Role of intercellular junctions in the biological behavior of SP cells of multiple myeloma / 药学实践杂志

Objective To observe the effects of the intercellular gap junction (GJIC) composed of connexin 43(Cx43) in mesenchymal stem cells (MSCs) from different sources and their signals on the biological behavior of multiple myeloma (MM) lateral population cells (SP cells), and to explore its possible mechanism. Methods Mesenchymal stem cells (MSCs) from different sources were isolated and cultured. SP cells of MM cell line RPMI 8266 were sorted by flow cytometry. RT-PCR and Western blot were used to detect the expression of Cx43 gene and protein in MSCs, RPMI 8266 and SP cells from different sources. The effects of MSCs from different sources on SP cell cycle, Cx43 protein expression, colony formation ability in vitro, stem cell related gene expression, cytokine secretion and drug resistance were observed. Results There was no significant difference in morphology and phenotype between MM-MSCs and ND-MSCs. Both MM-MSCs and RPMI 8266 cells expressed a higher level of Cx43. Co-culture with MM-MSCs induced more SP cells to enter G0 phase (P<0.001). The expressions of c-myc, Kif4 and Sox2 genes in SP cells were significantly up-regulated, while the expression of Oct-4 gene was down-regulated. After adding α-GA, c-myc, Kif4 and Sox2 were down-regulated in varying degrees, but there was no significant difference. The expression of Cx43 was up-regulated by (31.00±2)% and (39.00±2)%, respectively. The colony formation ability in vitro was up-regulated, and the addition of α-GA could partially inhibit this effect. A small amount of c-myc, Kif4, Sox2 and Oct-4 genes were expressed in RPMI 8266. These genes were significantly up-regulated in SP cell subpopulation. MM-MSCs secreted high levels of interleukin (IL)-6. After co-culture with SP cells, the expressions of IL-6, IL-10 and TGF-β in the supernatant of MM-MSCs were up-regulated (P=0.0072, P=0.037). bFGF and IL-17 had no significant change. After adding α-GA, the levels of IL-6, IL-10 and TGF-β in the supernatant decreased. MM cells were sensitive to bortezomib (BTZ) induced apoptosis, but SP cells were less sensitive. Co-culture with MM-MSCs significantly reduced BTZ-mediated apoptosis. The addition of α-GA partially restored the sensitivity of MM cells to bortezomib. Conclusion MM-MSCs and multiple myeloma SP cells up-regulate the expression of Cx43 protein, form more GJIC, and promote the proliferation and drug resistance of SP cells by changing the cytokine secretion profile of MSCs, which may be one of the reasons for the recurrence of MM.

The changes of follicular helper T cells and their associated molecules in patients with malignant lymphoid diseases and their significances / 白血病·淋巴瘤

Objective To investigate the changes of follicular helper T cells (Tfh cells) and Tfh cells associated molecules in the peripheral blood (PB) of patients with malignant lymphoid diseases (MLD) dynamically, and explore their roles on pathogenesis of the diseases. Methods Fifty-five patients with MLD were enrolled in this study,including 9 patients with acute lymphocyte leukemia (ALL), 30 patients with non-Hodgkin lymophoma (NHL) and 16 patients with multiple myeloma (MM), and 10 healthy controls (NC) of similar age were also enrolled. The percentage of CD4+CXCR5+cells (Tfh cells) and expression of ICOS+, PD1+among the T cells were detected by flow cytometry (FCM), while the levels of interleukin 21 (IL-21) in plasma were detected by ELISA tests. Results The percentage of Tfh cells and expression of ICOS and/or PD-1 in PB of all untreated patients were significantly higher than those of NC (all P 0.05), and apparently lower than those who achieved PR (P 0.05), and much higher than NC (P< 0.01). The concentration of IL-21 in patients were much higher than that in NC [(326.56±32.44) pg/ml] (P<0.01), and MM group

