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Virtual reality therapy (VRT) is a new psychotherapeutic approach integrating virtual reality technology and psychotherapy. This case series aimed to study effectiveness of VRT in treating psychological problems. We described four cases of first-line health care professionals with emerging clinically significant early psychological problems during the COVID-19 outbreak, and specifically received the VRT treatment. We compared the Patient Health Questionnaire 9 items (PHQ-9), Generalized Anxiety Disorder-7 (GAD-7), PHQ-15, and Athens Insomnia Scale to evaluate psychological symptoms and sleep quality before and after sessions. All four cases showed a reduction in scale comparison. General scores of the PHQ-9 reduced 65%, GAD-7 reduced 52.17%, PHQ-15 decreased 38.17%, and scores of the Athens Insomnia Scale reduced 67.44%. Meanwhile, a reduction in depression, anxiety, psychosomatic, and sleeping symptoms was also found, which decreased 76.92% in general. These results are highly significant statistically. This case series demonstrated the effectiveness of VRT on psychological problems as a promising approach to apply on various psychological distress and disorders.

Network Meta-analysis was conducted to compare the efficacy and safety of different Chinese medicine injections combined with conventional therapy in the treatment of stroke-associated pneumonia. CNKI, Wanfang, VIP, PubMed, Web of Science, and Cochrane Library were searched for the relevant literature pubslished from inception to April 1, 2022. Stata 17 was used for data analysis. After screening of 1 189 papers, 72 studies were finally selected, which involved 5 819 patients and 6 Chinese medicine injections(Tanreqing Injection, Xingnaojing Injection, Xuebijing Injection, Xiyanping Injection, Shenfu Injection, and Shenmai Injection). The network Meta-analysis ranked the injections as follows.(1) In terms of improving the total clinical effective rate, the surface under the cumulative ranking curve(SUCRA) followed the order of Xiyanping Injection + conventional therapygt; Xuebijing Injection + conventional therapygt; Tanreqing Injection + conventional therapygt; Shenmai Injection + conventional therapygt; Xingnaojing Injection + conventional therapygt; Shenfu Injection + conventional therapygt; conventional therapy.(2) In terms of recovering the National Institute of Health stroke scale(NIHSS) scores, the SUCRA followed the order of Xuebijing Injection + conventional therapygt; Xingnaojing Injection + conventional therapygt; Tanreqing Injection + conventional therapygt; Shenfu Injection + conventional therapygt; conventional therapy.(3) In reducing the average time to abatement of fever, the SUCRA followed the order of Xiyanping Injection + conventional therapygt; Tanreqing Injection + conventional therapygt; Xuebijing Injection + conventional therapygt; conventional therapy.(4) In terms of reducing the mean hospital stay, the SUCRA followed the order of Xiyanping Injection + conventional therapygt; Xubijing Injection + conventional therapygt; Tanreqing Injection + conventional therapygt; Shenmai Injection + conventional therapygt; conventional therapy. The clinical efficacy of Tanreqing Injection, Xuebijing Injection, Xiyanping Injection, Xingnaojing Injection, Shenmai Injection, or Shenfu Injection combined with conventional therapy was superior to that of conventional therapy alone. However, due to the limitations of the quality and methodology of different intervention measures, this conclusion needs to be verified by more high-quality and rigorously designed randomized controlled trial.

Background: It was designed to investigate whether umbilical cord matrix stem cells (CVMSC) could affect the functionality of trophoblasts to prevent preeclampsia. Methods: In this study, CVMSC-derived exosomes were isolated; its effect on trophoblasts was investigated based on the CCK8 assay, migration assay, and apoptosis detection. And the underlying mechanism of this effect was investigated using mRNA sequencing, western blot, co-immunoprecipitation, luciferase report assay, and ubiquitination assay. Findings: The results showed that CVMSC-derived exosomes promoted migration and proliferation of trophoblasts, and also reduced cell apoptosis. mRNA sequencing confirmed that after treatment of CVMSC-derived exosomes, TRIM72 expression was upregulated and P53 expression was downregulated, both significantly in trophoblasts. Subsequent study confirmed that TRM72 could directly interact with P53 and promoted P53 ubiquitination and proteasomal degradation, reducing apoptosis rate and elevating proliferation and migration in trophoblasts. Interpretation: Our study confirmed that CVMSC-derived exosomes promoted trophoblast migration and proliferation by upregulating TRIM72 expression, and subsequently advanced P53 ubiquitination and proteasomal degradation. Funding Statement: This study received no specific grant from public funding agencies, commercial, or not-for-profit sectors. Declaration of Interests: All authors declare there is no competing interest. Ethics Approval Statement: This study was approved by the Ethics Committee of the Affiliated Hospital of Qingdao University.

Subsequent microvascular decompression (MVD) might be affected by the previous two-isocentre gamma knife radiosurgery (GKS) due to the tissue changes caused by its higher dose radiation and larger treatment volume. This study aimed to evaluate the safety and efficacy of MVD as a second step treatment after two-isocentre GKS.Between December 2016 and May 2019, data from 19 consecutive trigeminal neuralgia (TN) patients who experienced MVD after failed two-isocentre GKS were collected. The clinical characteristics, intraoperative findings, surgical outcomes and complications were reviewed and compared with 158 patients who underwent MVD as an initial treatment.Fifteen patients (78.9%) achieved complete pain relief (Barrow Neurological Institute, BNI class I) immediately after surgery and nine patients (47.4%) maintained complete pain relief at the last follow-up, which was similar to patients who underwent initial MVD. The median follow-up period was 36 months. The incidence of new or worsened facial numbness showed no statistical significance between the groups. During surgery, trigeminal nerve atrophy was noted in 9 patients (47.4%), thickened arachnoid in 3 patients (15.8%), atherosclerotic plaque in 3 patients (15.8%) and neurovascular adhesion in 1 patient (5.3%).MVD remains an effective and safe rescue therapy for patients who elect the minimally invasive treatment with two-isocentre GKS for the first time, without an increased risk of facial numbness.

