细胞的重要效应因子,在SAA患者CD8+HLA-DR+效应T细胞的表达也增高。因此,上述三种途径可能均在SAA免疫发病过程中发挥了重要作用。
(2)SAA初治患者CD8+HLA-DR+效应T细胞能够明显增加缓解患者及正常人骨髓造血靶细胞凋亡率,提示CD8+HLA-DR+效应T细胞在SAA骨髓免疫损伤中机制中居于重要地位。而缓解SAA患者CD8+HLA-DR+效应T细胞功能并未降至正常,临床应用免疫抑制治疗中应注重足量、足疗程给药,以便彻底控制患者异常免疫状态,防止复发。
(3)SAA初治患者CD8+HLA-DR+效应T细胞能够诱导正常人CD34+、CD33+、CD14+细胞凋亡增加,对正常的骨髓造血干/祖、粒系、单核系细胞均具有杀伤作用；经检测SAA患者骨髓CD34+、CD33+、CD14+、GlycoA+细胞均表达Fas抗原,并可能因此被高表达FasL的CD8+HLA-DR+效应T细胞识别,故Fas/FasL系统介导的细胞凋亡在SAA免疫发病中发挥了重要作用；而SAA患者CD34+细胞Fas表达明显增加,可能是SAA免疫损伤的主要靶细胞。
(4)SAA患者mDC中凋亡相关蛋白、核糖体连接及泛素化相关蛋白表达异常,可能与mDC的激活相关。
Objective
To investigate the level of functional factors secreted from CD8+HLA-DR+effector T cells in peripheral blood (PB) of the patients with severe aplastic anemia(SAA), the damage on hematopoietic cells by this group of cells, and which were the target cells attacked by CD8+HLA-DR+effector T cells. Thus, we investigated the function of CD8+HLA-DR+cells, which were considered to be activated CTL, in peripheral blood to further explore the pathogenesis of SAA. Also, we explored the heterogenic proteins in myeloid dentritic cells between SAA patients and normal controls, which may be the pathogenic antigen in SAA.
Methods
Untreated SAA patients, remission paitents, as well as normal controls were enrolled in this study. Part Ⅰ The CD8+HLA-DR+cells were sorted from the PB of24untreated SAA patients and23normal controls by immunomagnetic separation. The mRNA expressions of perforin, granzyme B, tumor necrosis factor-β (TNF-β) and FasL of sorted cells were analyzed by RT-PCR. Part ⅡCD8+HLA-DR+T cells (effector cells) from untreated SAA patients, remission patients and normal controls were co-cultured with bone marrow mononuclear cells (with CD3+T cells depleted) from remission patients and normal controls (target cells). The effector T cells of untreated SAA were cocultured with the target cells of remission patients and normal controls, which were designated as SAA groups1and2, respectively. There were two control groups:one was the remission SAA group (effector cells from untreated SAA patients and target cells of remission patients), and the other was the normal control (effector cells from untreated SAA patients and target cells of normal controls). Apoptosis of target cells was detected by flow cytometry after staining with AnnexinⅤ. The levels of lactate dehydrogenase (LDH) in the supernatant were determined by automatic biochemistry analyzer. Part ⅢCD8+HLA-DR+T cells from untreated SAA patients, and normal controls were co-cultured with bone marrow mononuclear cells (with CD3+T cells depleted) from normal controls. Apoptosis of CD14+、CD33+、CD34+、GlycoA+cells was detected by flow cytometry after staining with AnnexinV. Meanwhile, the expression of Fas on the CD14+、CD33+、CD34+、 GlycoA+cells was detected by flow cytometry in untreated patients and normal controls respectively. Part Ⅳ Bone marrow mononuclear cells were isolated and inoculated in cell culture flask,2hours later the suspended cells were given up. The cells adhering to the wall cultured for7days and became myeloid dentritic cells. After that, the cells were purified by flow cytometry, and extracted total protein from them. Furtherly, explore the difference of the protein among untreated SAA, remission SAA and normal controls by two-dimensinal electrophoresis.
