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To analysis the specific protein markers of essential thrombocythemia (ET) based on proteomics technology, to explore and verify the differential protein related to platelet activation.Blood samples were obtained from ET patients and healthy people and a certain protein mass spectrometry was detected using label-free quantitative technology. The proteins relative abundance increased or down-regulated by 1.3 times in the disease group compared with the control group, and the protein abundance in the two groups t test P＜0.05 were defined as differential proteins. Bioinformatics analysis of the differential proteins was performed using GO and KEGG. The difference in the average protein abundance between the two groups was analyzed by t test and P＜0.05 was considered statistically significant. Differential proteins were selected for verification by parallel reaction monitoring (PRM) technology.A total of 140 differential proteins were found, of which 72 were up-regulated and 68 were down-regulated. KEGG enrichment showed that the differential protein expression was related to the platelet activation pathway. The differential proteins related to platelet activation were GPV, COL1A2, GP1bα, COL1A1 and GPVI. Among them, the expressions of GPV, GP1bα and GPVI were up-regulated, and the expressions of COL1A2 and COL1A1 were down-regulated. PRM verification of COL1A1, GP1bα, GPVI and GPV was consistent with LFP proteomics testing.Differential proteins in ET patients are related to platelet activation pathway activation.Differential proteins such as GPV, GPVI, COL1A1 and GP1bα can be used as new targets related to ET platelet activation.基于非标记定量蛋白组学对原发性血小板增多症患者血小板活化相关差异蛋白的分析.基于蛋白组学技术分析原发性血小板增多症（ET）特异性蛋白标志物，寻找与血小板活化相关特异性蛋白，并进行验证.应用非标记定量蛋白组学（LFP）检测ET患者和健康者血清蛋白质谱，选取疾病组蛋白丰浓度较对照组差异倍数上调或下调1.3倍及两组蛋白丰浓度经t检验P＜0.05的蛋白定义为差异蛋白。应用KEGG和GO分析软件对差异蛋白进行生物信息学分析。组间差异蛋白平均丰浓度的差异分析采用t检验，P＜005为差异有统计学意义。选取差异蛋白，应用平行反应监测进行验证.本研究发现差异蛋白共140个，其中72个蛋白表达上调，68个蛋白表达下调。KEGG富集显示差异蛋白表达与血小板活化通路相关，与血小板活化相关的差异蛋白是GPV、COL1A2、GP1bα、COL1A1和GPVI，其中GPV、GP1bα和GPVI表达上调，COL1A2和COL1A1表达下调。对COL1A1、GP1bα、GPVI和GPV进行PRM验证，其结果与LFP蛋白组学结果一致.ET患者差异蛋白与血小板活化通路激活相关，GPV、GPVI、COL1A1和GP1bα等差异蛋白可作为ET血小板活化相关的新靶点.