To analyze the polymorphism of the HPA1-5,15 system of the donors in Zhangjiakou area.DNA was extracted from the blood samples of the donors, PCR- SSP method was used to divide HPA1-6, 15 genotype. The gene frequency and genotype frequency were calculated, compared with the difference and regiahal specificity of the populations in our country and foregiens was compared other populations.The gene expression in the HPA-1, HPA-2 and HPA-4 systems were all homozygous aa, and the donors who expressed homozygous bb was not exessed. Among them, one heterozygous ab expression was found in both HPA-1 and HPA-4 systems (1%), and 14 cases of heterozygous ab expression were found in HPA-2 system (14%). The gene expression in the HPA-5 system was mainly homozygous aa (98%), and a very few expressed homozygous bb (2%) was found. The degree of heterozygosity of gene expression in the HPA-3 and HPA-15 systems was relatively high. The proprotion of the expression of aa, ab and bb in the HPA-3 system was respectively 46%, 40% and 14%, the proprotion of the expression of aa, ab and bb in the HPA-15 system was respectively 21%, 64% and 15%.The gene frequency of platelet-specific antigen HPA1-5,15 system in zhangjiakou region shows local characteristics. The heterozygosity degree of gene expression in the HPA-3 and HPA-15 systems are both high, suggesting that they are more likely to result in alloimmunization and ineffective platelet transfusion, which should be pays attention to.张家口地区机采血小板捐献者HPA分型研究.分析河北省张家口地区血小板捐献者血小板特异性抗原HPA1-5，15系统的基因多态性.采集血小板捐献者静脉血并提取DNA，采用聚合酶链-序列特异性引物（PCR-SSP）法进行血小板特异性抗原分型，计算基因频率、基因型频率并与国内外其他地区进行比较是否存在差异及是否表现地区特异性.HPA-1、HPA-2、HPA-4系统中基因表达均为纯合子aa，未出现表达纯合子bb的存在者，其中HPA-1和HPA-4系统各发现杂合子ab表达1例（1%），HPA-2系统发现杂合子ab表达14例（14%）； HPA-5系统基因表达主要为纯合子aa （98%），极少数表达纯合子bb（2%）； HPA-3和HPA-15系统基因表达杂合程度较高，HPA-3系统基因表达aa、ab、bb表达比例分别为46%、40%、14%，HPA-15系统基因表达aa、ab、bb表达比例分别为21%、64%、15.张家口地区血小板特异性抗原HPA1-5,15系统基因频率具有本地区特点； HPA-3和HPA-15系统基因表达杂合程度较高，引发同种免疫及血小板输注无效的可能性较大，因此应引起注意.

To investigate the expression of IL-9 and IL-6 in patients with BCR-ABL- bone marrow proli- ferative tumor (MPN), and to explore its role in the occurrence and development of MPN.A total of 71 newly diagnosis MPN patients treated in Tianjin Medical University General Hospital from 2018 to 2019 were selected, including 32 patients with polycythemia vera (PV) and 22 patients with primary thrombocytosis (ET), and 17 patients with primary myelofibrosis (PMF). Then 58 patients who retestine after treatment were selected as therapy group，and 20 healthy volunteers were recruited as control group. ELISA was used to detect the expression level of IL-6 and IL-9 in bone marrow supernatant, and the relative expression level of IL-6 and IL-9 mRNA in BMMNC was detected by real-time PCR. The proportion of Th9 cells in peripheral blood were detected by flow cytometry (FCM). The expression level of IL-6 mRNA and IL-9 mRNA of BMMNC and clinical indicators were analyzed, and the correlation between JAK2 gene mutation load and IL-9 level was further analyzed.The level of IL-6 in bone marrow supernatant and the expression of IL-6 mRNA in BMMNC were higher in the newly diagnosed group as compared with those in the treated group and the control group (P0.001). The expression level of IL-9 in bone marrow supernatant and the expression of IL-9 mRNA in BMMNC were lower in the newly diagnosed group as compared with those in the treated group and the control group (P0.05). The proportion of Th9 cells in peripheral blood was lower in the newly diagnosed group as compared with that in the treated group and the control group (P0.001). The level of IL-6 in bone marrow supernatant and the expression of IL-6 mRNA in BMMNC in JAK2The increased expression of IL-6 in MPN and hyperfunction may promote the progression of BCR-ABL- MPN disease. The expression of IL-9 in MPN decreases, and it negatively correlates with the mutation load of JAK2 gene, which may be related with the decrease of tumor environmental antitumor immune effect.IL-9和IL-6在BCR-ABL- MPN患者中的表达及其意义.检测BCR-ABL- 骨髓增殖性肿瘤（MPN）患者的IL-9和IL-6的表达，探讨其在MPN疾病中的意义.选取2018年至2019年间于天津医科大学总医院血液科就诊的MPN初治患者71例（初诊组），包括32例真性红细胞增多症（PV），22例原发性血小板增多症（ET）和17例原发性骨髓纤维化（PMF），治疗后复查患者58例作为治疗组，同时选取健康志愿者20名作为对照组。采用ELISA法检测骨髓上清液中IL-6和IL-9的水平，采用RT-PCR技术检测骨髓单个核细胞IL-6和IL-9 mRNA相对表达水平，应用流式细胞术(FCM)检测外周血Th9细胞的比例，将骨髓单个核细胞IL-6 mRNA和IL-9 mRNA表达水平与临床指标进行相关性分析，并进一步追踪分析JAK2基因突变负荷与IL-9水平之间的关系.初诊组骨髓上清液中IL-6水平及骨髓单个核细胞中IL-6 mRNA表达水平均高于治疗组和对照组（P0.001），初诊组骨髓上清液中IL-9水平及骨髓单个核细胞中IL-9 mRNA表达水平均低于治疗组和对照组（P0.05）；初诊组外周血Th9细胞比例低于治疗组和对照组（P0.001）；JAK2IL-6在MPN中表达水平增加，功能亢进，可能促进BCR-ABL- MPN疾病进展；IL-9在MPN中表达水平减低，且与JAK2基因突变负荷呈负相关关系，这可能与肿瘤微环境抗肿瘤免疫作用减低有关.