Results
Part I The mRNA of perforin, granzyme B of CD8+HLA-DR+T cells were (0.66±0.25)、(0.56±0.26) in untreated group, higher than these of controls (0.53±0.14)、(0.40±0.13)(P=0.042、0.012). The mRNA of FasL in CD8+HLA-DR+T cells of untreated SAA patients was (0.77±0.24),higher than that of controls (0.61±0.16)(P=0.011). The mRNA of TNF-β in CD8+HLA-DR+T cells of untreated SAA patients was (0.58±0.16),also higher than that of controls (0.46±0.15)(P=0.011).
Part II The apoptosis values in SAA group1(CD8+HLA-DR+T cells from untreated SAA patients and CD3-bone marrow mononuclear cells from remission patients), SAA group2(CD8+HLA-DR+T cells from untreated SAA patients and CD3-bone marrow mononuclear cells from normal controls), the remission group (CD8+HLA-DR+T cells and CD3-bone marrow mononuclear cells from remission patients) and the normal control (CD8HLA-DR+T cells and CD3-bone marrow mononuclear cells from normal controls) were41.12±24.84%,45.81±20.47%,35.03±22.09%,20.95±13.82%. There were no significant differences between SAA group1, SAA group2, and the remission group (P>0.05). However, the apoptosis in each of these groups was higher than in the normal control (P<0.05).
The LDH levels in SAA group1, SAA group2, the remission group and normal control were74.56±49.13U/L,62.61±31.76U/L,61.06±28.41U/L, and28.60±8.91U/L. There were no significant differences between SAA group1, SAA group2, and the remission group (P>0.05). However, the level of LDH in these groups was significantly higher than in the normal control (P<0.05).
Part Ⅲ The ratios of Fas in the CD34+cells from SAA groups was46.59±27.60%, significantly higher than that of normal control (8.89%±7.28%, P<0.01). The ratio of Fas in the CD14+、CD33+、GlycoA+cells from SAA groups were29.29±9.23%、46.88±14.30%，15.15±9.26%, significantly lower than that of normal control (51.25±38.36%，72.06±39.88%、50.38±39.88%, P<0.05). The apoptosis values of CD34+、CD33+、CD14+cells in SAA group (CD8+HLA-DR+T cells from untreated SAA patients and CD3-bone marrow mononuclear cells from normal contros) were55.43±20.50%、38.13±20.10%、61.87±21.65%, significantly higher than that in the normal control (35.02±13.95%、23.44±10.33%、37.04±22.41%,P<0.05). However, there were no significant differences of CD45-cells between SAA group and normal control (P>0.05).
Part IV Our studies about intracellular proteins in mDC have demonstrated that the expression of apoptotic protein (p53,caspase3,Fas), spliceosome-related protein, and ubiquitin-related protein were higher in SAA than normal controls, which seem to related to mDC activation.
Conclusions
(1) By RT-PCR, we found that the expression of perforin, granzyme B were elevated in the CD8+HLA-DR+T cells, suggested that CD8+HLA-DR+T cells and their functional products might contribute to bone marrow failure in SAA. We also found that the expression of FasL of CD8+HLA-DR+T cells were higher in the SAA than normal controls. Thus, FasL might take part in the pathogenesis of SAA, however, it might have no effect on CD8+HLA-DR+T cells. Moreover, the expression of tumor necrosis factor-beta (TNF-β) were elevated in the CD8+HLA-DR+T cells, which might be the important functional factor in the CD8+HLA-DR+T cells of SAA patients.
(2) The CD8+HLA-DR+T cells from untreated SAA and remission patients were more cytotoxic than those cells from healthy controls. These observations suggest that the induction of apoptosis of hematopoietic cells by CD8+HLA-DR+T cells might play a critical role in the bone marrow failure of SAA. In our research, the cytotoxicity of CD8+HLA-DR+T cells was reduced after IST treatment, but it did not reach the level of the control subjects as the normal state. Thus, in order to decrease the risk of relapse, it is important for SAA patients to maintain the IST treatment for a longer period of time, and to taper cyclosporine very slowly.
