Successful hematopoietic stem cell transplantation depends on T cells immunol reconstitution. At present, the T-cell receptor excision DNA circles(TRECs)is the reliable index to monitor recent thymic output function. Quantitative analysis of TRECs level can determine the recent thymic output function and evaluate T cells immune reconstitution after transplantation.

Objective To analyze the risk factors of clinically overt hemorrhagic cystitis(HC)(grade ≥Ⅱ) in 114 patients undergoing allogeneic hematopoietic stem cell transplantation(allo-HSCT) to predict the occurrence of HC.Methods We retrospectively analyzed 29 cases of clinically overt HC from a series of 114 patients subject to allo-HSCT from April 1997 to December 2004.The time of follow-up began from the day of initiating conditioning to day 180 post-transplant.The 11 clinical parameters were selected for univariate analysis using a Cox regression: age,sex,underlying disease,conditioning regimen,disease status at transplant,aGVHD,donor type,use of ATG,GVHD prophylaxis,platelet and neutrophil engraftment.Factors that were significant at the 0.1 level on univariate analysis were evaluated by multivariate analysis using a Cox regression.The cumulative incidence of grade ≥Ⅱ HC within the day 180 after transplantation was calculated by the method of Kaplan and Meier.Results Twenty-nine out of 114 patients(26 %) developed HC with grade Ⅱ in 12/29 cases(41.4 %),grade Ⅲ in 11/29 cases(37.9 %) and grade Ⅳ in 6/29 cases(20.7 %).The following factors were associated with an increased risk of HC by univariate analysis: male gender(RR=2.885,P=0.021),younger than 26 years(RR=3.265,P=0.002),gradeⅢ～Ⅳ aGVHD(RR=4.039,P=0.002),unrelated donor(RR=4.347,P=0.000),intense GVHD prophylaxis(RR=2.218,P=0.045),advanced disease(RR=2.668,P=0.009).These risk factors were entered into a multivariate model.Only male gender(RR=2.993,95 % CI 1.218-7.358;P=0.017) and unrelated donor(RR=4.478,95 % CI 2.049-9.786;P=0.000) were identified as being significantly associated with the occurrence of HC.Conclusion In multivariate analysis,patients were at increased risk of HC if they were male or had received grafts from unrelated donors.

Objective To evaluate the relationship between transplantation of allogeneic peripheral blood stem cells (allo-PBSCs) and low incidence of leukemic relapse. Methods Thirty-seven adult patients with acute myeloid leukemia in first remission ( n =16) and chronic myeloid leukemia in chronic phase ( n =21) underwent allo-PBSCT from June 1997 to December 1999. A median number of CD34 + cells, CD3 + cells, CD4 + cells and CD8 + cells infused was 7.3 ?10 6/kg, 364?10 6/kg, 210?10 6/kg and 137?10 6/kg, respectively. Busulfan and cyclophosphamide were used as conditioning regimen (BU-CY2). All patients received cyclosporine and methotrexate for GVHD prophylaxis. Results Engraftment of neutrophil and platelet was achieved at a median of day +13(9～28) and day +12(7～19) respectively. Acute GVHD (aGVHD) occurred in 18 of 37 patients ( 48.6% ) with grade II～IV aGVHD ( 24.3% ). Chronic GVHD (cGVHD) developed in 22 (extensive 8, limited 14) out of 32 evaluable patients ( 68.8% ). 28 patients were still alive in complete remission at a median follow-up of 950 days (range 600～ 1 500 days). The main causes of death were aGVHD in two patients, CMV-IP in four patients, cGVHD in two patients, and relapse only in one patient. The probability of disease free survival at 3 years was 75.7% . Conclusion In our series, leukemia recurrence was seen only in one patient ( 2.7% ), suggesting that a high incidence of cGVHD may have an enhanced graft-versus-leukemia effect and hence reduce the incidence of relapse after transplantation.

