[Selection of reference genes of Siraitia grosvenorii by real-time PCR].

Siraitia grosvenorii is a traditional Chinese medicine also as edible food. This study selected six candidate reference genes by real-time quantitative PCR, the expression stability of the candidate reference genes in the different samples was analyzed by using the software and methods of geNorm, NormFinder, BestKeeper, Delta CT method and RefFinder, reference genes for S. grosvenorii were selected for the first time.

[Extraction of total RNA and cloning of sgDHAR gene from Siraitia grosvenorii].

Total RNA was isolated from Siraitia grosvenorii fruit by the method of modified Trizol, according to S. grosvenorii fruit characteristics of rich phenols, polysaccharide, oil and proteins. The OD260/280, OD260/230, RNA integrity (RIN) and yield of the total RNA with this method were 2.

[Retrospective analysis on therapeutic effect of autologous hematopoietic stem cell transplantation in multiple myeloma patients].

Objective: To evaluate the efficacy and prognostic factors of autologous hematopoietic stem cell transplantation (ASCT) in multiple myeloma (MM) patients.

Methods: Retrospective analysis was performed in 27 MM patients undergoing ASCT at our hospital from May 2004 to August 2011. After comparing with 28 patients achieving very good partial response (VGPR) or better outcome and not undergoing ASCT, the impact on the extent of response, progression-free survival (PFS) and overall survival (OS) as well as related prognostic factors of MM patients were analyzed.

[Correlation between clinical outcome and WT1 detection after hematopoietic stem cell transplantation in acute leukemia].

Objective: To evaluate preliminarily the significance of detecting the Wilms' tumor (WT1) gene level on monitoring minimal residual disease (MRD) and predicting the clinical outcome in patients of acute leukemia following hematopoietic stem cell transplantation (HSCT).

Methods: The mRNA expression levels of WT1 and house-keeping gene ABL were dynamically measured with Real-time quantitative reverse transcription polymerase chain reaction (RQ-RT-PCR) on 326 bone marrow samples from 63 post-HSCT patients in our hospital from December 2001 to September 2009. After comparing the WT1 levels of patients with different post-transplantation outcomes, the investigators used the receiver operating characteristic (ROC) curve to determine the WT1 threshold so as to predict their clinical relapses.

[The incidence of TET2 gene mutation and its clinical significance in acute myeloid leukemia patients].

Objective: To evaluate the prevalence of TET2 gene mutation in acute myeloid leukemia (AML) patients, and analyze their clinical characteristics and prognosis.

Methods: Polymerase chain reaction (PCR) and direct sequencing were used to sequence exon 3 to 11 of TET2 gene.

Results: Among 96 AML patients, TET2 gene mutation was detected in 13 (13.

[The role of intracellular signal pathway of mTOR/S6 in CD3+ T lymphocytes of refractory/relapsed aplastic anemia patients].

Objective: To explore the activation status of signal pathway of mTOR/S6 in bone marrow (BM) T lymphocytes of refractory/relapsed aplastic anemia patients (AA), and the effects of rapamycin (RAPA) and CTLA-4 immunoglobulin (CTLA-4Ig) on this pathway.

Methods: BM was collected from 13 refractory/relapsed AA patients, 8 newly diagnosed severe AA (SAA) patients and 10 iron deficiency anemia (IDA) (as controls) patients, and cocultured with RAPA and CTLA-4 Ig. The expression of p-mTOR, p-S6 and Interferon gamma (IFN-gamma) in CD3(+)T cells was measured by flow cytometry (FCM).

[5-azacytidine treatment induces tumor suppressor gene XAF1 expression and inhibits proliferation in myeloma cells].

Objective: To investigate the effect of 5-azacytidine on XAF1 expression in myeloma cell lines RPMI8226 and XG-7 and the in vitro anti-myeloma activity of 5-azacytidine.

Methods: XAF1 mRNA and protein expression was detected by semi-quantitative reverse transcriptase PCR and Western blot, respectively. Methylation specific PCR (MSP) was used to detect methylation status of XAF1 promoter CpG islands.

[Prevalence and clinical significance of FLT3 mutations in acute promyelocytic leukemia].

Objective: To evaluate the prevalence of Fms-Like tyrosine kinase 3 (FLT3) mutations including internal tandem duplication (ITD) of juxtamembrane region and point mutation in the second tyrosine kinase domain (TKD) in acute promyelocytic leukemia (APL) and its clinical significance.

Methods: Bone marrow mononuclear cells from 160 newly diagnosed APL patients were analyzed. Polymerase chain reaction (PCR) was used to detect FLT3-ITD mutations, FLT3-ITD positive samples were further analyzed for the ITD allelic ratio (ITD-AR, mutant-wild type ratio).

[A comparison of clinical outcomes between unrelated donor and HLA-haploidentical donor hematopoietic stem cell transplantation].

Objective: To compare the clinical outcomes between unrelated donor hematopoietic stem cell transplantation (URD-HSCT) and HLA-haploidentical (Hi)-HSCT.

Methods: Twenty-five patients with hematologic malignancies received URD-HSCT and thirty patients received Hi-HSCT. The conditioning regimen consisted of modified BUCY or modified total body irradiation (TBI) plus CY.

[Role of CD28/CTLA-4 co-stimulators in immune pathophysiology of aplastic anemia].

Objective: To explore the possible role of CD28/CTLA-4 co-stimulators in immune pathophysiology of acquired aplastic anemia(AA).