(3) The apoptosis of CD34+、CD33+、CD14+cells from the normal persons increased after coculture with the CD8+HLA-DR+T cells from SAA patients, which indicated that the CD8+HLA-DR+T cells from SAA patients may lead to the excessive apoptosis of hematopoietic cells in normal controls. The expression of Fas on the CD34+、CD33+、CD14+、GlycoA+cells were all positive, which may induce the damage by CD8+HLA-DR+T cells. And the expression of Fas on the CD34+cells markly increased, which may be the main target cell in the pathogenesis of SAA.
(4) Abnomal expression of apoptotic protein, spliceosome-related protein,and ubiquitin-related protein in SAA might be involved in the activation of mDC.
通过研究重型再生障碍性贫血(Severe Aplastic Anemia, SAA)患者CD8+HLA-DR+效应T细胞功能因子表达水平、对骨髓造血细胞的损伤及其作用的靶细胞,证实CD8+HLA-DR+效应T细胞与SAA患者骨髓衰竭之间的“因果效应”及“因果方式”,进一步阐明SAA免疫损伤骨髓造血的机制；同时采用差异蛋白质组学技术,对SAA患者与正常人髓样树突状细胞(myeloid dendritic cells,mDC)的蛋白质成份进行分析,筛选激活SAA免疫反应可能的抗原物质,即导致SAA发病可能的自身抗原,为开发靶向治疗药物提供依据。方法
研究对象为天津医科大学总医院自2010年4月至2012年4月初治SAA患者、治疗后缓解的SAA患者及正常对照者。第一部分研究效应T细胞损伤骨髓造血细胞的途径,采用免疫磁珠分选24例SAA初治患者及23名健康正常人外周血CD8+HLA-DR+效应T细胞,半定量RT-PCR检测分选细胞效应因子穿孔素、颗粒酶B、肿瘤坏死因子-p(tumor necrosis factor beta, TNF-β)、FasL mRNA表达情况。第二部分验证SAA患者效应T细胞对骨髓造血细胞的损伤作用,以免疫磁珠阳性分选12例SAA初治患者、12例SAA缓解患者及12名正常健康人CD8+HLA-DR+T细胞作为效应细胞,阴性分选去除SAA缓解患者、正常健康人骨髓单个核细胞中CD3+T细胞后作为靶细胞,将SAA初治患者效应细胞分别与SAA缓解患者及正常健康人靶细胞混合培养(分别为实验组1、实验组2),同时设SAA缓解患者效应T细胞与SAA缓解患者骨髓靶细胞混合培养组及正常健康人效应T细胞与正常健康人靶细胞混合培养组作为对照(分别为对照组1、对照组2),72h后通过流式细胞术以膜联蛋白V(Annexin V)检测靶细胞凋亡状况,全自动生化分析仪检测培养体系上清液乳酸脱氢酶(lactate dehydrogenase, LDH)水平。第三部分明确SAA患者靶向损伤的骨髓造血细胞,分别将SAA患者、正常人CD8+HLA-DR+效应T细胞与正常人的CD3-细胞(免疫磁珠分选获得)混合培养,72h后采用流式细胞术检测培养系中CD14+、CD33+、CD34+,GlycoA+细胞凋亡状况；以单克隆抗体标记SAA患者和正常人骨髓CD34+、GlycoA+、CD33+、CD14+细胞,比较其Fas蛋白的表达量。第四部分探索导致SAA免疫激活的抗原物质,采用贴壁培养法将SAA患者、缓解患者及正常人单核细胞,体外诱导、分化为成熟的mDC,流式细胞仪分选得到高纯度mDC,提取总蛋白,进行双向电泳分离蛋白质,取差异蛋白质点进行比对,明确SAA患者mDC内与正常人不同的蛋白质成分,即为激活mDC,引起SAA一系列自身免疫反应可能的抗原物质。结果
第一部分SAA初治组CD8+HLA-DR+效应T细胞穿孔素、颗粒酶B mRNA的表达量分别为(0.66±0.25)、(0.56±0.26),正常对照组表达量为(0.53±0.14)、(0.40±0.13),前者明显高于后者(P值分别为0.042、0.012)；SAA初治组CD8+HLA-D时效应T细胞FasL nRNA的表达量为(0.77±0.24),明显高于正常对照组(0.61±0.16)(P=0.011)；SAA初治组CD8+HLA-DR+效应T细胞TNF-β mRNA的表达量为(0.58±0.16),亦明显高于正常对照组(0.46±0.15)(P=0.011)。