Efficacy and safety of anagrelide in treatment of essential thrombocythemia: multicenter, randomized controlled clinical trial / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the efficacy and safety of anagrelide in essential thrombocythemia (ET).</p><p><b>METHODS</b>Patients who diagnosed as ET according to the World Health Organization classification were enrolled. Each patient was assigned to take anagrelide hydrochloride capsule or hydroxyurea tablet by random 1∶1 ratio. Dose of anagrelide started at 2 mg/d, then increased gradually and the maximum dose was 10 mg/d until the platelet counts dropped to (100-400) × 10⁹/L, one month later gradually reduced to maintain dose. The dose of hydroxyurea was 1000 mg/d at beginning, then increased gradually, when platelet counts dropped to (100-400)×10⁹/L and kept for one month, reduced to maintain dose as 10 mg·kg⁻¹·d⁻¹. The observation period was 12 weeks.</p><p><b>RESULTS</b>A total of 222 patients were enrolled in seventeen centers (including 113 patients treated with anagrelide and 109 with hydroxyurea). Therapy efficacy can be evaluated in 198 patients (including 97 patients administered with anagrelide and 101 with hydroxyurea). At 12th weeks of therapy, the hematologic remission rate was 87.63% (85/97) in anagrelide group and 88.12% (89/107) in hydroxyurea group, the differences between the two groups were not significant (P=0.173). Treatment with anagrelide lowered the platelet counts by a median of 393 (362-1 339) × 10⁹/L from a median of 827 (562-1657) × 109/L at the beginning of the observation to 400(127-1130)×10⁹/L after 12 weeks (P<0.001), which were similar to the treatment result of hydroxyurea by a median drop of 398 (597-1846)× 10⁹/L (P=0.982). The median time to achieving response of anagrelide group was 7 (3-14) days, superior to that of hydroxyurea for 21 (14-28) significantly (P=0.003). Frequency of anagrelide related adverse events was 65.49 % (74/113), including cardiopalmus (36.28% ), headache (21.24% ), fatigue (14.16% ) and dizzy (11.50% ).</p><p><b>CONCLUSION</b>Anagrelide was effective in patients with ET which had similar hematologic remission rate to hydroxyurea and could take effect more quickly than hydroxyurea. Incidence of adverse events was undifferentiated between anagrelide and hydroxyurea, but anagrelide treatment had tolerable adverse effects and no hematologic toxicity.</p>

Recent progress of myelodysplastic syndrome: reports from the 56th American Society of Hematology annual meeting / 白血病·淋巴瘤

The myelodysplastic syndrome (MDS),which are characterized by the presence of ineffective hematopoetic stem cells.Research on pathogenesis and related epigetics has made significant progress in recent years,more and more abnormal cytogenetics and gene mutations are used for prognosis and treatment options for MDS.In the 56th American Society of Hematology (ASH) annual meeting,lots of gene mutations were introduced.

Investigation of the pathogenesis of cell fusion in multiple myeloma bone disease / 白血病·淋巴瘤

Objective To discuss function of the fusion cells of human bone marrow stromal cells (BMSCs) and human myeloma cell RPMI 8226 in the pathogenesis of multiple myeloma bone disease.Methods The cells,labeled by cell tracer green fluorescent probe (CMFDA) and red fluorescent probe (CMTMR),respectively,were induced into fusion by chemical polyethylene glycol (polyethyleneglycol,PEG-1000),and cell fusion model was set up.Whether fusion cells had nucleus fusion was determined by Karyotype analysis.The expressions of stemness-related genes,SIRP α gene and DC-STAMP gene in fusion cells were identified.Results Polyethylene glycol (PEG-1000) could mediate the integration of BMSCs and RPMI 8226 cells.The number of chromosomes in more than 80 ％ the hybrid cells was about 80.Fusion cells not only showed that BMSCs,stemness-related genes of c-myc,Klf-4 and OCT-4 genes expressed positively,but also the fusion-related genes SIRPα and DC-STAMP expressed positively.Conclusion BMSCs and RPMI 8226 cells can form fusion cells,and the cells have the potential for further integration,which is one of the important reasons for the promotion of muhiple myeloma bone destruction.

Emerging agents for treatment of the relapsed/refractory multiple myeloma: reports from the 56th American Society of Hematology annual meeting / 白血病·淋巴瘤

Relapse/refractory multiple myeloma must be the main tough for the current clinical therapy,thus at the 56th American Society of Hematology annual meeting the latest treatment studies for the disease in 2014 were described in detail,including the joint application of drugs,the new drug clinical research,the safety and efficacy that could be inspiring.This chapter shows the mechanism of these new drug action,as well as the methods and side effects.

