Effects of fluoride on skeletal development of zebrafish and its correlation with OPG/RANKL pathway / 中华地方病学杂志

Objective:To study the effects of fluoride exposure on skeletal development of zebrafish larvae and its possible molecular mechanisms.Methods:Six hours post fertilization(6 hpf) wild-type zebrafish embryos were selected and exposed to sodium fluoride [NaF, control group (0 mg/L NaF), low fluoride group (25 mg/L NaF) and high fluoride group (100 mg/L NaF)] for 9 days. Fluorine ion selective electrode was used to detect the overall fluorine content of zebrafish larvaes, and the death and development of zebrafish larvaes were observed and counted. Bone mineralization and chondrogenesis of the zebrafish larvaes were analyzed by alizarin red staining and alcin blue staining, respectively. The expression levels of sry-related-high-mobilty-group box 9a (Sox9a), osteprotegerin (OPG) and receptor-activator of nuclear factor kappa beta ligant (RANKL) were analyzed by real-time quantitative PCR.Results:Compared with control group [(0.12 ± 0.01) μg/140 larvaes], the overall fluorine contents of zebrafish larvaes in low fluoride group [(0.28 ± 0.03) μg/140 larvaes] and high fluoride group [(0.64 ± 0.10) μg/140 larvaes] were significantly higher, and the differences were statistically significant ( P < 0.05). Compared with control group, zebrafish larvaes in high fluoride group had shorter body length, higher swim bladder loss rate and higher spinal curvature rate ( P < 0.05). The alizarin red staining area, integrated optical density (IOD) and the number of mineralized vertebrae were higher in low fluoride group, while the alcin blue staining area of cartilage formation was lower ( P < 0.05). In the high fluoride group, alizarin red staining area, IOD and the number of mineralized vertebrae were lower, while the alcin blue staining area of cartilage formation was higher ( P < 0.05). Compared with control group, the expression levels of OPG mRNA and OPG/RANKL mRNA in low fluoride group were higher ( P < 0.05); the expression level of RANKL mRNA was higher in high fluoride group, while the expression level of OPG/RANKL mRNA was lower ( P < 0.05). Conclusion:A short period of fluoride exposure from zebrafish embryo to zebrafish larvae can cause abnormal bone development of zebrafish larvae, which may be related to endochondral osteogenesis and OPG/RANKL pathway.

SIRT1 deficiency in CD4+T cells induces acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation / 中南大学学报(医学版)

To study the relationship between acute graft-versus-host disease (aGVHD) and the SIRT1 expression in peripheral blood CD4+T cells from patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT).
 Methods: We collected 40 patients who underwent allo-HSCT from human leukocyte antigen (HLA)-identical sibling donors. SIRT1 expression level in CD4+T cells was measured by real-time PCR and Western blot. Acetylation and phosphorylation of STAT3 in CD4+T cells were detected by Western blot. The binding level between SIRT1 and STAT3 in CD4+T cells was analyzed by co-immunoprecipitation and Western blot. Over-expression of SIRT1 in aGVHD CD4+T cells, as well as STAT3 acetylation and phosphorylation were measured by Western blot. The mRNA levels of RORγt, IL-17A, IL-17F related to Th17 were detected by real-time PCR.
 Results: SIRT1 expression was significantly down-regulated, while STAT3 expression, acetylation and phosphorylation levels were significantly up-regulated in patients with aGVHD compared with patients without aGVHD. The STAT3 acetylation was positively correlated with STAT3 phosphorylation (r=0.69, P<0.01). Less SIRT1-STAT3 complexes were found in CD4+T cells from patients with aGVHD compared with patients without aGVHD. After SIRT1 over-expression in aGVHD CD4+T cells, the STAT3 acetylation and phosphorylation, and the expression of RORγt, IL-17A, and IL-17F related to Th17 were significantly down-regulated (P<0.05).
 Conclusion: SIRT1 deficiency in CD4+T cells plays a crucial role in up-regulation of STAT3 acetylation and phosphorylation, the increase of Th17 related gene expression, and induction of aGVHD after allogeneic hematopoietic stem cell transplantation.