第二部分SAA初治患者效应细胞与SAA缓解患者靶细胞混合培养组(实验组1)、SAA初治患者效应细胞与正常健康人靶细胞混合培养组(实验组2)靶细胞凋亡比例分别为(41.12±24.84)%、(45.81±20.47)%,SAA缓解患者效应细胞与SAA缓解靶细胞混合培养组(缓解组)、正常人效应细胞与正常人靶细胞混合培养组(正常组)靶细胞凋亡比例分别为(35.03±22.09)%、(20.95±13.82)%,实验组1、实验组2、缓解组三组间两两比较差异无统计学意义(P>0.05),实验组1、实验组2、缓解组均明显高于正常组(P<0.05)；实验组1、实验组2培养体系上清液LDH水平分别为(74.56±49.13)U/L、(62.61±31.76)U/L,明显高于正常组的LDH水平(28.60±8.91)U/L(P<0.05),与缓解组LDH水平(61.06±28.41)U/L比较差异无统计学意义(P>0.05),缓解组LDH水平明显高于正常组(P<0.05)。
第三部分SAA组骨髓CD34+细胞Fas蛋白的表达量为46.59±27.60%,明显高于对照组(8.89±7.28%,P<0.01)；SAA组CD14+、CD33+、GlycoA+细胞Fas蛋白的表达量分别为29.29±9.23%、46.88±14.30%、15.15±9.26%,明显低于对照组(51.25±38.36%、72.06±39.88%、50.38±39.88%,P<0.05)。SAA初治患者效应细胞与正常健康人靶细胞(实验组)CD34+、CD33+、CD14+细胞凋亡率分别为55.43±20.50%、38.13±20.10%、61.87±21.65%,均明显高于正常人效应细胞与正常人靶细胞混合培养组(对照组)35.02±13.95%、23.44±10.33%、37.04±22.41%(P均<0.05)；实验组CD45-细胞凋亡率为26.43±24.99%,与对照组22.76±7.62%无明显差异(P>0.05)。
第四部分SAA患者mDC细胞内蛋白成分与缓解后患者及正常人之间进行对比,其凋亡相关蛋白(p53、caspase3、Fas),核糖体连接蛋白、泛素化相关蛋白表达增高,与SAA免疫发病机制中mDC细胞激活相关。结论
(1)SAA患者外周血CD8+HLA-DR+效应T细胞穿孔素、颗粒酶表达水平均较正常人明显升高,提示穿孔素、颗粒酶可能在SAA患者细胞毒性T淋巴细胞(cytotoxic lymphocyte, CTL)损伤骨髓造血过程起到了重要的作用；SAA患者CD8+HLA-DR+T效应细胞FasL表达亦明显增高,可能参与了骨髓造血靶细胞的凋亡损伤,同时高表达的FasL也可能参与了辅助性T细胞(helper T cell,Th)亚群的调节,但对CTL本身可能没有作用；此外,TNF-β也是参与CTL损伤骨髓造血靶细胞的重要效应因子,在SAA患者CD8+HLA-DR+效应T细胞的表达也增高。因此,上述三种途径可能均在SAA免疫发病过程中发挥了重要作用。
(2)SAA初治患者CD8+HLA-DR+效应T细胞能够明显增加缓解患者及正常人骨髓造血靶细胞凋亡率,提示CD8+HLA-DR+效应T细胞在SAA骨髓免疫损伤中机制中居于重要地位。而缓解SAA患者CD8+HLA-DR+效应T细胞功能并未降至正常,临床应用免疫抑制治疗中应注重足量、足疗程给药,以便彻底控制患者异常免疫状态,防止复发。
(3)SAA初治患者CD8+HLA-DR+效应T细胞能够诱导正常人CD34+、CD33+、CD14+细胞凋亡增加,对正常的骨髓造血干/祖、粒系、单核系细胞均具有杀伤作用；经检测SAA患者骨髓CD34+、CD33+、CD14+、GlycoA+细胞均表达Fas抗原,并可能因此被高表达FasL的CD8+HLA-DR+效应T细胞识别,故Fas/FasL系统介导的细胞凋亡在SAA免疫发病中发挥了重要作用；而SAA患者CD34+细胞Fas表达明显增加,可能是SAA免疫损伤的主要靶细胞。
(4)SAA患者mDC中凋亡相关蛋白、核糖体连接及泛素化相关蛋白表达异常,可能与mDC的激活相关。
Objective
To investigate the level of functional factors secreted from CD8+HLA-DR+effector T cells in peripheral blood (PB) of the patients with severe aplastic anemia(SAA), the damage on hematopoietic cells by this group of cells, and which were the target cells attacked by CD8+HLA-DR+effector T cells. Thus, we investigated the function of CD8+HLA-DR+cells, which were considered to be activated CTL, in peripheral blood to further explore the pathogenesis of SAA. Also, we explored the heterogenic proteins in myeloid dentritic cells between SAA patients and normal controls, which may be the pathogenic antigen in SAA.