BackgroundMitochondrial Lon is a chaperone and DNA-binding protein that functions in protein quality control and stress response pathways. The level of Lon regulates mitochondrial DNA (mtDNA) metabolism and the production of mitochondrial reactive oxygen species (ROS). However, there is little information in detail on how mitochondrial Lon regulates ROS-dependent cancer immunoescape through mtDNA metabolism in the tumor microenvironment (TME).MethodsWe explored the understanding of the intricate interplay between mitochondria and the innate immune response in the inflammatory TME.ResultsWe found that oxidized mtDNA is released into the cytosol when Lon is overexpressed and then it induces interferon (IFN) signaling via cGAS-STING-TBK1, which upregulates PD-L1 and IDO-1 expression to inhibit T-cell activation. Unexpectedly, upregulation of Lon also induces the secretion of extracellular vehicles (EVs), which carry mtDNA and PD-L1. Lon-induced EVs further induce the production of IFN and IL-6 from macrophages, which attenuates T-cell immunity in the TME.ConclusionsThe levels of mtDNA and PD-L1 in EVs in patients with oral cancer function as a potential diagnostic biomarker for anti-PD-L1 immunotherapy. Our studies provide an insight into the immunosuppression on mitochondrial stress and suggest a therapeutic synergy between anti-inflammation therapy and immunotherapy in cancer.

JAK2, MPL and CALR gene mutations play an important role in the onset of myeloproliferative disease(MPD). The latest researches show that the difference of ATP binding ability between the wild type JAK2 protein and mutated JAK2 protein can help us understand the pathogenesis of the MPD further, and the clinical manifestation is related to the mutation burden of the JAK2. In some ET and PMF patients, research find the expression of MPL mutation, which can affects the progress of the disease by collaborating with the JAK2 mutation. CALR mutation is a gene related with the MPD that has been found recently. The pathogenesis of the CALR is similar to that of the JAK2, while there are some features in clinical manifestation comparing with the other mutations.

To evaluate the efficacy and safety of high-dose intravenous mecobalamin (HDIME) for the treatment of bortezomib-induced peripheral neuropathy(BIPN) in the patients with multiple myeloma (MM).A total of 65 newly diagonsed patients with multiple myeloma receiving bortezomib in Tianjin Medical University General Hospital were enrolled in this single-centre randomized clinical trial from July 2012 to May 2016. Out of 65 patients 38 in control group received bortezomib-based chemotherapy and 27 patients in HDIME group received the additional high-dose intravenous mecobalamin.The incidence of BIPN in HDIME group was lower than that in control group(29.63% vs 55.26%, χHDIME has a good efficacy for the prophylaxis BIPN and and without serious side effects.

To investigate the expression pattern of HOXA9 in myelodysplastic syndrome (MDS) patients and its relation with clinical characteristics and treatment response.The mRNA and protein expression levels of HOXA9 in bone marrow cells from 33 cases of MDS, 12 cases of AML, 20 cases of ITP and 18 normal controls were detected by real-time guautitative PCR(RT-PCR) and flow cytometry, respectively.The percentage of HOXA9(+)/CD34(+) and HOXA9(+)/CD34(+)CD38(-) in MDS patients were significantly higher than that in control group (P0.05), and the mRNA and protein expression of HOXA9 in MDS patients had a similar trend. The percentages of HOXA9(+)/CD34(+) and HOXA9(+)/CD34(+)CD38(-) before decitabine treatment were (50.64±27.59)% and (55.67±28.57)% respectively, which were both higher than those in control group (P0.05). After decitabine treatment, expression of HOXA9 significantly decreased (P0.05).HOXA9 is overexpressed in MDS patients and associated with several clinical characteristics. The detection of HOXA9 expression may have guide roles for diagnosis and treatment of MDS patients.

To explore the effect of costimulatory signaling molecules (CD80, CD86) expression on the quantity and function of B lymphocytes in peripheral blood (PB) of the patients with immune thrombocytopenia (ITP).A total of 55 ITP patients (30 cases were newly diagnosed and 25 cases were in remission), 25 healthy volunteers as controls were enrolled in this study. The levels of CD19(+)CD5(+), CD19(+)CD80(+), CD19(+)CD86(+), CD41a(+)IgG, CD41a(+)IgM and IgG, IgM in CD19(+)B cells were measured by flow cytometry (FCM). The correlation of CD19(+)B cells with clinical parameters of ITP patients was analyzed.The level of B1 (CD19(+)CD5(+)) of newly diagnosed ITP patients was significantly higher than that of remitted ITP patients and controls (P0.05). The level of CD19(+)CD80(+) of newly diagnosed ITP patients was significantly higher than that of remitted ITP patients and controls (P0.05). And the expression of IgG and IgM in CD19(+)B cells of newly diagnosed ITP patients was significantly higher than that of remitted ITP patients and controls (P0.05). The levels of IgG and IgM in remitted ITP patients after treatment were significantly lower than that before treatment (P0.05). The level differences of IgG and IgM before and after treatment in refractory ITP patients were not statistically significant (P0.05). The expression of CD19(+)CD80(+) in newly diagnosed ITP patients positively correlated with the level of Th1 and Th1/Th2 (r=0.502, r=0.471, P0.05). The expression of CD19(+)CD80(+) of newly diagnosed ITP patients positively correlated with the level of IgG and IgM in CD19(+)B cells (r=0.552, r=0.467, P0.05), and negatively correlated with PB platelet count (r= -0.424, P0.05). The levels of IgG and IgM in CD19(+)B cells of newly diagnosed ITP patients negati- vely correlated with PB platelet count (r=-0.658, r=-0.526, P0.05).The enhacement of costimulatory signaling pathway of CD19(+)B cells in ITP patients results in the abnormal activation of B lymphocytes, thereby mediates the dysfunction of immune system and involves in the pathogenesis of ITP.

This study was aimed to investigate the expression of microRNA-21 and its correlation with PTEN in diffuse large B cell lymphoma (DLBCL) paraffin-embedded tissues, and evaluate its potential relevance with clinical characteristics. The expression levels of miR-21 in 26 primary DLBCL and 10 normal lymph node tissue specimens were examined by real-time polymerase chain reaction. The expression of PTEN was detected by immunohistochemical staining. The results indicated that the expression of miR-21 was significantly higher in tumor tissues [6.586(1.10,38.22)] than that in normal tissues [0.791 (0.35,2.87)] (P0.05). Among 26 patients with DLBCL the expression of PTEN protein was positive in 6 patients (23%), and was negative in 20 patients (77%). In patients with DLBCL, the expression level of miR-21 was negatively correlated with the level of PTEN protein. The high expression of miR-21 was positively correlated with the level of serum LDH. The expression level of miR-21 in patients with Ann Arbor III-IV stage was obviously higher than that of patients with Ann Arbor I-II stage, but did not correlate with the subtype of patients in clinic (P0.05). It is concluded that the expression of miR-21 is high in DLBCL and its overexpression may be related with poor prognosis of DLCBL. These findings suggest that PTEN is possibly one of the targets of miR-21 in DLBCL.