Regulation of DNA demethylation of STAT3 promoter in CD4+ T cells from aGVHD patients by HMGB1/GADD45A / 中南大学学报(医学版)

To study the molecular mechanism for DNA hypomethylation of STAT3 promoter in CD4+ T cells from acute graft-versus-host disease (aGVHD) patients.
 Methods: We collected CD4+ T cells from peripheral blood of 42 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) from HLA-identical sibling donors. GADD45A expression level in CD4+ T cells was measured by real-time PCR and Western blot. The binding level between HMGB1 and GADD45A in CD4+ T cells was analyzed by co-immunoprecipitation, while the binding levels of HMGB1/GADD45A with STAT3 promoter were detected by chromatin immunoprecipitation-quantitative real-time PCR (ChIP-qPCR). After overexpression of HMGB1 and knockdown of GADD45A in normal CD4+ T cells, STAT3 expression and DNA methylation were measured by Western blot and bisulfite sequencing PCR, respectively.
 Results: GADD45A expression was significantly up-regulated in patients with aGVHD compared with that in the patients without aGVHD. More HMGB1-GADD45A complexes were found in CD4+ T cells from patients with aGVHD compared with that in patients without aGVHD. The bindings of HMGB1/GADD45A with STAT3 promoter were significantly increased, and the binding levels of HMGB1/GADD45A were negatively correlated with STAT3 promoter DNA methylation. The expression of STAT3 was significantly reduced and the DNA methylation of STAT3 promoter was significantly increased in CD4+ T cells with overexpression of HMGB1 and knockdown of GADD45A compared with CD4+ T cells only with overexpression of HMGB1.
 Conclusion: The increased expression of HMGB1/GADD45A plays an importent role in STAT3 promoter DNA hypomethylation, thereby promoting STAT3 expression in CD4+ T cells from aGVHD patients.

Efficacy and safety of IA regimen containing different doses of idarubicin in de-novo acute myeloid leukemia for adult patients / 中华血液学杂志

Objective@#To investigate the efficacy and safety of IA regimen which contains idarubicin (IDA) 8 mg/m2, 10 mg/m2 or 12 mg/m2 as induction chemotherapy for adult patients with de-novo acute myeloid leukemia (AML) .@*Methods@#A total of 1 215 newly diagnosed adult AML patients, ranging from May 2011 to March 2015 in the First Affiliated Hospital of Soochow University and other 36 clinical blood centers in China were enrolled in the multicenter, single-blind, non-randomized, clinical controlled study. To compare the response rate of complete remission (CR) , adverse events between different dose idarubicin combined with cytarabine (100 mg/m2) as induction chemotherapy in newly diagnosed patients of adult AML.@*Results@#Of 1 207 evaluable AML patients were assigned to this analysis of CR rate. The CR rates of IDA 8 mg/m2 group, IDA 10 mg/m2 group and IDA 12 mg/m2 group were 73.6% (215/292) , 84.1% (662/787) and 86.7% (111/128) , respectively (P<0.001) . After adjusted for age, blast ratio of bone marrow, FAB classification and risk stratification, the odds ratios (95% CI) of IDA 10 mg/m2 group and IDA 12 mg/m2 group were 0.49 (0.34-0.70) and 0.36 (0.18-0.71) , as compared with the IDA 8 mg/m2 group (P<0.001, P=0.003) . In the intermediate and favorable groups, CR rates was 76.5% (163/213) , 86.9% (506/582) and 86.1% (68/79) in different doses of IDA (P=0.007) . Interestingly, IA regimen with IDA 10 mg/m2 was the only beneficial factor affecting CR in this group after adjusted for age, blast ratio of bone marrow and FAB classification[OR=0.47 (95% CI 0.31-0.71) , P<0.001]. CR rates in adverse group was 50.0% (18/36) , 60.6% (43/71) and 81.8% (18/22) respectively (P=0.089) . However, the odds ratios (95% CI) of IDA 12 mg/m2 when compared with the IDA 8 mg/m2 was 0.22 (0.06-0.80) , after adjusted for age, blast ratio of bone marrow and FAB classification. The median time (days) of neutrophil count less than 0.5×109/L in IDA 8 mg/m2 group, IDA 10 mg/m2 group and IDA 12 mg/m2 group were 14 (11-18) , 15 (11-20) and 18 (14-22) , respectively (P=0.012) and of platelet count lower than 20×109/L were 14 (7-17) , 15 (11-20) and 17 (15-21) , respectively (P=0.001) . The incidences of lung infection in the three groups were 9.8%, 13.5% and 25.2%, respectively (P<0.001) .@*Conclusions@#For young adult patients (aged 18-60 years) with AML in China, intensifying induction therapy with idarubicin 10 mg/m2 is clinically superior to IDA 8 mg/m2 and IDA 12 mg/m2 in favorable intermediate AML subgroup. However, idarubicin 12 mg/m2 is more suitable to adverse AML subgroup.