AIM:To explore the impact of granulocyte colony-stimulating factor (G-CSF) on acute graft-ver-sus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in a murine model and its possible mechanisms.METHODS:Male C57BL/6 (H-2b) and BALB/c (H-2d) mice were used as the allogeneic and syngeneic donor mice , respectively .Moreover , female BALB/c mice were used as recipient mice .The recipient mice were conditioned by a single dose ( 8 Gy ) of total body irradiation ( TBI ) .The recipient mice were randomly divided into 7 groups:TBI group, Syn-BMST control group, post-Syn-BMST G-CSF administration (Syn-BMST+G-CSF) group, allo-BMT control group, post-allo-BMT G-CSF administration (allo-BMT+G-CSF)group, allo-BMST control group and post-al-lo-BMST G-CSF administration (allo-BMST+G-CSF) group.The mice in control groups and G-CSF administration groups were subcutaneous injected with 0.1 mL normal saline (NS) and 0.1 mL NS containing 2μg G-CSF per day from 1st day, respectively.The effect of G-CSF on aGVHD was evaluated by clinical manifestations and pathological changes , as well as survival time of the mice in different groups .The serum levels of IL-2, IL-4, IFN-γand TNF-αin allo-BMST and allo-BMST+G-CSF groups were detected by ELISA at 10th day.Flow cytometry was used to analyze the immunophenotypes of splenocytes at 10th day.RESULTS:The mice in TBI group were all died for hematologic failure on 9~15 d after TBI.No effect of G-CSF on the survival of the mice underwent Syn-BMST and transplantation of single allogeneic marrow cells was observed.The mean survival days in allo-BMST group and allo-BMST+G-CSF group were (34.8 ±4.5) d and (19.8 ± 6.1) d’respectively (P<0.01).Moreover, post-transplant administration of G-CSF increased the spleen total nucleated cells count (SpTNC), NK cells subset, and DC1/DC2 ratio in the spleen with over 99%of donor chimerism rate at 10th day.No difference in the levels of serum IL-2, IL-4, IFN-γand TNF-αbetween the 2 group at 10th day was found.CON-CLUSION:The administration of G-CSF after allo-BMST significantly aggravates mouse aGVHD .The expansion of NK cells stimulated by G-CSF may be involved in the mechanism of generating alloreactivity against host cells .These results imply there may be potential risk of evoking or aggravating acute GVHD if G-CSF is administered in the early stage of clini-cal allo-HSCT.

AIM:To differentiate bone marrow mesenchymal stem cells(BMSCs) into functional insulin-producing cells to produce sufficient pancreatic islet cells for transplantation.METHODS:Recombinant adenovirus vectors carrying PDX1 and NKX6.1 genes were constructed and the bone marrow mesenchymal stem cells were infected by the recombinant adenovirus combined with several cytokines for differentiation.The expressions of PDX1,NKX6.1 and insulin and C-peptide in the differentiated bone marrow mesenchymal stem cells were detected by RT-PCR and Western blotting.After the differentiated bone marrow mesenchymal stem cells were transplanted into subrenal capsule of diabetic mice,cell morphology of the grafts as well as their secretion of insulin and C-peptide were detected.Besides,regulating capacities of grafts on the blood glucose level of the diabetic mice were also detected.RESULTS:BMSCs induced by recombinant adenovirus(pAdxsi-CMV-PDX1/CMV-NKX6.1) and several cytokines showed positive dithizone staining and the expressions of ?-cell related molecule such as insulin and glucose transporter 2 were detected by RT-PCR,which showed a sustaining and stable expression.The similar results were showed by Western blotting,immunohistochemical staining and indirect immunofluorescence.The insulin secretions in the cells stimulated with glucose at concentrations of 5.5 and 25 mmol/L in the experimental group were(1 240.4?109.3) mU/L and(3 539.8?245.1) mU/L,respectively,and were significantly higher than those in control group.Moreover,transplantation of the cells to STZ mice in treatment group made serum glucose recover to normal level.CONCLUSION:PDX1 and NKX6.1 gene-modified bone marrow mesenchymal stem cells differentiate into insulin-producing cells in vitro.When these cells transplanted into STZ induced diabetic mice,their serum glucose could return to the normal level and they could live well.Thus this is a promising method for diabetes treatment.

Objective To investigate the incidence, risk factors, treatment and clinical outcome of extramedullary EM) relapse following allogeneic hematopoietic stem cell transplantation (alloHSCT), and explore the possible pathogenesis. Methods We retrospectively analyzed the medical records of 164 patients who underwent allogeneic HSCT. The 10 clinical parameters were selected for Cox univariate analysis: gender, age, underlying disease, donor type, disease status at transplant,HLA disparity, acute GVHD, chronic GVHD, EM involvement prior to transplantation and conditioning regimen. Factors that were significant at the 0. 10 level on univariate analysis were evaluated by multivariate analysis using a Cox regression. The therapeutic options for EM relapse included local radiation, surgical removal, chemotherapy, donor lymphocyte infusion (DLI), second HSCT. Results 164 recipients had sustained engraftment. EM relapse occurred in 9 patients(5.5 %), with a median time to EM relapse of 7.5 months (2.3 to 42.6 months). Ninety-fourpatients (57. 3 % ) developed acute GVHD and 83 (50. 6 % ) chronic GVHD respectively. Four patients died of EM relapse. The following factors were associated with an increased risk of EM relapse by univariate analysis: gender, donor type, disease status at transplant, chronic GVHD, EM involvement prior to transplantation. Only advanced stage of the disease (P＜ 0. 05), absence of chronic GVHD (P＜0. 01) and EM involvement prior to transplantation (P＜0. 01) were identified as being significantly associated with the occurrence of EM relapse by multivariate analysis using a Cox regression. Conclusion Many factors may be involved in the pathogenic mechanism of EM relapse,and among them, immune escape might play a major role. Advanced stage of the disease, absence ofchronic GVHD and EM involvement prior to transplantation were independently associated with an increased risk of EM relapse. EM relapse frequently followed by bone marrow involvement has poor prognosis, and therefore, prevention of leukemic cells spreading from EM sites to bone marrow is vital for long-term survival.