To explore the changes in telomere length and gene expression of complex shelterin (composed of 6 core components: TRF1, TRF2, POT1, TIN2, TPP1 and RAP1) in severe aplastic anemia (SAA).Bone marrow samples were obtained from 20 SAA patients and 10 normal controls. CD3(+)T cells were sorted by immunomagnetic separation. Telomere length was tested by Southern blot and the gene expressions of TRF1, TRF2, POT1, TIN2, TPP1 and RAP1 were detected by reverse transcription-PCR(RT-PCR).Telomeres of CD3(+)T cells were found significantly shorter in SAA untreated ((4.4 ± 1.1) kb, n = 9) and recovering groups((5.8 ± 1.0) kb, n = 11) than control group ((9.2 ± 3.3) kb, P0.05). Telomere length of CD3(+)T cells shortened with TH/S decreasing (r = 0.564, P = 0.029). The mRNA expression of POT1 decreased in untreated SAA patients (0.16(0.02-0.29)) and over-expressed in recovering patients (1.17(0.82-1.86), P0.05). The mRNA expression of RAP1 was significantly higher in untreated patients (4.14 (1.93-6.92)) than that in recovering group (0.87 (0.30-1.73) ) and controls (0.62 (0.45-4.07) , both P0.05).Changes in telomere length and shelterin gene expression occur in CD3(+)T cells of SAA patients and may be correlated with disease severity.

To study the quantity and function of bone marrow (BM) T follicular helper (Tfh) cells of the cytopenia patients with positive bone marrow mononuclear cells (BMMNC)- Coombs test (also known as immuno-related pancytopenia, IRP), and explore the role of Tfh cells in the pathogenesis of IRP.Forty- three untreated IRP patients, 47 recovered IRP patients and 25 healthy donors were enrolled in this study. The percentages of Tfh cells, Tfh-related molecules ICOS, CD40L, IL-21 and Bcl-6 in BM were investigated by flow cytometry and semiquantitive RT-PCR.The ratio of CD4⁺CXCR5⁺/CD4⁺ cells of untreated IRP patients [(28.79 ± 19.70)%] was significantly higher than that of recovered IRP patients [(21.15 ± 12.81)% ] and normal controls ([ 13.42 ± 6.72)% ](P0.05). The ratio of CD4⁺CXCR5⁺ICOS⁺/CD4⁺CXCR5⁺ cells of untreated IRP patients [(5.05 ± 4.71)% ] was significantly higher than that of recovered IRP patients [(2.96 ± 2.89)% ] and normal controls [(2.99 ± 2.23)% ] (P0.05). The ratio of CD4⁺CXCR5⁺CD40L⁺/CD4⁺CXCR5⁺ cells of untreated IRP patients [(5.87 ± 4.14)%] and recovered IRP patients [(6.52±5.47)%] were significantly higher than that of normal controls [(2.93 ± 2.92)%] (P0.05). The ratio of intracytoplasmic CD4⁺CXCR5⁺IL-21⁺/CD4⁺CXCR5⁺ cells of untreated IRP patients [(8.20 ± 7.41)% ] and recovered IRP patients [(6.30 ± 6.03)% ] were significantly higher than that of normal controls [(3.43 ± 3.40)%] (P0.05). The relative expressions of Bcl-6 mRNA in BMMNC were 0.625 ± 0.248, 0.485 ± 0.253, 0.306 ± 0.210 in three groups, respectively. The differences between untreated IRP patients, recovered IRP patients and normal controls were significant (P0.05).There exists increased quantity and hyperfunction of Tfh cells in the IRP patients, they may play important role in the pathogenesis of IRP. Tfh cells and their related effector molecules could be a potential therapeutic target for the disease.