To investigate the expressions of microRNAs in peripheral blood of patients with primary immune thrombocytopenia(ITP) and its correlation with the imbalance of Th1/Th2 cells.Thirty patients with ITP (ITP group) and 15 healthy people (control group) were enrolled．Real-time polymerase chain reaction (RT-PCR) was used to detect the expressions of six miRNAs (miR-107,miR-205-5p,miR-138-5p,miR-326,miR-1827,miR-185-5p) and Th1-specific transcription factor T-bet mRNA and Th2-specific transcription factor GATA-3 mRNA in the peripheral blood of the two groups. Th1 and Th2 cells were detected by flow cytometry. The expressions of Th1-cytokines TNF-α and IFN-γ and Th2-cytokines IL-4 and IL-10 were detected by AimPlex multiple immunoassays for Flow. The expression difference of miRNAs, mRNA, Th1, Th2 cells and cytokines of the two groups were compared, and the correlations of miRNAs to mRNA, Th1, Th2 cells and cytokines were analyzed in ITP group.The expressions of miRNAs（miR-107, miR-205-5p, miR-138-5p, miR-326, miR-1827, miR-185-5p）and Th2-specific transcription factor GATA-3 mRNA of the patients in ITP group were significantly decreased (P0.05) as compared with those in control, while the expressions of Th1 cells and Th1-specific transcription factor T-bet mRNA and Th1-cytokines TNF-α were significantly increased (P0.05), also for the ratios of T-bet mRNA/GATA-3 mRNA and Th1/Th2 cells were significantly increased (P0.05). The relative expressions of miR-107, miR-205-5p, miR-138-5p in ITP patients were negatively correlated with Th2 cells (r=-0.411, r=-0.593, r=-0.403,P0.05) and the relative expression of miR-1827 was negatively correlated with TNF-α (r=-0.390).The relative expressions of the six miRNAs in peripheral blood of patients with ITP are significantly decreased, which result in the increasing ratio of T-bet mRNA/GATA-3 mRNA, then lead to the imbalance of Th1/Th2.原发性免疫性血小板减少症患者外周血microRNAs的表达及与Th1/Th2失衡的关系.探讨原发性免疫性血小板减少症（ITP）患者外周血micoRNAs的表达量与Th1/Th2失衡的关系.纳入30例ITP患者(ITP组)及15例健康人(对照组)，采用实时定量 PCR(RT-PCR)法检测2组外周血中6种miRNA（miR-107、miR-205-5p、miR-138-5p、miR-326、miR-1827、miR-185-5p）和Th1特异性转录因子T-bet、Th2特异性转录因子GATA-3的表达水平，采用流式细胞术检测Th1、Th2细胞，AimPlex流式细胞术高通量技术检测Th1细胞因子TNF-α、IFN-γ以及Th2细胞因子IL-4、IL-10含量。比较2组间miRNAs、mRNA、Th1、Th2细胞及其分泌的主要细胞因子的差异,分析ITP患者miRNAs与Th1、Th2细胞及其分泌的主要细胞因子的相关性.与对照组相比，ITP组miRNA（miR-107、miR-205-5p、miR-138-5p、miR-326、miR-1827、miR-185-5p）和Th2细胞特异性转录因子GATA-3的表达水平均显著降低(P0.05)；Th1细胞、Th1细胞特异性转录因子T-bet及Th1细胞因子TNF-α表达水平均显著升高(P0.05)，T-bet/GATA-3和Th1/Th2比值均显著升高（P0.05）。ITP患者miR-107、miR-205-5p、miR-138-5p相对表达量与Th2细胞均呈负相关（P＜0.05，r=-0.411； r=-0.593； r=-0.403）；miR-1827相对表达量与TNF-α呈负相关（r=-0.390）.ITP患者外周血6种miRNAs表达水平显著降低，导致T-bet mRNA/GATA-3 mRNA比值升高，Th1/Th2失衡.

To investigate the damaging of human umbilical vein endothelial cells (HUVEC) induced by antiplatelet integrin β3 antibodies in vitro.The serum from 36 chronic ITP patients were collected, flow cytometry and monoclonal antibody specific immobilization of platelet antigen (MAIPA) assay were used to collect antiplatelet integrin β3 antibodies from the serum of the patients. After HUVEC were treated by ITP patient serum (PS) containing anti-integrin β3 antibodies, the cell damage was detected by Lactate dehydrogenase (LDH) assay, cell apoptosis was detected by flow cytometry, the expression of apoptosis-related gene Bax was detected by Reverse transcription-Quantitative real-time PCR (RT-qPCR), and expression of Apoptosis-related signaling pathway protein Akt and related protein Bax were detected by Western blot. HUVEC were treated by PS combined with Akt activator SC79, the cells damage were detected by LDH assay, apoptosis of the cells were detected by flow cytometry, the expression of apoptosis-related gene Bax was detected by RT-qPCR.Among 36 cases of serum from the chronic ITP patients, 5 patients' serum containing anti-integrin β3 antibodies were collected. After HUVEC was treated by PS, the viability of LDH was significant increased（P0.05）, so as for the apoptosis of the cells（P0.05）, the expression of gene and protein of Bax was increased up-regulated（P0.05）, the protein expression of pAkt was down-regulated（P0.05）. Comparing with HUVEC cultured with PS alone, the viability of LDH of HUVEC treated by PS combined with SC79 was significantly reduced（P0.05）, so as for the apoptosis of the cells（P0.05）, and gene expression of Bax was significantly decreased（P0.05）.Anti-integrin β3 serum can cause the damage and apoptosis of HUVEC through Akt signaling pathway，the apoptotic effects of anti-integrin β3 antibodies to HUVEC was effectively reversed by SC79.抗血小板整合素β3抗体诱导内皮细胞凋亡机制的研究.探讨抗血小板整合素β3抗体对人脐静脉血管内皮细胞（HUVEC）的损伤及其相关机制.收集36例慢性免疫性血小板减少症患者的血清，通过流式细胞术和单克隆抗体特异性俘获血小板抗原技术（MAIPA）筛选出含抗整合素β3抗体的患者血清。用抗整合素β3血清处理HUVEC后，乳酸脱氢酶(LDH)活性测定法检测HUVEC损伤情况，流式细胞术检测HUVEC凋亡情况，逆转录实时荧光定量PCR(RT-qPCR)检测HUVEC凋亡相关基因Bax表达情况，Western blot检测细胞凋亡相关信号通路蛋白Akt和相关蛋白Bax的表达情况。用抗整合素β3血清结合Akt激活剂SC79处理HUVEC后，LDH活性测定法检测HUVEC损伤情况，流式细胞术检测HUVEC的凋亡情况，RT-qPCR检测HUVEC凋亡相关基因Bax的表达情况.从36份患者血清中筛选出5份含抗整合素β3抗体血清，用这些血清处理HUVEC后，细胞外LDH活性增加（P0.05），细胞凋亡比例增加（P0.05），Bax蛋白和mRNA表达水平上调（P0.05），pAkt蛋白表达下调（P0.05）。抗整合素β3血清结合SC79处理HUVEC后，与单独使用抗整合素β3血清处理组相比，细胞外LDH活性显著降低（P0.05），HUVEC凋亡比例显著下降（P0.05），Bax基因表达下降（P0.05）.抗整合素β3抗体在体外能够引起HUVEC的损伤和凋亡，其作用机制可能与抑制Akt信号通路有关，且Akt激活剂SC79能够在体外抑制抗整合素β3抗体引起的HUVEC损伤和凋亡.