Effect of rosiglitazone on the expression of HIF1α and IGF1 mRNA in myeloma / 中华血液学杂志

<p><b>OBJECTIVE</b>To observe the effect of rosiglitazone (RGZ) on the mRNA expression of HIF1α and IGF1 genes in the myeloma cells and explore possible mechanism of angiogenesis inhibition.</p><p><b>METHODS</b>Human myeloma cell line RPMI8226 and primary myeloma cells from five patients enriched by using CD138 immunomagnetic beads were treated with different concentrations (10, 20, 40 μmol/L) of RGZ. The mRNA expression of HIF1α and IGF1 was analyzed in cells treated with RGZ after 48h by RT-PCR, The levels of phosphorylated AKT and ERK proteins were detected by Western blotting. Bone marrow mononuclear cells from five patients with iron deficiency anemia were regarded as control.</p><p><b>RESULTS</b>Higher mRNA expression of HIF1α and IGF1 genes in RPMI8226 and in primary myeloma cells was showed as compared to those in control. Treated with RGZ of 20 μmol/L after 48 h, the mRNA expression of HIF1α (1.21 ± 0.08 vs 0.75 ± 0.06) and IGF1 (0.62 ± 0.06 vs 0.32 ± 0.04) in RPMI8226 cells was declined as compared to those without RGC treatment. The same declination was also seen in primary myeloma cells (HIF1α: 2.02 ± 0.16 vs 0.53 ± 0.04; IGF1: 1.92 ± 0.13 vs 0.58±0.03). RGZ could inhibit the expression of pAKT and pERK, nor the total AKT and ERK proteins, in RPMI8226 cells in a dose-dependent manner at the concentration of 10 μmol/L, 20 μmol/L, and 40 μmol/L.</p><p><b>CONCLUSION</b>RGZ could inhibit the mRNA expression of HIF1α and IGF1. Inhibition of angiogenesis by RGZ may be associated with down-regulation of pAKT and pERK expression.</p>

The separation of side population cells in multiple myeloma cell line PRIM8226 and their biological characteristics / 白血病·淋巴瘤

Objective To detect and separate the side population cells(SP) from multiple myeloma (MM) cell lines PRIM8226,and to study their biological characteristics.Methods Fluorescence activated cell sorter (FACS) and Hoechst33342/PI dye were used to sort SP cells of PRIM8226.The multiplication capacity was tested by the growth curve and MTT test,SP cells proportion and the cell cycle were analyzed by flow cytometry,the colony-formtion ability was compared in terms of colony forming experiment,the expression of c-myc,KIF4,SOX2,OCT4 was tested by RT-PCR.The oncogenicity of the cells was analyzed by nude mouse transplantation tumor experiment in vivo.Results The ratio of SP cells in PRIM8226 was (1.78±0.89) ％.More SP cells in the G0 / G1 period,(44.34±3.09) ％ vs (28.49±1.97) ％,P ＜ 0.05,and fewer cells in S phase than MP cells,(38.83±3.69) ％ vs (51.49±4.62) ％,P ＜ 0.05.There were no difference in the expression of CD38 and CD138 between SP cells and MP cells,respectively,(78.5±8.5) ％ vs (82.0±4.0) ％ and (72.3±15.7) ％ vs (84.3±11.9) ％,P ＞ 0.05.Colony formation assay showed that the colony forming efficiency of the SP cells was higher than the MP cells,single cell clone diameter,the number of clone forming,the clone formtion rate were significantly higher than MP cells,0.280±0.016 vs 0.118±0.019,1 722±127 vs 358±14,(86.1±3.46) ％ vs (17.9±1.88) ％,P ＜ 0.05.The mRNA expression levels of c-myc,KIF4,SOX2,OCT4 in SP cells were higher than those in MP cells,c-myc (29.90±3.73) ％ vs (16.84±2.35) ％,KIF4 (29.97±2.89) ％ vs (19.06±1.23) ％,SOX2 (40.00±4.58) ％ vs (16.62±2.09) ％,OCT4 (32.96±0.92) ％ vs (23.27±0.92) ％,all P ＜ 0.05.Nude mouse transplantation tumor experiment in vivo showed the oncogenicity of the SP cells was more stronger than MP cells (5×103 vs 5×l05).Conclusion There are notably difference in the cell cycle,colony formation assay,the mRNA expression levels and oncogenicity,but no difference in the expression of CD38 and CD138,the proliferation ability between SP cells and MP cells.