To investigate the mechanisms underlying bone marrow damage by iron overload in pancytopenic patients with positive BMMNC-Coombs test (IRP).Twenty-one iron overloading, 26 non-iron overloading IRP patients and 10 normal controls were enrolled in this study. The expressions of ROS, Bcl-2, Caspase-3 and apoptosis of BMMNC were analyzed by flow cytometry (FCM). Antioxidants were added to iron overloading IRP BMMNC, and then the changes of indices above were detected by FCM. The number and apoptosis of T lymphocytes of IRP patients were also detected.ROS and apoptosis of BMMNC, myelocytes, erythrocytes and stem cells of iron overloading IRP patients were significantly higher than that of non-iron overloading IRP ones and normal controls (P0.05). The expressions of Bcl-2 on BMMNC, erythrocytes and stem cells of iron overloading IRP patients were significantly lower than those of non-iron overloading IRP ones (P0.05). The levels of Caspase-3 on myelocytes, erythrocytes and stem cells of iron overloading IRP patients were significantly higher than those of non-iron overloading IRP ones and normal controls (P0.05). After treatment with antioxidants, the expressions of ROS, Caspase-3 and apoptosis of iron overloading IRP BMMNC significantly decreased, but opposite for Bcl-2. The percentages of CD4(+) lymphocytes [ ( 40.86 ± 8.74）%] and CD4(+)/CD8(+) (1.44 ± 0.36) in PB of iron overloading IRP patients were significantly higher than that of non-iron overloading IRP ones [(35.96 ± 7.03)% and 1.14 ± 0.37] and normal controls [(28.00 ± 6.73)% and 0.79 ± 0.21], respectively (P0.05), as opposite for CD8(+) lymphocytes (P0.05). The apoptosis of CD8(+) lymphocytes [(27.35 ± 10.76)%] and the ratio of CD8(+) apoptosis/CD4(+) apoptosis (2.51 ± 0.81) in BM of iron overloading IRP patients were significantly higher than those of non-iron overloading IRP ones [(15.47 ± 8.99)%] and normal controls (1.39 ± 0.47), respectively (P0.05). The apoptosis of erythrocytes and stem cells coated with auto-antibodies in BM of iron overloading IRP patients were significantly higher than those of non-iron overloading IRP and normal controls.Mechanisms underlying bone marrow damage by iron overload might be through the follows: ①The increased ROS induced by excessive iron deposition affected the expressions of Caspase-3 and Bcl-2, which caused more BMMNC apoptosis; ②The abnormal number and ratio of T lymphocytes caused by iron overload aggravated the abnormality of immunity of IRP; ③Iron overload may increase the damage to erythrocytes and stem cells coated with auto-antibodies.目的 探究铁过载对骨髓单个核细胞（BMMNC）Coombs试验阳性全血细胞减少症（又称免疫相关性全血细胞减少症，IRP）患者骨髓造血损伤的机制。方法 采用流式细胞术检测21例铁过载IRP患者、26例非铁过载IRP患者及10名正常对照者骨髓造血细胞活性氧自由基（ROS）、Bcl-2、Caspase-3水平及凋亡情况；并检测铁过载组BMMNC经N-乙酰半胱氨酸处理后上述指标的变化；同时检测IRP患者T细胞数量、比例及凋亡率。结果 铁过载组BMMNC、粒系、红系和干细胞ROS水平及凋亡率均明显高于非铁过载组及正常对照组（P0.05）；铁过载组BMMNC、红系和干细胞Bcl-2水平均明显低于非铁过载组（P0.05）；铁过载组粒系、红系和干细胞Caspase-3水平均明显高于非铁过载组及正常对照组（P0.05）。铁过载组经N-乙酰半胱氨酸处理后，BMMNC ROS、Caspase-3水平及凋亡率均较前明显下降，Bcl-2水平较前明显升高。铁过载组外周血CD4+细胞、CD4+/CD8+细胞比值分别为（40.86±8.74）%及1.44±0.36，均明显高于非铁过载组[（35.96±7.03）%及1.14±0.37]和正常对照组[（28.00±6.73）%及0.79±0.21]（P0.05），铁过载组CD8+细胞百分率明显低于非铁过载组和正常对照组（P0.05）。铁过载组骨髓CD8+细胞凋亡率、CD8+细胞凋亡率/CD4+细胞凋亡率比值分别为（27.35±10.76）%及2.52±0.81，均明显高于非铁过载组[（15.47±8.99）%及1.39±0.47]（P0.05）。在骨髓有核红细胞膜抗体或干细胞膜抗体阳性IRP患者中，铁过载组骨髓红系或干细胞凋亡率均高于非铁过载组（P0.05）。结论 铁过载可能通过以下机制影响IRP患者骨髓造血功能：①过量铁沉积可诱导骨髓造血细胞ROS生成增加，影响Bcl-2、Caspase-3凋亡信号的表达，引起IRP患者骨髓造血细胞凋亡增加；②铁过载可通过影响T细胞数量及比例加重IRP患者免疫异常；③铁过载可增加IRP患者被覆自身抗体的骨髓有核红细胞和干细胞的破坏。

To investigate the function of dendritic cells (DC) of patients with immune related pancytopenia (IRP) and explore the role of DC in IRP.The expression of CD80 and CD86 on myeloid DC (mDC, Lin-HLA-DR(+) CD11c(+) cells) and plasmacytoid DC (pDC, Lin-HLA-DR(+) CD123(+) cells) of 65 IRP (37 untreated and 28 remitted) patients and 17 healthy controls were analyzed by flow cytometry.The expression of CD86 on pDC was (82.47 ± 13.17)% in untreated group and (60.08 ± 14.29)% in remission group, which were significantly higher than that of controls (47.95 ± 18.59)% (P0.05), while the expression in untreated group was higher than that of remission group (P0.05). The expression of CD80 on pDC was (6.31 ± 4.49)% in untreated group, which was significantly higher than that of remitted patients (3.09 ± 2.93)% and controls (2.33 ± 2.25)% (P0.05). The expression of CD86 on mDC was (97.06 ± 4.82)% in untreated group and (91.35 ± 12.20)% in control group, while the expression in untreated group was higher than that of control group (P0.05). The expression of CD80 on mDC was (6.20 ± 5.44)% in untreated group and (3.97 ± 3.24)% in remission group, which were significantly higher than that of controls (1.86 ± 1.73)% (P0.05). The expression of CD86 on pDC was negatively correlated to Th1/Th2 (r = -0.733, P0.05), it was positively correlated to the antibody on membrane of BMMNC (r = 0.283, P0.05) and the quantity of CD5(+)B cells (r = 0.436, P0.05), while it was negatively correlated to the level of hemoglobin, platelets and white blood cells (r = -0.539, P0.05; r = -0.519, P0.05; r = -0.567, P0.05, respectively). The expression of CD80 on pDC was negatively correlated to the level of hemoglobin and platelets (r = -0.431, P0.05; r = -0.464, P0.05).The function of pDC in PB of IRP were strengthened, which was relevant to the immunopathogenesis of IRP.

To observe the relationship between erythropoietin receptor (EPOR) and autoantibodies-IgG/IgM (auto-Ab) on the membrane of erythropoietic cells of the patients with bone marrow mononuclear cells (BMMNC)-Coomb's test(+) hemocytopenia (immunorelated pancytopenia (IRP)) and explore the probable autoantigens of auto-Ab in IRP.A total of 46 newly diagnosed IRP patients (15 with auto-Ab on erythropoietic cells and 31 without) and 18 healthy controls were enrolled. The EPOR expressions on their nuclear erythrocytes were tested with flow cytometry (FCM) to observe the relationship between EPOR and auto-Ab. EPOR mRNA was detected by reverse transcription (RT)-PCR. Stat5 and P-Stat5 proteins in nucleated erythrocytes were measured by Western blot. EPOR expressions on nucleated erythrocytes membrane were re-tested after stripping autoantibodies with glycine buffer.(1) EPOR of auto-Ab(+) group (1.6% ± 0.9%)was significantly lower than that of auto-Ab(-) group (4.6% ± 4.1%, P0.01)and the latter was significantly higher than that of normal controls (2.3% ± 1.8%, P0.05). EPOR of IRP patients was inversely correlated with their auto-Ab (r = -0.543, P = 0.000). (2) EPOR mRNA of auto-Ab(+) group (0.68 ± 0.14)was significantly higher than that of auto-Ab(-) group (0.55 ± 0.12, P0.01) and normal controls (0.58 ± 0.12, P0.05). (3) Protein Stat5 of auto-Ab(+) group (1.45 ± 0.94) was significantly higher than that of normal controls (0.54 ± 0.36, P0.05). While P-Stat5 of auto-Ab(+) group (0.42 ± 0.18)was significantly lower than that of normal controls (0.85 ± 0.38, P0.05). (4) EPOR expression increased significantly after auto-Ab stripping.The auto-Ab of some IRP patients blocks or competitively inhibits EPOR on the membrane of erythropoietic cells. And EPOR may be one of autoantigens in IRP.