A prospective study of the efficacy and safety of maintenance therapy with recombinant human thrombopoietin in patients with primary immune thrombocytopenia: a multicenter study / 中华血液学杂志

Objective@#To evaluate the efficacy and safety of maintenance therapy with reduced dose of rhTPO in the patients with primary immune thrombocytopenia (ITP) who attained stable platelet (PLT) counts after daily administration of rhTPO.@*Methods@#Treatment was started with a daily administration of rhTPO (300 U/kg) for 2 consecutive weeks. Patients who attained stable PLT≥50×109/L were enrolled to maintenance therapy starting with every other day administration of rhTPO, then adjusted dose interval to maintain platelet count (30-100) ×109/L.@*Results@#A total of 91 eligible patients were enrolled. Fourteen patients discontinued the study due to noncompliance (12/14) and investigator decision (2/14) . Among 77 patients who completed the study, 38 patients with the administration of rhTPO at every other day or less could maintain PLT≥30×109/L for 12 weeks. The percentage of patients with a platelet response (PLT≥30×109/L) at 4th week, 8th week and 12th week of maintain therapy was 92.6% (63/68) , 82.7% (43/52) and 85.0% (34/40) , respectively. Median platelet counts remained in the range of (70-124) ×109/L. The overall incidence of rhTPO-related adverse events was 7.7%. All the adverse events were generally mild.@*Conclusion@#Extending the dose interval of rhTPO is feasible to maintain stable platelet count in the patients with ITP, but the optimal dose interval is uncertain and might vary with individuals.

Relationship between the methylation status of STAT3 promoter DNA in peripheral blood CD4+ T cells from patients after allo-HSCT and aGVHD / 中南大学学报(医学版)

Objective:To study the relationship between acute graft versus host disease (aGVHD) and the methylation status of the STAT3 promoter in peripheral blood CD4+ T cells from patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods:We collected 40 patients who underwent allo-HSCT from HLA-identical sibling donors.Serum IL-10,TGF-β1,IL-17A and IL-17F levels were detected by ELISA.Foxp3 cytotoxic T-lymphocyte-associated protein 4 (CTLA4),IL-10,TG F-β 1,RO Rγt,IL-17A and IL-17F mRNA levels in CD4+ T cells were measured by real-time PCR.STAT3 expression levels were detected by real-time PCR and Western blot,and promoter DNA methylation was analyzed by bisulfite sequencing PCR (BSP).Results:IL-10 and TGF-β1 levels were significantly down-regulated,while IL-17A and IL-17F levels were significantly up-regulated in patients with aGVHD compared with patients without aGVHD.Foxp3,CTLA4,IL-10,TGF-β1 mRNA levels were significantly down-regulated,while RORγt,IL-17A,IL-17F mRNA levels were significantly up-regulated in patients with aGVHD compared with patients without aGVHD.STAT3 expression was increased,while STAT3 promoter DNA was hypomethylated in patients with aGVHD compared with those without aGVHD.The STAT3 mRNA level was negatively correlated with STAT3 promoter DNA methylation.Conclusion:The imbalance of Treg/fh17 in CD4+ T cells from patients after allo-HSCT is a key factor for triggering aGVHD,and the DNA hypomethylation of STAT3 promoter could promote its expression in CD4+ T cells and contribute to the imbalance.

Analysis of laboratory examination, diagnosis and treatment of 57 cases of acute promyelocytic leukemia / 中国医师杂志