Therapeutic potential of non-adherent bone marrow-derived stem cells for acute radiation injury / 中国组织工程研究

BACKGROUND: Effective treatment for severed acute radiation sickness (over 8 Gy) has not been obtained at present. Mesenchymal stem cells, which are shown to secrete hematopoietic cytokines and support hematopoietic progenitors, play an important role in cute radiation sickness. OBJECTIVE: To investigate the therapeutic potential of non-adherent bone marrow-derived stem cells in the treatment of acute radiation injury induced by 8.5 Gy X-ray irradiation, as wel as the mechanisms involved. METHODS: Non-adherent marrow-derived stem cells from the long bone of fetal limbs were col ected for analyzing surface antigens, cel cycle, osteogenic and adipogenic differentiation potential, and expressions of vascular endothelial growth factors and Annexin A2. After being exposed to 8.5 Gy total body irradiation, BALB/C mice were randomly assigned into transplantation group and control group. Mice in the transplantation group were given 3×106 CFDA-SE labeled human non-adherent bone marrow-derived stem cells, and those in the control group were given 0.3 mL normal saline. Then, the survival rate, peripheral white blood cells at different time, pathologic change and angiogenesis of the bone marrow were observed. RESULTS AND CONCLUSION: After X-ray irradiation, transplanted non-adherent mesenchymal stem cells appeared to have a homing to the site of injury. The survival rate of mice in the transplantation group was much higher than that in the control group. Compared with the control group, the white blood cells in the transplantation group decreased more slowly while recovered more rapid: the nadir appeared at day 14 after transplantation while it recovered within 30 days. The bone marrow of mice in the transplantation group regenerated more actively and had more hematopoietic islands than those in the control group on day 21. In addition, bone marrow angiogenesis of the transplantation group was more obvious than that of the control group. In conclusion, human fetal non-adherent bone marrow-derived stem cells could promote bone marrow angiogenesis in a mouse model of acute radiation injury, through which they could play an important role in tissue regeneration of acute radiation injury.

Impairment in immunoregulatory capability of mesenchymal stem cells from multiple myeloma patients and role of it in the pathogenesis of myeloma bone disease / 中华微生物学和免疫学杂志

Objective To deplore the immunoregulatory function changes of mesenchymal stem cells(MSCs)from multiple myeloma(MM)patients and its effects on the pathogenesis of myeloma bone disease.Methods MSCs from MM patients and normal controls were isolated and the immunophenotype was detected.Real-time PCR was performed to detect the expressions of TGF-β1,TGF-β2,TGF-β3,IL-6,IL3,TNF-α,FasL and RANKL of MSCs.Transwell coculture systems were performed between MSCs and T cells.Lymphocyte proliferative assay was employed to detect the effect of MSCs on T cell proliferation.The effect of MSCs on T cell cycle and T cell activation markers CD25 and CD69 expression were analyzed by flow cytometry.Cleaved caspase 3 protein by western blot and hoechst 33258 staining were employed to detect the apotosis of T cells.Influence of T cells on the osteogenesis potential of MSCs were detected by Von kossa stain,real-time PCR and Western blot.Results MSCs from both MM patients and normal controls possessed similar morphology and immunophenotypes.MM derived MSCs exhibited increased expressions of TGF-β1,IL-6,IL-3,TNF-α and RANKL and decreased expression of TGF-β2,TGF-β3 and FasL.The inhibitory effect of MM derived MSCs on T cell proliferative ability was attenuated compared to control MSCs.MSCs from normal controls silence more T cells in Go/G1 phase than those from MM patients.The daupening effect of MM derived MSCs on activation-induced T apoptosis seemed to be enhanced.Expression of T cell activation markers were significantly inhibited by MSCs from normal controls.Both T cells cocultured with MM deprived MSCs and T cells directly from MM patients inhibited osteogenesis potential of MSCs from normal controls.Conclusion MSCs from MM patients showed impaired immunoregulatory capability on T cells.The activated T cells,in turn,inhibited the osteogenesis potential of MSCs.This may participate in the pathogenesis of myeloma bone disease.

The role of co-stimulatory molecules CD28/B7 in abnormal T cells activation in acquired aplastic anemia / 中华微生物学和免疫学杂志