This study was aimed to investigate the quantity and subtypes of dendritic cells (DC) in patients with immune related pancytopenia (IRP) and to explore the role of DC in pathogenesis of IRP. The quantity of plasmacytoid dendritic cells (pDC, Lin(-)HLA-DR(+) CD123(+) cells) and myeloid dendritic cells (mDC, Lin(-)HLA-DR(+) CD11c(+)cells) in peripheral blood of 65 patients with IRP (37 new diagnosed and 28 remitted) and 17 healthy controls were analyzed by flow cytometry. The results indicated that the ratio of pDC in peripheral blood mononuclear cells (PBMNC) was (0.91 ± 064)% in new diagnosed group, which was significantly higher than that in remission group (0.39 ± 0.11)% and control group (0.29 ± 0.13)% (P0.01), while this ratio of pDC in remission group was higher than that in control group (P0.05). The ratio of mDC in PBMNC was (0.21 ± 0.20)% in new diagnosed group and (0.34 ± 0.21)% in remission group respectively, there was no statistical difference as compared with control group (0.29 ± 0.09)% (P0.05). The ratio of pDC to mDC in new diagnosed group was 6.75 ± 7.11, which was significantly higher than that in remission group (1.55 ± 0.93) and control group (1.07 ± 0.43, P0.01), there was no statistical difference between the ratio of remission group and control group (P0.05). The ratio of pDC in PBMNC of IRP group negatively correlated to ratio of Th1/Th2 (r = -0.347, P0.05), and positively correlated to the ratio of auto-antibody on membrane of BMMNC (r = 0.606, P0.05) and to the quantity of CD5(+)B cells (r = 0.709, P0.05), while it negatively correlated to the levels of hemoglobin (r = -0.381, P0.01) and platelets (r = -0.343, P0.01). The ratio of mDC in PBMNC positively correlated to the ratio of Th1/Th2 (r = 0.595, P0.05) and the level of hemoglobin (r = 0.292, P0.05). The ratio of pDC/mDC negatively correlated to ratio of Th1/Th2 (r = -0.395, P0.05), it positively correlated to the level of antibody on membrane of BMMNC (r = 0.421, P0.05) and the quantity of CD5(+)B cells (r = 0.423, P0.05), while it negatively correlated to the levels of hemoglobin (r = -0.304, P0.05) and platelets (r = -0.287, P0.05). It is concluded that the quantity of pDC in peripheral blood of IRP patients increases, which may be related to the immunopathogenesis of IRP.

To study the quantity and function of bone marrow (BM) Th17 cells in the blood cytopenia patients with positive BMMNC-Coombs test (IRP) and to explore the role of Th17 cells in the pathogenesis of the disease.Forty-three untreated IRP patients, 34 recovered IRP patients and 13 healthy donors were enrolled in this study. The ratio of IL-23R(+)CD4(+)/CD4(+)cells in BM were examined by flow cytometry(FCM), the levels of IL-6, IL-23, IL-17 by ELISA, and the expressions of RORγt mRNA, STAT3 mRNA in BMMNC by semiquantitive RT-PCR.The ratio of IL-23R(+)CD4(+)/CD4(+)cells [(3.6 ± 3.1)%], the levels of IL-6[(26.21 ± 14.55) µg/L], IL-23[(2.23 ± 0.99) µg/L], IL-17[(2.54 ± 1.33) µg/L] and the expressions of RORγt mRNA (0.25 ± 0.08) and STAT3 mRNA (1.10 ± 0.16) in BMMNC of untreated IRP patients were significantly higher than those of recovered IRP patients[(2.0 ± 1.0)%, (9.08 ± 6.36) µg/L, (0.91 ± 0.76) µg/L, (1.28 ± 0.18) µg/L, 0.12 ± 0.08, 0.97 ± 0.12 respectively] (P0.05); there was no significiant difference between those of recovered IRP patients and normal controls [(1.9 ± 1.4)%, (14.63 ± 7.66) µg/L, (1.19 ± 0.98) µg/L, (1.50 ± 0.28) µg/L, 0.07 ± 0.05, 0.95 ± 0.13, respectively] (P0.05). In IRP group, there were significantly positive correlations between the ratios of IL-23R(+)CD4(+)/CD4(+)cells and CD5(+)CD19(+)/CD19(+) (P0.05), there were significantly positive correlations between the levels of IL-17 and CD5(+)CD19(+)/CD19(+), the quantity of BMMNC-antibody (r = 0.494 and 0.377, respectively) (P0.05); and so did between the expressions of RORγt mRNA and the ratio of CD5(+)CD19(+)/CD19(+), the quantity of BMMNC-antibody (r = 0.741 and 0.541, respectively) (P0.05), and between the ratio of IL-23R(+)CD4(+)/CD4(+) and the level of IL-17, the expression of STAT3 mRNA (r = 0.438 and 0.448, respectively) (P0.05).There exists increased qunantity and hyperfunction of Th17 cells in the IRP patients which induce B cells hyperfunction and production of autoantibodies against the BM hematopoietic cells. Th17 cells might be a potential new therapeutic target of IRP.