Single cell sequencing technology is different from traditional sequencing method, which is based on population cell average level. It has been widely used in many fields and made great progress in the application of malignant hematological diseases. In this review, the principle, methodology and application of single cell sequencing technology in malignant hematological diseases are summarized briefly, including the study of the pathogenesis in myelodysplastic syndrome, the mechanism of transformation into leukemia, accurate diagnosis and classification, differential diagnosis, evaluation of targeted drug therapeutic efficacy and exploration of biomarkers; specific diagnostic indicators for myeloproliferative diseases, progression of disease monitoring and epidemiological studies; moreover, the pathogenesis and drug resistance of acute myeloid leukemia (AML), which can provide reference for the diagnosis and research of malignant hematological diseases.单细胞测序技术在恶性血液病诊疗中的应用.摘要 单细胞测序技术与基于群体细胞平均水平的传统测序方法不同，已经在多个领域得到广泛应用，在恶性血液病中的应用也取得很大进展。本文对单细胞测序技术的原理、方法学以及在多种恶性血液病的应用进行综述，涉及到对骨髓增生异常综合征发病机制、转白机制、精确诊断与分型、鉴别诊断、靶向药疗效评价、生物标志物的探索；以及骨髓增生性疾病的特异性诊断指标的确立、疾病进展监测、流行病学相关研究；还有急性髓系白血病的发病机制以及耐药机制研究，为恶性血液病的诊断和研究提供参考.

To analyze the consistency of gene mutation sites between bone marrow DNA (BM-tDNA) and perepheral plasma circulating tumor DNA (PP-ctDNA) in patients with myelodysplastic syndrome (MDS).The simultaneous sampled BM and PP from 19 patients (SBPP) was detected by NGS-127 gene panel, and the consistency of VAF between BM-tDNA and PP-ctDNA was analyzed. The peripheral blood cell tumor DNA (PC-tDNA) of 5 out of 19 patients was detected randomly, the consistency of VAF among PC-tDNA，BM-tDNA and PP-ctDNA was analyzed. The non simultaneous sampled BM and PP from 13 patients (NBPP) was detected, and the difference value of VAF between BM-tDNA and PP-ctDNA in SBPP and NBPP was analyzed.The average concentration of PP-ctDNA in SBPP was 0.59 ng/µl and 0.604 ng/µl in NBPP. The median concentration of PP-ctDNA in SBPP and NBPP was 0.330 ng/µl and 0.338 ng/µl, respectively. The study showed a good consistency of VAF between BM-tDNA and PP-ctDNA in the SBPP (RPP can replace BM as a biological sample for genes mutation detection in patients with MDS due to its stable concentration, high degree of consistency with bone marrow in clinical significant mutation sites and easy collection.