Objective To investigate the laboratory examination,diagnosis and treatment of 57 cases of Acute Promyelocytic Leukemia.Methods The clinical manifestation,laboratory morphology-immunology-cytogenetics-molecular (MICM) classification,treatment and follow-up were analyzed retrospectively among 57 newly diagnosed cases of acute pmmyelocytic leukemia (APL) patients from January 2011 to December 2013 in Xiangya Hospital,Central South University.Results In 57 patients,the first symptom was given priority to with bleeding (73.7％),fever (26.3％),and the organ infiltration accounted for 29.8％.The phenotype of CD13 + CD33 + CD34-HLD-RA-was 59.6％ in 57 patients,CD13 + CD33 +CD34 + HLD-RA + was 8.8％,and the remaining 18 cases were not classic phenotypes.The positive percentage of PML/RARa was 93.0％ by fluorescence in situ hybridization (FISH),and the remaining 4 cases were other rare abnormal.Except one case gave up the treatment,the other 56 cases of APL patients reached 100％ complete remission rate under early screening and diagnosis,relatively standardized and effective treatment.Conclusions Hemorrhage is the most common clinical symptoms of M3.MICM classification,especially the FISH technology,is the main laboratory basis for diagnosis of M3.After the treatment of early specifications currently,M3 can almost be cured.

Interpretation of Chinese and international guidelines of acute promyelocytic leukemia / 白血病·淋巴瘤

The therapy of acute promyelocytic leukemia (APL) with all-trans retinoic acid and arsenic trioxide was first discovered in China, which made a great contribution worldwide to APL treatment. However, foreign guidelines did not include the Chinese chemotherapy regimens, and our regimens were inconsistent with foreign guidelines. Therefore, it is necessary to interpret the home and international guidelines and to explore standard treatment of APL by analyzing APL guidelines of the China, Europe and the United States. Owing to several discrepancies between domestic and foreign APL guidelines, unifying the APL's diagnosis and treatment standard is desperately needed at present according to the evidence-based medicine. It is hoped that Chinese chemotherapy regimens will be more acceptable to other countries of the world, and would benefit the diagnosis and treatment of human APL.

Detecting chromosomal aberrations in myelodysplastic syndrome with fluorescence in situ hybridization and conventional cytogenetic analysis / 中南大学学报(医学版)

OBJECTIVE@#To detect chromosomal abnormalities in myelodysplastic syndrome (MDS) patients by fluorescence in situ hybridization (FISH) and conventional cytogenetic analysis (CCA).@*METHODS@#FISH and CCA were performed in 100 patients who were diagnosed with MDS by conventional detection of bone marrow smear and bone marrow biopsy, and were followed up.@*RESULTS@#Forty-eight (48%) patients showed chromosomal abnormalities. The positive rate of -5/5q-, 20q-, +8, -7/7q-, and -Y was 16%, 15%, 12%, 11%, and 5%, respectively, and that of CCA was 11%. The positive rate of molecular genetics abnormalities detected by FISH was obviously higher than that of CCA (P<0.01) and the combination of FISH and CCA increased the detection rate to 49%. The follow-up showed that the prognosis of patients with normal FISH results was significantly better than the abnormal ones. A correlation between complex karyotypes and poor prognosis was observed. Abnormality of -7/7q- was found closely correlated with the higher risk of acute leukemia and death.@*CONCLUSION@#Chromosomal abnormalities have been found in 49 MDS patients. Common chromosomal abnormalities in MDS patients include -5/5q-, 20q-, +8 and -7/7q-. FISH combined with CCA can improve the detection rate of chromosomal aberrations in MDS. FISH is more sensitive than CCA for detection and can be used as an important basis for prognostic assessment for MDS.

Effect of cryopreservation on umbilical blood cells and its mechanism / 中南大学学报(医学版)

Objective:To evaluate the effect of cryopreservation on clonogenic ability and apoptosis rate of mono-nuclear cells and CD34+cells in umbilical blood (UB), and to choose the index to present the freezing injury and optimize the cryopreservation of UB. Methods:hTe mono-nuclear cells (MNC) and CD34+cells were separated from UB and frozen.Atfer 30 days, they were thawed in warm water. Clonogenic capacity and clonogenic recovery before and atfer the cryopreservation was compared. We also used Annexin V-FITC-PI to investigate the apoptosis rate of the cells before and atfer the cryopreservation of these 2 types of cells. Results:hTe number of colony forming unit-granulocyte/monocyte (CFU-GMs) was not changed atfer freezing and thawing in both MNCs and CD34+cells, while the number of colony forming unit-granulocyte, erythrocyte, monocyte and megakaryocyte (CFU-GEMM) was obviously reduced after freezing in CD34+cells. The 2 types of cryopreserved cells had certain degree of apoptosis before the cryopreservation. MNC-type cryopreservation increased the cells apoptosis a little, while CD34+-type cryopreservation increased more. Conclusion:hTe cells have certain degree of apoptosis before the cryopreservation. hTe freezing and thawing procedure does affect the early stage progenitor cells-CFU-GEMM in the CD34+-type cryopreserved cells in UB. hTe damage may be induced by the cell apoptosis.