To explore the mechanism of 'erythroblast island (EI)' formation in the bone marrow of patients with immun-related hemocytopenia (IRP).The category of BM-auto antibody (au Ab) in 48 patients with IRP was detected with FCM. The BM-au Ab in the 'EI' of these cases were explored with immuonhistofluorescence (IF). Clinical and laboratory characteristics of these cases were also analyzed retrospectively.IgG could be detected in the 'EI' on the BM smear of 14 cases (29.17%), BM-au Ab mainly deposited at the edge/membranes between macrophage and erythroblasts rather than cyto plasm. Positive reaction were seen in all the cases with GlycoAIgG. The red blood cell count [(1.8 ± 0.5) × 10(12)/L] and hemoglobin level [(59.6 ± 16.2)g/L] were significantly lower than that in the IF(-) group [(2.5 ± 0.9) × 10(12)/L and (83.4 ± 25.0) g/L] (P0.05). The percentage of reticulocyte [(2.0 ± 0.8)%], serum level of IBIL [(9.4 ± 4.7) µmol/L], percentage of erythroblats in sternum BM (0.441 ± 0.139) and response rate to therapy (85.7%) in IF(+) group were significantly higher than that in IF(-)group [(1.3 ± 1.0)%, (6.6 ± 6.7)µmol/L, 0.298 ± 0.082, 61.3%, respectively] (P0.05).Macrophage was connected with erythroblasts through autologous IgG in the 'EI's of some patients with IRP. 'EI' were the places where macrophages devoured and destroyed erythroblasts rather than erythroid development and differentiation. The pathogenetic mechanism of IRP might be associated with macrophages phagocytosing and destroying BM hematopoietic cells.

To investigate the quantity and function of regulatory T cells in immune related pancytopenia (IRP) and explore the significance of Treg cells in the pathogenesis of IRP.Sixty-six IRP patients and 24 healthy donors were enrolled. The levels of IL-2 and TGF-β were detected by ELISA. The ratios of CD4(+)CD25(+)/CD4(+) and CD4(+)CD25(+)CD127(low)/CD4(+) in bone marrow were examined with flow cytometry. The expressions of Foxp3 and galectin-10 mRNA in BMMNC were measured by semi-quantitative RT-PCR.The levels of IL-2 and TGF-β in bone marrow of untreated or recovered IRP patients [(5.6 ± 1.7), (6.2 ± 2.5) µg/L, (1.8 ± 0.7), (1.9 ± 0.8) µg/L]were significantly lower than those of healthy controls [(7.9 ± 3.7), (2.5 ± 0.9) µg/L, all P0.05]. The ratio of CD4(+)CD25(+)/CD4(+) cells in bone marrow of untreated IRP patients (22.5% ± 9.5%) was significantly lower than that of recovered IRP patients or healthy controls (27.1% ± 7.1%, 30.6% ± 8.6%, both P0.05). The ratio of CD4(+)CD25(+)CD127(low)/CD4(+) cells in bone marrow of untreated IRP patients was significantly lower than that of recovered IRP patients or healthy controls (7.2% ± 2.7% vs 9.1% ± 4.7%, 10.4% ± 3.2%, both P0.05). The relative mRNA expressions of Foxp3 and galectin-10 were 0.34 ± 0.25, 0.69 ± 0.51, 0.82 ± 0.66 and 0.66 ± 0.11, 0.74 ± 0.11, 0.76 ± 0.09 in three groups respectively. The expressions of two factors in untreated IRP patients were significantly lower than those in recovered IRP patients or normal controls (all P0.05).The abnormalities in quantity and function of Treg cells in IRP patients might play an important role in the pathogenesis of IRP.

To explore the expression of antigen activated of macrophages (MPhi) of bone marrow and its clinical significance in pancytopenia patients with positive bone marrow mononuclear cells (BMMNC)-Coombs test (immunorelated pancytopenia, IRP).Sixty-one IRP patients, 10 severe aplastic anemia (SAA) patients and 13 healthy controls were enrolled in this study. The categories of auto-antibodies (IgG, IgM) on BMMNC (CD(34)(+)/CD(15)(+)/GlycoA(+) hematocytes), the quantity (CD(68)(+)/CD(45)(+))% and expression of antigen activated (CD(69)) of MPhi (CD(68)(+)CD(69)(+)/CD(+)(68))% in bone marrow of all cases and controls were measured by fluorescence activated cell sorting (FACS).The quantity and expression ratio of activated antigen of bone marrow (BM) MPhi in IRP patients [(0.57 +/- 0.30)% and (40.30 +/- 18.49)%] were respectively significantly higher than those in SAA [(0.46 +/- 0.08)% and (32.44 +/- 19.37)%] and healthy controls [(0.44 +/- 0.69)% and (29.71 +/- 11.67)%] (both P0.05). The quantity presented high-positive correlation with the expression ratio of activated antigen of BM MPhi (r = 0.89, P0.01). Patients with IRP were classified into two subgroups according to the quantity of MPhi: Group A (MPhi/= 0.5%, 34 cases) and Group B (MPhi0.5%, 27 cases). Thirty-two cases (94.12%) were with auto antibody (IgG) in Group A, while only 2 (7.41%) with auto antibody (IgG) in Group B. There was significant difference in expression ratio of activated antigen of BM MPhi between Group A (49.19 +/- 16.63)% and Group B (29.11 +/- 14.30)% (P0.05), but no difference was found between Group B and the control group (P0.05). Total curative rates at 3 and 6 month (47.06% and 79.41%) of Group A were better than those of Group B (22.22% and 51.85%). Thirty-four IRP patients with autoantibody (IgG)(+) were divided into two subgroups according to the quantity of MPhi: high level group (/= 0.75%, 9 cases) and low level group (0.75%, 25 cases), 24 cases (96%) in MPhi low level group were found auto-antibody (IgG) on one hemotopoietic cell lineage, 1 on two lineages, while 8 (88.89%) in MPhi high level group were detected auto-antibody (IgG) on two cell lineages, and 1 on three cell lineages. Expression ratio of activated antigen (56.12 +/- 15.11)% was much higher in MPhi high level group than that in MPhi low level group (44.58 +/- 18.16)% (P0.05). The count of red blood cell concentration of hemoglobin and platelet in peripheral blood in MPhi high level group were respectively lower than those in MPhi low level group, while the percentage of Ret, the level of total bilirubin and indirect bilirubin, the ratio of erythroid of sternal bone marrow in MPhi high level group were higher than those in MPhi low level group.The expression of activated antigen of BM MPhi was enhanced in IRP especially with auto-antibody (IgG), which might be involved in damage process of hemotopoietic cell.