Methods
Untreated SAA patients, remission paitents, as well as normal controls were enrolled in this study. Part Ⅰ The CD8+HLA-DR+cells were sorted from the PB of24untreated SAA patients and23normal controls by immunomagnetic separation. The mRNA expressions of perforin, granzyme B, tumor necrosis factor-β (TNF-β) and FasL of sorted cells were analyzed by RT-PCR. Part ⅡCD8+HLA-DR+T cells (effector cells) from untreated SAA patients, remission patients and normal controls were co-cultured with bone marrow mononuclear cells (with CD3+T cells depleted) from remission patients and normal controls (target cells). The effector T cells of untreated SAA were cocultured with the target cells of remission patients and normal controls, which were designated as SAA groups1and2, respectively. There were two control groups:one was the remission SAA group (effector cells from untreated SAA patients and target cells of remission patients), and the other was the normal control (effector cells from untreated SAA patients and target cells of normal controls). Apoptosis of target cells was detected by flow cytometry after staining with AnnexinⅤ. The levels of lactate dehydrogenase (LDH) in the supernatant were determined by automatic biochemistry analyzer. Part ⅢCD8+HLA-DR+T cells from untreated SAA patients, and normal controls were co-cultured with bone marrow mononuclear cells (with CD3+T cells depleted) from normal controls. Apoptosis of CD14+、CD33+、CD34+、GlycoA+cells was detected by flow cytometry after staining with AnnexinV. Meanwhile, the expression of Fas on the CD14+、CD33+、CD34+、 GlycoA+cells was detected by flow cytometry in untreated patients and normal controls respectively. Part Ⅳ Bone marrow mononuclear cells were isolated and inoculated in cell culture flask,2hours later the suspended cells were given up. The cells adhering to the wall cultured for7days and became myeloid dentritic cells. After that, the cells were purified by flow cytometry, and extracted total protein from them. Furtherly, explore the difference of the protein among untreated SAA, remission SAA and normal controls by two-dimensinal electrophoresis.
Results
Part I The mRNA of perforin, granzyme B of CD8+HLA-DR+T cells were (0.66±0.25)、(0.56±0.26) in untreated group, higher than these of controls (0.53±0.14)、(0.40±0.13)(P=0.042、0.012). The mRNA of FasL in CD8+HLA-DR+T cells of untreated SAA patients was (0.77±0.24),higher than that of controls (0.61±0.16)(P=0.011). The mRNA of TNF-β in CD8+HLA-DR+T cells of untreated SAA patients was (0.58±0.16),also higher than that of controls (0.46±0.15)(P=0.011).
Part II The apoptosis values in SAA group1(CD8+HLA-DR+T cells from untreated SAA patients and CD3-bone marrow mononuclear cells from remission patients), SAA group2(CD8+HLA-DR+T cells from untreated SAA patients and CD3-bone marrow mononuclear cells from normal controls), the remission group (CD8+HLA-DR+T cells and CD3-bone marrow mononuclear cells from remission patients) and the normal control (CD8HLA-DR+T cells and CD3-bone marrow mononuclear cells from normal controls) were41.12±24.84%,45.81±20.47%,35.03±22.09%,20.95±13.82%. There were no significant differences between SAA group1, SAA group2, and the remission group (P>0.05). However, the apoptosis in each of these groups was higher than in the normal control (P<0.05).