In vitro selection of single strand deoxyribonucleic acid aptamers binding to cells from patients with acute myeloblastic leukemia / 中南大学学报(医学版)

OBJECTIVE@#To screen aptamers binding CD33+/CD34- cells from patients with acute myeloblastic leukemia M2 subtype (AML-M2).@*METHODS@#CD33+/CD34- cells from patients with AML-M2 were taken as targeted cells, CD33+/ CD34- cells from normal people were taken as anti-selecting cells, and aptamers in the single strand deoxyribonucleic acid (ssDNA) library were then selected repeatedly by cell-systematic evolution of ligands by exponential enrichment (C-SELEX) technology, and amplified by polymerase chain reaction (PCR) to generate sub-ssDNA library. During the experiment, PCR amplification with fluorescently labeled primer and flow cytometry were performed to analyze the aptamers'enrichment of sub-library, and the final round product of the sub-ssDNA library was cloned. After the sequencing, the primary and secondary structures of the aptamers were analyzed.@*RESULTS@#Electrophoresis indicated that the product of PCR amplification for each round subssDNA library was able to see a clear DNA band in the agarose gel. After 13 rounds of screening, the fluorescence intensity of the sub-ssDNA library binding the cells ranged from 2.14% to 51.12%, reaching a steady state at the 13th round. A total of 30 clones were selected and sequenced, 22 of which contained 1 of the 4 conserved sequences of AAGTA, TATCT, AGATG and AAATT in their primary structure, but the remained eight aptamers contained none of the conserved sequence. Secondary structure analysis indicated that four stem-loops and loop simulation convex structures existed in the aptamers.@*CONCLUSION@#C-SELEX technology can be used to screen the aptamers binding primary cells from patients with leukemia. The aptamers selected from the CD33+/CD34- cells from the patients of AML-M2 subtype might be used for the diagnosis and treatment for leukemia.

Detection of chromosomal aberrations in multiple myeloma with fluorescence in situ hybridization / 中南大学学报(医学版)

OBJECTIVE@#To detect of chromosomal abnormalities in multiple myeloma (MM) patients with fluorescence in situ hybridization (FISH).@*METHODS@#FISH was performed in 20 MM patients using 5 specific DNA probes. The difference in chromosomal abnormalities was compared by FISH and other routine cytogenetic tests.@*RESULTS@#Eighteen of the 20 patients showed chromosomal abnormalities (90%). The positive rates of t(14q32), del(13q14), dup(1q21), and p53 gene were 65% (13 in 20), 55% (11 in 20), 25% (5 in 20), and 15%(3 in 20), respectively. The abnormal rate of the conventional chromosome examination was 15% only.@*CONCLUSION@#FISH is more sensitive than traditional chromosomal tests and can be used as an index in prognostic evaluation for MM. Del(13q14) and t(14q32) are the most common chromosomal abnormalities in MM patients.

Effect of trichostatin alone and combination with imatinb on the proliferation and apoptosis of the K562R cells / 中南大学学报(医学版)

OBJECTIVE@#To explore the effect of trichostatin A (TSA) alone and combination with imatinib on the proliferation and apoptosis of K562R cells.@*METHODS@#3-(4,5-dimethylthiazol-2-yl) -5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) assay was used to observe the proliferation of K562R cells and apoptosis was analyzed by annexin-V/propidium iodide(PI) staining.@*RESULTS@#After exposure to TSA, the proliferation of K562R cells was inhibited, and the effect was in both time- and dose-dependent manner. The apoptosis was induced. Combined with imatinib, the effect of proliferation inhibition was better.@*CONCLUSION@#TSA combined with imatinib can inhibit the proliferation of K562R cells and induce its apopotosis. TSA may become a new drug for imatinib-resistant patients.