Objective To explore the role and possible mechanism of CD28/B7 molecules in T cell ab-normal activation by establishing a mouse model of the autoimmune aplastic anemia. Methods Unmanipulated B6D2F1 or CByB6F1 hybrid mice were infused with about 40 × 106 lymph node (LN) cells from their C57BL/6 (B6) parent. Distribution of the injected T cells was assayed by CFDA-SE fluorescent staining. Anti-D80 and anti-CD86 monoclonal antibodies were used to block CD28/B7 signal transduction pathways and to test the change of peripheral blood of F1 mice at different times. Damage was assessed by histological staining. Bone marrow (BM) cells and LN iymphocytes were cultured to observe the effect of different number of lymphocytes in the LN on BM cells' hematopoiesis by the count of hematopoietic colony-forming cells, and to test the effect of cyclosporine A of different concentration on BM cells' hematopoiesis. Results Infusion of about 40 × 106/mouse B6 LN cells led to the development of BM failure in the fifth day: anemia, neutropenia and thrombocytopenia. At 21st day recipients began to appear death. Frozen section revealed the injected lymph node major in myeloid tissue. Pathological sec-tion revealed obvious immune-induced marrow destruction and tissue destruction. There was similar performance of the above in the recipients infused with anti-D80 and anti-CD86 monoclonal antibodies. B6 LN five times the num-ber of lymphocytes in the blood cells F1, CFU-E and CFU-G colony formation of a blank group difference was not significant (P >0.05); When B6 LN 10 times the number of lymphocytes in the blood cells F1, CFU-E colony forming significantly reduce the number of (P < 0.05) ; When B6 LN lymphocytes 50 times in F1 hematopoietic cells, not observed CFU-E colony formation. CFU-E and CFU-G colony formation reducing the number of lympho-cytes showed a dose-dependent. Cyclosporine A can significantly increase the CFU-E and CFU-G colony forming rate. Conclusion This mouse model indicates that activated lymphocytes play important roles in marrow destruc-tion in lymphocyte infusion-induced BM failure. Only blocking the CD28/B7 signal transduction can not block the abnormal T-cells activated.

Clinical Features of 110 Cases of Primary Extranodal Non-Hodgkin's Lymphoma / 中国肿瘤临床

Objective: To evaluate the incidence, clinical features, diagnosis and treatment of primary ex-tranodal non-Hodgkin' s lymphoma (PE-NHL). Methods: The data of 110 patients diagnosed as PE-NHL be-tween January 2001 and May 2008 were reviewed. Results: These PE-NHL patients counted 60.11% of the 183 malignant lymphoma patients at the same period. The primary sites affected were the gastrointestinal tract 21.82% (24/110), Waldeyer ring 10.91% (12/110), nasal cavity 9.10% (10/110), soft tissue 9.10% (10/110), mediastinum 7.27% (8/110) and other unusual sites 41.82% (46/110). Symptoms and signs of PE-NHL were not specific, and 77.27% of these cases had a swelling organ or lump of the primary organ affected. Ac-cording to the International Prognosis Index (IPI), the percentage of patients in low, intermediate, and high group was 41.11%, 44.44% and 14.44%, respectively. Immunophenotype was assayed in 93 cases. The per-centage of B-cell lymphoma was 69.90% while that of T-cell lymphoma was 30.10%. For those 95 cases treat-ed, the effective rate including complete remission (61.05%) and part remission (16.84%) was 77.89%, the median survival was 30 months, and the 5-year overall survival (OS) was 27%. While, for patients with prima-ry gastrointestinal non-Hodgkin' s lymphomas (PGIL), the complete remission rate, part remission rate and the effective rate was 65.21%, 17.39% and 82.80%, respectively. The median survival was 24 months, and the 5-year overall survival (OS) was 30%. Conclusion: PE-NHL is more common than nodal lymphoma. The symptoms and signs of PE-NHL of different sites are quite different. To improve the curative strategies of PE-NHL, it is important to make an allround understanding of PE-NHL and follow reasonable mode of diagno-sis and therapy.

Bone marrow mesenchymal stem cells promote the regeneration of irradiation-induced acute skin injury / 中国组织工程研究

BACKGROUND:Irradiation-induced skin injury is difficult to treat and easy to recurrence.Thus,irradiation-induced skin injury has been a difficulty in clinic.With the development of stem cell engineering and tissue engineering,practical application of bone marrow mesenchymal stem cells(BMSCs) has received much attention.In particular,the potential of multi-directional differentiation displays the application prospect in field of trauma repair.OBJECTIVE:To explore the effect of BMSCs on repair of irradiation-induced acute skin injury.DESIGN,TIME AND SETTING:The randomized,grouping design,comparison study was performed at the Laboratory of Hematopathy,Second Affiliated Hospital,Soochow University from June to August 2008.MATERIALS:A total of 46 male Kunming mice,weighing(25?2) g were used.Of them,40 were used to establish models of irradiation-induced local skin injury at grade Ⅲ.METHODS:A total of 40 mice were randomly selected and anesthetized with 3.6% chloral hydrate.Mouse skin of the buttock(2.0 cm?1.5 cm) was radiated using 4 MeV electron beam(1 Gy/min),total dose of 40 Gy.Mice were randomly assigned to experimental and control groups(n = 20).Mice in the experimental group were injected with CFDA SE-labeled BMSCs(0.1 mL)(2?1010/L) through the caudal vein immediately following irradiation.Mice in the control group were treated with an equal volume of saline.The remaining 6 rats were not radiated,but infused with an equal volume of BMSCs through the caudal vein.MAIN OUTCOME MEASURES:Wound surface injury in mice,pathological changes were observed 2 and 3 weeks following irradiation in the experimental and control groups.New vessels of skin were analyzed using immunohistochemistry.BMSC distribution in mouse skin and other tissues was observed at various time points in the experimental group.RESULTS:Following BMSC transplantation through the caudal vein,the speed of skin healing was faster 4-6 days in the experimental group compared with the control group.The trauma area was smaller in the experimental group compared with the control group,with the pathological presence of mild damage in epidermis,hair follicle,sesbaceous glands and collagen fiber.There were many new blood capillary in skin wound surface.BMSCs distributed widely in injured skin and multiple organs,and the number of BMSCs was significantly greater in the experimental group compared with the control group.CONCLUSION:BMSCs promoted the regeneration of irradiation-induced acute skin injury,which might contribute the interaction between BMSCs and wound microenvironment.