The LDH levels in SAA group1, SAA group2, the remission group and normal control were74.56±49.13U/L,62.61±31.76U/L,61.06±28.41U/L, and28.60±8.91U/L. There were no significant differences between SAA group1, SAA group2, and the remission group (P>0.05). However, the level of LDH in these groups was significantly higher than in the normal control (P<0.05).
Part Ⅲ The ratios of Fas in the CD34+cells from SAA groups was46.59±27.60%, significantly higher than that of normal control (8.89%±7.28%, P<0.01). The ratio of Fas in the CD14+、CD33+、GlycoA+cells from SAA groups were29.29±9.23%、46.88±14.30%，15.15±9.26%, significantly lower than that of normal control (51.25±38.36%，72.06±39.88%、50.38±39.88%, P<0.05). The apoptosis values of CD34+、CD33+、CD14+cells in SAA group (CD8+HLA-DR+T cells from untreated SAA patients and CD3-bone marrow mononuclear cells from normal contros) were55.43±20.50%、38.13±20.10%、61.87±21.65%, significantly higher than that in the normal control (35.02±13.95%、23.44±10.33%、37.04±22.41%,P<0.05). However, there were no significant differences of CD45-cells between SAA group and normal control (P>0.05).
Part IV Our studies about intracellular proteins in mDC have demonstrated that the expression of apoptotic protein (p53,caspase3,Fas), spliceosome-related protein, and ubiquitin-related protein were higher in SAA than normal controls, which seem to related to mDC activation.
Conclusions
(1) By RT-PCR, we found that the expression of perforin, granzyme B were elevated in the CD8+HLA-DR+T cells, suggested that CD8+HLA-DR+T cells and their functional products might contribute to bone marrow failure in SAA. We also found that the expression of FasL of CD8+HLA-DR+T cells were higher in the SAA than normal controls. Thus, FasL might take part in the pathogenesis of SAA, however, it might have no effect on CD8+HLA-DR+T cells. Moreover, the expression of tumor necrosis factor-beta (TNF-β) were elevated in the CD8+HLA-DR+T cells, which might be the important functional factor in the CD8+HLA-DR+T cells of SAA patients.
(2) The CD8+HLA-DR+T cells from untreated SAA and remission patients were more cytotoxic than those cells from healthy controls. These observations suggest that the induction of apoptosis of hematopoietic cells by CD8+HLA-DR+T cells might play a critical role in the bone marrow failure of SAA. In our research, the cytotoxicity of CD8+HLA-DR+T cells was reduced after IST treatment, but it did not reach the level of the control subjects as the normal state. Thus, in order to decrease the risk of relapse, it is important for SAA patients to maintain the IST treatment for a longer period of time, and to taper cyclosporine very slowly.
(3) The apoptosis of CD34+、CD33+、CD14+cells from the normal persons increased after coculture with the CD8+HLA-DR+T cells from SAA patients, which indicated that the CD8+HLA-DR+T cells from SAA patients may lead to the excessive apoptosis of hematopoietic cells in normal controls. The expression of Fas on the CD34+、CD33+、CD14+、GlycoA+cells were all positive, which may induce the damage by CD8+HLA-DR+T cells. And the expression of Fas on the CD34+cells markly increased, which may be the main target cell in the pathogenesis of SAA.
(4) Abnomal expression of apoptotic protein, spliceosome-related protein,and ubiquitin-related protein in SAA might be involved in the activation of mDC.
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[5]Marsh JC, Ball SE, Cavenagh J, et al. Guidelines for the diagnosis and management of aplastic anaemia. Br J Haematol.2009; 147:43-70.
[6]Wassim Y, Almawi, Ohannes K, et al. Clinical and mechanistic differences between FK506(tacrolimus) and cyclosporin A. Nephrol Dial Transplant,2000,15: 1916-1918.
[7]Alsuhan A,Goldenberg NA,Kaiser N,et al.Tacrolimus as an alternative to cyclosporine in the maintenance phase of immunosuppressive therapy for severe aplastie anemia in children[J]. Pediatr Blood Cancer,2009,52(5):626-630.
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