Clinical analysis of 104 patients with hematological malignancy after allogeneic hemotopoietic stem cell transplantation / 中南大学学报(医学版)

OBJECTIVE@#To study the efficacy of allogeneic hemotopoietic stem cell transplantation (allo-HSCT) for hematological malignancy.@*METHODS@#A total of 104 patients with hematological malignancy, who underwent allo-HSCT in Xiangya Hospital from December 1999 to January 2010, were retrospectively analyzed. Of the patients, the transplantation related mortality (TRM), relapse rate (RR), 5-year overall survival (OS) and disease free survival (DFS) were estimated by Kaplan-Meier analysis. The unfavorable prognostic factors were also statistically examined.@*RESULTS@#Hematopoietic reconstitution was achieved in 101 patients. At the last data of follow-up, the incidences of severe acute graft versus host disease (aGVHD) and extensive chronic GVHD were 15.38% and 25.53%, and the TRM and RR were 15.66% and 21.76%, respectively. The estimated 5-year OS and DFS for all patients were (73.49±4.59)% and (63.10±5.32)%, respectively. Those for acute myeloid leukemia (AML) patients were (63.00±9.51)% and (49.30±9.96)%, and those for chronic myeloid leukemia (CML) patients were (83.87±5.06)% and (74.55±6.79)%, respectively. The survival analysis suggested the poor prognostic factors for allo-HSCT recipients including female sex, severe aGVHD and refractory hematological malignancy. Further multivariate analyses revealed that severe aGVHD and refractory hematological malignancy were the independent risk factors of poor prognosis for the recipients (P<0.05). The 5-year DFS of severe aGVHD and refractory hematological malignancy patients was (48.22±12.69)% and (42.09±12.31)%, respectively. The TRM of severe aGVHD, HLA-mismatched graft and unrelated donor transplant was significantly higher than that of the corresponding control groups (57.14% vs. 4.81%, 33.33% vs. 10.41%, 26.09% vs. 9.28%; P<0.05). The RR of refractory hematological malignancy was significantly higher than that of the control group (41.09% vs. 15.63%, P<0.05).@*CONCLUSION@#The treatment of allo-HSCT can improve the disease free survival of patients with hematological malignany and is an important therapeutic method for hematological malignancy. Severe aGVHD and refractory hematological malignancy are the independent risk factors of poor prognosis for the allo-HSCT recipients with hematological malignancy.

Analysis of autologous hemopoietic stem cell transplantation for 61 patients with hematological malignancies / 白血病·淋巴瘤

Objective To evaluate the clinical outcome of autologous hemopoietic stem cell transplantation (AHSCT) for hematological malignancies. Methods Data of 61 patients with hematological malignancies who underwent AHSCT in Xiangya Hospital from April 1994 to August 2008 were retrospectively analyzed. There were 30 acute non-lymphoblastic leukemias (ANLL), 25 non-Hodgkin lymphoma (NHL), 3 Hodgkin lymphoma (HL), and 3 plasmacytoma. Mel 160 mg/m2 + Ara-C 2.0/2.5 g × 2 +Cy 1.8 g/m2 × 2, or TBI 8-10 Gy + Cy 1.8 g/m2 × 2 were mainly included in pretreatment regimens. Results All patients had rapid hemopoietic reconstitution. There was one patient who died of heart failure during the transplantation process. The rate of AHSCT related death was 1.6 %. The median follow up duration was 52(2-211) months. Forty-seven of 61 patients were still alive during the analysis. The probabilities of disease free survival (DFS) at 5 years were significantly different between these two groups: (77.5±5.5) % for AHSCT groups and (31.6±7.3) % for synchronous intensive chemotherapy groups(P ＜0.01). Conclusion AHSCT can be safely performed as an important treatment constituent for hematological malignancies.

Effect and mechanism of glycosylphosphatidilinoditol- specific phospholipase D on the adhesion function of bone marrow mononuclear cells separated from myeloid leukemia patients / 中国组织工程研究