CD40 ligandization promotes IL-6 and Flt3 ligand production of bone marrow stromal cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze the expression of the CD(40) and its ligand (CD(40)L) on bone marrow stromal cells (BMSC), and investigate interleukin-3 (IL-3), IL-6, Flt3 ligand (FL) and stem cell factor (SCF) production of BMSC after stimulated with CD(40) agonistic monoclonal antibody (5C11) and its role in the regulation of hematopoiesis.</p><p><b>METHODS</b>BMSC were freshly isolated from adult bone marrow. The expression of CD(40) on these cells was determined with flow cytometry, the concentrations of IL-3, IL-6, FL and SCF in the supernatant at 24, 48 and 72 hours after BMSC cultured with 5C11 at a dose of 20 micro g/ml were determined by the ELISA assay. After BMSC incubated with 5C11 for 24 hours, the supernatant was collected and cultured with purified cord blood CD(34)(+) cells for 7 days under 37 degrees C in a fully humidified atmosphere supplement with 5% CO(2). Colony formation assay and Annexin V assay were employed to determine the proliferation and apoptosis of the CD(34)(+) cells.</p><p><b>RESULTS</b>BMSC expressed CD(40) and the production of IL-6 and FL increased after stimulated with 5C11 while IL-3 and SCF had no change. The supernatant collected from the stimulated BMSC promoted proliferation of CD(34)(+) cells, increased the CFU-GM yields and had anti-apoptosis effects.</p><p><b>CONCLUSION</b>CD(40) ligandization on BMSC increased the production of IL-6 and FL and promoted the proliferation of CD(34)(+) cells. The couple CD(40)/CD(40)L may be involved in the control of hematopoiesis via modulation of the cytokine network in the bone marrow.</p>

Effects of dendritic cells on T cells oligoclonal expansion of patients with aplastic anemia / 中国免疫学杂志

Objective:To investigate the effects of dendritic cells(DCs)in pathogenesis of aplastic anemia,especially its roles in T cell abnormal activation.Methods:Direct immunofluorescence assays and flow cytometry(FCM)were used to determined proportion and kinds of DCs in bone marrow sample of patients with AA.The DCs were got from standard method by culturing mononuclear cells isolated from peripheral blood of normal person in medium with recombinant human granulocyte macrophage colony stimulating factor(rhGM-CSF)and interleukin 4(IL-4)in vitro.The phenotype of DCs were determined by FCM.After pulsed with purified cord blood CD34+ cells and stimulated with recombinant human CD40 ligand(rhCD40L),the ability of those cultured CDs to activate and promote auto T lymphocytes to proliferation was tested.The changes of TCR V? gene repertorie of those T cells were also investigated by real time quantitative PCR(RQ-PCR).Results:The CD1a+CD11c+ and CD11c+CD83+ double positive cells increased in bone marrow of patients of AA,especially the proportion of CD11c+CD83+ cells increased significantly in those patients.After primed with CD34+ cells,DCs induced from peripheral monocytes could activate T lymphocytes and promote T cells to proliferation.The restricted usage of TCR V? genes was confirmed by RQ-PCR in those activated T cells by DC as those in AA patients.Conclusion:DCs,especially with phenotype of CD11c+CD83+,may have great impact on oligoclonal expansion of lymphocytes in patients AA,but the antigen which recognized by auto T cells and stimulated it to proliferation are still needed to further investigation.