To investigate the role of regulatory B cells (Breg) in pathogenesis of immune thrombocytopenia(ITP) and its clinical significance.A total of 40 ITP patients and 20 normal controls were enrolled in this study. The content of Breg, Th1, Th2, Th17 and Treg cells were detected by flow cytometry (FCM). The expression level of IL-10,TGF-β, CD40 and CD40L was detected by AimPlex Flow High Throughput Screening Technology.The of Breg cells in ITP patients was significantly lower than that in normal controls (P0.05)，the expression levels of IL-10,TGF-β and CD40L in ITP patients were also significantly lower than those in normal controls (P0.05). The contents of Th1 cells in ITP patients were significantly higher than that in normal controls (P0.05), whereas the contents of Th2, Th17 and Treg cells in ITP patients were significantly lower than those in normal controls (P0.05).The Breg cells may play an important role in the pathogenesis of ITP.

To analyze the imbalance of pro-inflammatory and anti-inflammatory cytokines in the patients of immune thrombocytopenia (ITP).Thirty-five patients with ITP were enrolled in ITP group, while 28 healthy persons were included in control group. The expressions of IL-8, IL-17A, IL-22, TNF-α, IFN-γ, IL-4, CD40, CD40L, TGF-β and IL-10 were detected by flow cytometry with aimPlex multiple immunoassay Flow.The expressions of pro-inflammatory factors IL-8, IL-17A, IL-22, IFN-γ and TNF-α in ITP group all were significantly higher than that in the control group (P0.05), however the expressions of anti-inflammatory factors IL-4, CD40L, TGF-β and IL-10 in ITP group all were significantly lower than that in the control group (P0.05). The expression of CD40 was not significantly different between ITP group and control group (P0.05). Expressions of TNF-α significantly related with platelet counts in both ITP group and control group (ITP group, r=0.64, P0.05; control group, r=-0.41, P0.05). However the expression of CD40, TGF-β, CD40L, IL-8, IL-17A, IL-22, IL-10, IL-1β, IFN-γ and IL-4 significantly did not relate with platelet counts in both ITP and control group.The secretory imbalance between pro-inflammatory and anti-inflammatory cytokines exists in the patients of ITP. The decrease of Plt regulated may be regulated by the abnormal expression of TNF-α.

In recent years, significant progresses have been got in study on pathogenesis, treatment and prognosis of myelodysplastic syndromes (MDS), especially on use of new technology, that has great importance for cytogenetics of MDS. Recently, the progress of cytogenetic detection in MDS is very remarkable. Based on the metaphase cytogenetics (MC) method, prognostic significance of cytogenetics in MDS was clarified gradually. For example, people have known the prognostic significance of 12 p-, 11 q-, +21, t(11(q23)), although these genetic abnormalities are rare in the MDS. In addition, chromosome mutation emerged in the process of MDS may indicate the poor prognosis. On the other hand, with the use of SNP-A and aCGH in the study of genetics, MDS cytogenetic abnormality detection rate has been further improved and can reach to 78%. At the same time, some of MDS patients with the "normal karyotype" detected by MC have new hidden aberrations through the SNP or CGH detection, and these patients have a poorer prognosis. In this review, the advances of study on cytogenetic detection for MDS based on MC and SNP-A or aCGH methods are summarized.

The aim was to investigate the immunophenotypes of NK series leukemia. Immunophenotypes of 297 cases of acute leukemia (AL) were measured by flow cytometry, and these immucopenotypic features were analyzed. The results showed that 43 out of 297 cases of AL (14.5%) were CD56 positive. 6 cases were NK series leukemia and 37 cases were acute myelogenous leukemia with CD56 expressed. One patient has been diagnosed as myeloid/NK cell precursor acute leukemia, two patients were blastic NK cell leukemia, one was supposed to be NK-like T-cell lymphoma/leukemia, while another one was large granular lymphocyte leukemia (LGLL). It is concluded that almost all of CD56 positive leukemia were acute myelogenous leukemia with CD56 expressed. The immunophenotypes of NK series leukemia were antigens from hematopoietic stem cells to T/NK progenitor cells with meyloid antigen positive, and through NK progenitors to mature NK cells. The immunophenotypes of heterogeneous NK leukemia cells are different, that should be carefully distinguished.