BACKGROUND: There still is rarely report about the effect of glycosylphosphatidilinoditol-specific phospholipase D (GPI-PLD) on the adhesion function of leukemic cells through screening Medline and CNKI databases.OBJECTIVE: To observe the effect of GPI-PLD on the adhesion function of bone marrow mononuclear cells separated from myeloid leukemia patients, and to investigate the related mechanism. DESIGN, TIME AND SETTING: This study addressing cytology in vitro was conducted at the Hematological Laboratory of Xiangya Hospital from January to June 2004.MATERIALS: Bone marrow was collected from myeloid leukemia patients at the Department of Hematology, Xiangya Hospital, China.METHODS: The GPI-PLD activity of bone marrow mononuclear cells separated from myeloid leukemia patients was measured by using GPI-anchored placental alkaline phosphatase as substrate and Triton-X114 partition. By use of 1,10-phenanthroline, the activity of GPI-PLD was inhibited, the experiment was divided into 2 groups: treatment group adding phenanthroline to achieve a final concentration of 1 mmol/L, while control group adding the same amount of phosphate buffered saline. The adhesion rate to the fibronectin and CD24 expression of these cells were measured by MTT and immunohistochemical method, respectively.MAIN OUTCOME MEASURES: GPI-PLD activity of myeloid leukemic cells, cell adhesion rate, CD24 expression were all measured. RESULTS: The GPI-PLD activity of bone marrow mononuclear cells separated from myeloid leukemia patients was inhibited significantly after these cells were treated by 1 mmol/L 1,10-phenanthroline for 5 hours compared with control groups [(5.40±2.96)%, (42.08±7.21)%, P < 0.01]. At the same time, the adhesion rate of these cells were increased after the GPI-PLD activity was inhibited [(61.19±29.14)%, (49.78±26.73)%, P < 0.01], and the CD24 expression was also up-regulated [(18.5±11.14)%, (16.02±9.68), P < 0.01].CONCLUSION: The adhesion rate of bone marrow mononuclear cells separated from myeloid leukemia patients can be promoted by inhibiting GPI-PLD activity. At the same time, the CD24 expression of GPI-anchored proteins on bone marrow mononuclear cells is improved.

Glycosylphosphatidilinoditol-specific phospholipase D expression in bone marrow mononuclear cells derived from acute leukemia patients / 中国组织工程研究

BACKGROUND: The correlation of gycosylphosphatidilinoditol-specific phospholipase D (GPI-PLD) activity, mRNA expression to leukemia type, hepatosplenomegaly and/or lymphadenopathy has been rarely reported. OBJECTIVE: To explore the correlation of GPI-PLD expression to leukemia type and hepatosplenomegaly and/or lymphadenopathy of acute myeloid leukemia (AML) patients. METHODS: Fresh bone marrow specimens were obtained from 43 newly diagnosed AML patients, 28 acute lymphocytic leukemia (ALL) patients, and 21 normal persons. Bone marrow mononuclear cells were harvested by density gradient centrifugation. GPI-anchored human placent alkaline phosphatase was used as substrate. GPI-PLD activity was determined bytriton-X114 phase partitioning procedure. GPI-PLD mRNA expression was detected by semi-quantitative RT-PCR. The relationship of GPI-PLD activity, mRNA expression and leukemia type, hepatosplenomegaly and/or lymphadenopathy was analyzed. RESULTS AND CONCLUSION: Compared with control group, GPI-PLD activity and mRNA expression in bone marrow mononuclear cells were significantly higher in AML group (P < 0.01), while they were significantly lower in the ALL group (P < 0.01). Of 43 patients with AML patients, 13 patients had hepatosplenomegaly and/or lymphadenopathy. The GPI-PLD activity (%) and mRNA expression were significantly higher in AML patients without hepatosplenomegaly and lymphadenopathy than those patients with hepatosplenomegaly and/or lymphadenopathy (P < 0.05). These results demonstrated that GPI-PLD activity alteration is consistent with GPI-PLD mRNA expression in AML patients, and the expression levels correlate to leukemia type and hepatosplenomegaly and/or lymphadenopathy of AML patients.

Clinicopathologic features and diagnosis of 18 patients with angioimmunoblastic T-cell lymphoma / 中南大学学报(医学版)

OBJECTIVE@#To explore the clinical and pathologic features of angioimmunoblastic T-cell lymphoma(AITL) and provide evidence for diagnosis.@*METHODS@#Eighteen AITL patients (9 males and 9 females aged from 14 to 70 years) were retrospectively analyzed in Xiangya Hospital of Central South University from July 2002 to September 2007.@*RESULTS@#Characteristic features at the presentation of AITL included generalized lymphadenopathy, fever, splenomegaly, and skin rashes with polyclonal hyper-gammaglobulinemia and other hematological abnormalities (such as Coombs-positive hemolytic anemia), which often involved the bone marrow and had well-described histologic features. The positive rate for CXCL13 was 93.3%.@*CONCLUSION@#Repeated lymphadenbiopsy is helpful for AITL diagnosis. Routine histological and immunohistochemical examinations (especially including CXCL13) play significant role in the diagnosis and differential diagnosis of AITL.