To explore the quantity and function of bone marrow (BM) macrophages in patients with BMMNC-Coombs Test(+) pancytopenia (BCTP).Sixty-one patients with BCTP, 10 with severe aplastic anemia (SAA) and 13 normal controls were enrolled in this study. The quantity of BM macrophages was measured by FACS and their function was evaluated by phagocytosis test.The number of macrophages, phagocytosis ratio and index of cock's red blood cells (CRBC) in BCTP patients were (0.57 +/- 0.30)%, (37.56 +/- 15.20)%, and 0.75 +/- 0.34, respectively, being significantly higher than those in SAA group \[0.46 +/- 0.08)%, (28.26 +/- 10.46)%, and in 0.59 +/- 0.39\] and in normal control \[0.44 +/- 0.69)% (25.63 +/- 14.75)%, and 0.55 +/- 0.16\] (P0.05). The BCTP patients were classified into two subgroups according to the quantity of macrophages: Group A (M(Phi)or = 0.5%, 34 cases) and Group B (M(Phi)0.5%, 27 cases). There were 32 cases (94.12%) with BMMNC-IgG(+) in Group A and only 2 cases (7.41%) in Group B. There were significant differences in phagocytosis ratio and index of macrophages between Group A \[46.62 +/- 13.38)% and 0.91 +/- 0.36\] and Group B \[(28.67 +/- 12.59)% and 0.61 +/- 0.30\] (P0.05), while no statistical differences between group B and other two control groups (P0.05). Thirty-four BMMNC-IgG(+) patients were further divided into two subgroups: High level (HL) group \[or = 0.75%, 9 cases (26.47%)\] and Low level (LL) group \[0.75%, 25 cases (73.53%)\]. Only one lineage of BMMNC-IgG could be detected in LL group. Among 9 patients in HL group, 8(23.53%) had two lineages of BMMNC-IgG and 1(2.94%) had three lineages. Phagocytosis ratio and index of macrophages were significantly higher in HL group \[(60.22 +/- 12.51)% and 1.23 +/- 0.23\] than in LL group \[(43.32 +/- 9.24)% and 0.84 +/- 0.24\] (P0.05). The level of peripheral blood(PB) RBC, HGB and PLT in HL group were significantly lower than in LL group (P0.05), while the percentage of Ret, the level of TBIL and the ratio of erythroid of sternal BM in HL group were significantly higher than in LL group (P0.05).More quantity and stronger function of macrophages are observed in BCTP patients with BMMNC-IgG(+). One of the mechanism of hematocytopenia might be that macrophages activated by IgG autoantibodies phagocytose hematopoietic cells in BM. Macrophages do not involve in damage process of BM in BCTP with IgM or cold autoantibodies.

To investigate the variation of bone marrow complement level in cytopenia patients with positive BMMNC-Coombs test (CBCPC), and probe the role of complement in destroying hematopoietic cells of CBCPC patients.One hundred and twenty-four patients with CBCPC and twenty-three healthy donors as controls were enrolled in this study. The levels of CH50, C3, C4, C5b-9 were tested with ELISA. The auto-antibodies on bone marrow hematopoietic cells (BMHC) were examined with flow cytometry.The level of C5b-9 in bone marrow (BM) of untreated CBCPC patients [(119.8+/-54.0) microg/L] was significantly higher than that of recovered patients [(100.7+/-33.4) microg/L] or normal controls [(93.9+/-28.8) microg/L] (P0.05). The levels of CH50 in BM of untreated or recovered CBCPC patients [(33.3+/-11.5) kU/L, (30.8+/-10.3) kU/L] were significantly higher than that of normal controls [(24.1+/-6.4) kU/L] (P0.05). The level of C3 in BM of untreated or recovered CBCPC patients [(4.9+/-2.2) mg/L], (5.0+/-3.5) mg/L] was significantly lower than that of normal controls [(7.0+/-5.6) mg/L] (P0.05). The level of complement in peripheral blood was consistent with that in BM. CH50 in BM of CBCPC patients was negatively correlated with their C3 (r=-0.303, P=.0007) and positively correlated with their C5b-9 (r=0.241, P=0.003) levels. The level of C5b-9 in BM of CBCPC patients was higher in the BMHC-IgM positive group [(117.6+/-55.7) microg/L] than in the BMHC- IgM negative group [(99.2+/-26.2) microg/L] (P0.05). The positive rate of CD34(+)-IgG or CD34(+)-IgM of CBCPC patients was positively correlated with their C5b-9 level (r=0.593, P=0.000, r=0.326, P=0.049). The reticulocyte percentage (r=0.421, P=0.000) and serum indirect bilirubin level (r=0.230, P=0.032) of CBCPC patients were positively correlated with their CH50 level.The hematocytopenia of CBCPC patients might be related to the hematopoietic cells destruction caused by auto-antibody activated complements.

To investigate the effects of c-myc antisense oligodeoxynucleotide (ASODN) on the telomerase activity and the induction of apoptosis in HL-60 cells, and to explore the relationship between the telomerase activity and the expression of c-myc gene in HL-60 cells, after treatment by c-myc ASODN, the expression of c-myc was detected by RT-PCR, the apoptosis, cell cycle were detected with agarose gel electrophoresis and flow cytomety, and the telomerase activity was determined with TRAP-ELISA. The results showed that after blocking c-myc gene with ASODN for 72 hours, it is obvious that the expression of c-myc gene was inhibited. The percentage of S phase HL-60 cells decreased from 55.6% to 30%, the early apoptosis peak appeared (the percentage of apoptosis cells were 25.2%) and the DNA ladders were shown. OD(450 - 690) were 2.648 +/- 0.42, 2.324 +/- 0.36, 2.162 +/- 0.38, 1.952 +/- 0.14, 1.805 +/- 0.40, 1.616 +/- 0.41 and 2.466 +/- 0.29, respectively, as the cells were treated with 0, 1, 2, 3, 4, 5 micromol/L ASODN and 5 micromol/L SODN for 72 hours. The difference was significant when compared 3, 4, 5 micromol/L groups with 0 micromol/L ASODN group respectively (P0.05), but the difference was no significant when compared 1, 2 micromol/L ASODN and 5 micromol/L SODN groups with 0 micromol/L ASODN group (P0.05). It is concluded that the c-myc gene ASODN may induce the apoptosis of cells, inhibit cells from G(1) phase into S phase and regulate the telomerase activity down in HL-60 cells by blocking the expression of c-myc gene.