0.05).There was a positive correlation between the tempo of the engraftment and the progressive increase of the doses of MNC in the range of (3～5.99)?108/kg,but which was not found in the range of 6?108/kg.Conclusion These results indicate that MNC taken as an index for the measurement of the hematopoietic stem cell/progenitor content can reliably predict hematopoietic reconstitution after both HLA-matched and-mismatched sibling donor peripheral blood stem cell transplantation,with the rate and tempo of engraftment comparable with that in CD34+ cell group,and however,the target dose was reached with one apheresis in all 83 donors in MNC group(100%),as compared with 14 donors in CD34+ cell group(37.84%),suggesting that MNC may replace CD34+ cell as an independent index for the assessment of the stem cell/progenitor content in clinical practice.

AIM: To construct a recombinant plasmid vector containing human pancreatic duodenal homeobox1(Pdx1) and neurogenic differentiation 1(NeuroD1) genes,and to detect its effective expression in eukaryocytes and the ability to induce differentiation of hepatocytes.METHODS: Using human embryo pancreas mRNA as template,Pdx1 and NeuroD1 genes were amplified by RT-PCR and cloned into the two different multiple cloning sites(MCSA and MCSB) of plasmid pIRES.The recombinant plasmid pI/Pdx1/NeuroD1 was transfected into L02 cells.The expression of Pdx1 and NeuroD1 in transfected cells was detected by immunocytochemistry,IFA,RT-PCR and Western blotting,respectively.RESULTS: The length and sequence of cloned segments were correct.Pdx1 and NeuroD1 were expressed in eukaryocytes.Furthermore,the hepatic cells were induced to express glucose transporter 2(GLUT2) and eukaryocyte transcription factor Nkx6.1,which were functionally correlated to ? cells.CONCLUSION: pI/Pdx1/NeuroD1 plasmid is successfully constructed and expressed in human eukaryocytes,with which the cells express the eukaryocyte transcription factor and GLUT2,indicating the transfected cells functionally correlates to ? cells.The results suggest that Pdx1 and NeuroD1 genes can induce the differentiation the cells from hepatic cells to pancreatic endocrine secretion cells.

AIM: To investigate the expression of SCL (stem cell leukemia) gene in bone marrow stromal cells (BMSCs) and bone marrow hematopoietic cells from patients with aplastic anemia (AA) and normal individuals. METHODS: Bone marrow stromal cells from AA (9 cases) and normal individuals (33 cases) were amplified by long-term in vitro culture. The adherent and nonadherent cells were collected respectively. RT-PCR-ELISA assay was then performed to detect the expression of SCL gene and the housekeeping gene ?_2 microglobulin (?_2M). The expression ratio of SCL gene were analyzed and its expression level was normalized by ?_2M gene acting as an internal calibration for the purpose of semi-quantitative analysis. RESULTS: The expression ratio of SCL gene was lower in BMSCs from AA (22.2%) than that in normal controls (69.7%, P

AIM: To evaluate the effect and safety of Rhodamine 123 (Rh123)-mediated photodynamic treatment (PDT) on acute graft versus host disease. METHODS: An acute graft versus host disease (aGVHD) mice model was established using C57B/6 mice as donors and BALB/c mice as recipients. Mixed lymphocytic cells were cultured with Rh123 (50 nmol/L) and irradiated by argon laser 30 mW/cm2 for 3 min, then transplanted to BALB/c recipient mice mixed with donor bone marrow. Hepatopoietic recovery, aGVHD occurrence, survival time after transplantation and pathological changes were observed. In addition, CD3+CD69+ positive rates of MLC were examined by flowcytometry. RESULTS: Occurrence of aGVHD decreased, degree of pathological manifestation became milder, survival rates were higher than non PDT groups. CD3+CD69+ rates of MLC cells treated with photodynamic therapy (PDT) and cultured for 24 h significantly decreased. CONCLUSION: Rh123-mediated PDT can effectively prevent aGVHD of allogeneic bone marrow transplantation in mice.