Effect of Dahuang Zhechong pills on arterial thrombosis in rabbits / 中南大学学报(医学版)

OBJECTIVE@#To investigate the effect of Dahuang Zhechong pills (DZ) on arterial thrombotic model in vivo.@*METHODS@#Sixty-five rabbits were randomly divided into 7 groups: normal, model (collagen encapsulated thread-drawing),model+aspirin (ASA), model+clopidogrel (CP),model+ASA+CP, model+ low dosage DZ (DZL), and model+high dosage DZ (DZH). All rabbits except the normal group were fed with the drugs repectively for 8 days,and sacrificed at 2 hours after the last feeding, obtained aortae. The pathological changes in the aortae were observed under microscope,and the level of FDP, D-dimer and tissue factor (TF) were measured by enzyme-linked immunosorbent assay (ELISA).@*RESULTS@#The vascular vessels were filled with thrombi in the model group and the elastic membranes of the vessel wall were seriously injured. The arterial thrombi were observed around the vascular wall in the DZL group, but some of the thrombi were dissolved. The number of thrombi was remarkably decreased in the DZH group, and most thrombi were dissolved and the vascular intimal membranes were intact. Compared with the model group, the dry and wet weight of the thrombi and the level of D-dimer, FDP, and TF in the plasma were significantly attenuated (P0.05). The pathological changes in the vascular vessel and the elevation of plasma parameters in the DZL group were similar to those in the ASA and CP groups (P>0.05). The dry and wet weight, D-dimer, FDP, and TF in the plasma in the DZH group were significantly lower than those in the DZL group (P<0.01 or P<0.05, separatively), and closed to those in the ASA+CP group.@*CONCLUSION@#Dahuang Zhechong pills are potential novel anti-thromobotic agent for arterial thrombosis.

In vitro differentiation of umbilical cord blood CD34+ cells into mature megakaryocytes and generation of plateletss / 中国组织工程研究

BACKGROUND: There still was not any report about inducing stem cells into matured cells to form products in vitro.OBJECTIVE: To induce CD34+ cells of umbilical cord blood to differentiate into mature megakaryocytes, and to investigate the mechanism of production of platelets.DESIGN, TIME AND SETTING: This cytology in vitro study was conducted at the Central Laboratory of Xiangya Hospital and Xiangya Third Hospital from 2004 to 2006. MATERIALS: Umbilical cord was collected from healthy full-term pregnant puerperants at the Xiangya Hospital.METHODS: The CD34+ cells were isolated from umbilical cord blood by magnetic activated cell sorting (MACS) and then cultured in 24-well culture plate at 5x107/L in StemPro-34 serum-free medium, supplemented with L-glutamine, saturated human transferrin, CaCl2, insulin, deionized bovine serum albumin and recombinant human thrombopoietin at 37℃, under 0.05 volume fraction CO2 saturated humidity to be differentiated into megakaryocytes for 14-21 days. Cell medium was absorbed, and centrifuged to obtain supernatant. Samples were centrifuged again, and then supernatant was removed. The remaining was platelet-like particles in cell culture plate. Platelet was isolated from normal platelet-rich plasma.MAIN OUTCOME MEASURES: The following parameters were measured: morphological changes in cultured cells and platelet-like particles in supematant; results of immunohistochemistry; observation results under a microscope; platelet aggregation; CD41 expression.RESULTS: At day 10, silk-like substances were found in megakaryocyte culture medium, with the presence of platelet-sized particles. The production of platelet-sized particles reached a peal at day 16. Cultured cells were strongly positively for platelet-specific antigen GP Ⅱb Ⅲa. Under the optical microscope, mature megakaryocytes were detected, with the presence of some immature megakaryocytes, and platelet-sized particles were found surrounding megakaryocytes. Under the electron microscope, a majority of mature megakaryocytes and a few apoptotic megakaryocytes were detected, and platelet-sized particles in the supernatant had the same size and structure with the platelet in the platelet-rich plasma. Some platelet surfaces were smooth or irregular. Platelet-sized particles in the supematant aggregated in response to thrombin as platelets in normal platelet-rich plasma. Flow cytometry demonstrated that the cultured platelets had the same high expression rate of CD41 as the platelets from platelet rich plasma.CONCLUSION: Umbilical cord blood CD34+ cells can be induced to differentiate into pudfied and mature megakaryocytes and platelets in vitro.