Methods: By FACS, the percentages of CD28, CTLA-4 expressing CD3+ CD4+ T cells and the level of Th1, Th2 in bone marrow were detected in 23 AA patients at active phase, 10 at recovery phase and 15 normal controls. The relationship between the co-stimulators, Th1, Th2, and absolute neutrophil counts (ANC) was evaluated.

[Study on relationship between pretransplantation host thymic recent output function and prognosis in HLA-matched sibling hematopoietic stem cell transplantation].

Objective: To study the relationship between pretransplantation host thymic recent output function and prognosis in HLA-matched sibling bone marrow transplantation (MSD-BMT) and determine whether pretransplantation host thymic recent output function can act as a marker for predication of prognosis after HSCT.

Methods: T-cell receptor excision circle (TREC) in DNA of pretransplantation peripheral blood mononuclear cells from 64 patients underwent MSD-BMT was detected by real-time quantitative PCR. The content of TREC in 70 normal donors was detected as well.

[Prediction of T-cell immune reconstitution by investigating T cell receptor excision circle and T-cell receptor beta-chain variable region clonal repertoire in leukemia patients after allogeneic hematopoietic stem cell transplantation].

Objective: To predict T-cell immune reconstitution by investigating T cell receptor excision circles (TREC) and T-cell receptor beta-chain variable region (TCRBV) clonal repertoire in leukemia patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT).

Methods: Real-time quantitative PCR was used to detect the TREC in 43 leukemia patients undergoing matched sibling donor bone marrow transplantation (MSD BMT), matched unrelated donor (MUD) BMT, or haploidentical-stem cell transplantation (HID-SCT), and in 70 normal individuals. RT-PCR was used to amplify 24 subfamily genes of T-cell receptor beta-chain variable region (TCRBV) in 24 of the 43 patients and 5 normal donors as control.

[Preliminary study of the effect of HLA-Cw on haploidentical hematopoietic stem cell transplantation].

Objective: To investigate the effect of HLA-Cw on haploidentical hematopoietic stem cell transplantation (HHSCT) without T-cell depletion.

Methods: HLA-Cw were detected with PCR-SSP, the clinical data of 21 cases of haploidentical hematopoietic stem cell transplantation, including 8 standard risk and 13 high risk cases from July 2002 to March 2006 were summarized, and the effect of HLA-Cw in HHSCT was analyzed.

Results: Twenty patients achieved sustained, full-donor-type engraftment.

[The molecular characteristics of T-cell immune reconstitution in leukemia patients after allogeneic hematopoietic stem cell transplantation].

Objective: To study the molecular characteristics of CDR3 repertoires of T cell receptor beta chain variable region (TCRBV) of T lymphocytic clones in leukemia recipients after allogeneic hematopoietic stem cell transplantation ( allo-HSCT).

Methods: RT-PCR was used to amplify 24 subfamily genes of TCRBV from peripheral blood (PB) lymphocytes in twenty-four leukemia patients underwent three kinds of allo-HSCT and in five normal donors as control. The PCR products were further analyzed by genescan to evaluate the clonality of BV subfamily and characteristics of CDR3 and calculate usage rate of BV subfamily.

[Analysis of NPM1 gene mutations in acute myeloid leukemia].

Objective: To evaluate the prevalence of nucleophosmin (NPM1) gene exon 12 mutations in adults with acute myeloid leukemia (AML) and its clinical characteristics.

Methods: Genomic DNAs from 101 AML adults were screened by PCR and sequencing or capillary electrophoresis (CE) for NPMI mutations.

Results: NPM1 exon 12 mutations were present in 31.

[A clinical study of 31 patients with malignant hematopoietic disease treated with non-T cell-depleted HLA-haploidentical hematopoietic stem cell transplantation].

Objective: To investigate the effects and prognosis of malignant hematological disease after HLA haploidentical hematopoietic stem cell transplantation (H-HSCT) without T-cell depletion.

Methods: The clinical data of 31 cases with malignant hemopoietic disease treated with H-HSCT from July 2002 to July 2006 were analyzed, including 11 cases of standard risk and 20 of high risk.

Results: 30 patients achieved engraftment of a median of 13 and 22 days for neutrophil and platelet, with an accumulative incidence of II - IV grade acute graft-versus-host disease (GVHD) 61.

Patterns of T-cell reconstitution by assessment of T-cell receptor excision circle and T-cell receptor clonal repertoire after allogeneic hematopoietic stem cell transplantation in leukemia patients--a study in Chinese patients.

Objective: Successful allogeneic hematopoietic stem cell transplantation (HSCT) requires reconstitution normal T-cell immunity. Measurement of T-cell receptor excision circles (TRECs) and T-cell receptor beta (TCRBV) CDR3 repertoire is a means of quantifying recent thymic T-cell production and reflecting antigen-specific T-cell clones proliferation.

Methods: We used real-time quantitative PCR to detect TRECs from 43 Chinese patients who underwent three kind of allo-HSCT without T-cell depletion.

[Molecular characteristics of T-cell receptor of clonal expansion of T lymphocytes in leukemia patients after haploidentical bone marrow transplantation].

Background & Objective: Previous clinical and experimental results indicate that T-cell immune reconstitution is slow after hematopoietic stem cell transplantation. Immune reconstitution after haploidentical bone marrow transplantation (BMT) is closely related to clinical events. This study was to analyze the repertoire of T-cell receptor beta chain variable region (TCRBV) and the molecular characteristics of T-cell clones during immune reconstitution in leukemia patients after haploidentical BMT.

[Quantitative analysis of donor chimerism in adoptive immunotherapy following allogeneic hematopoietic stem cell transplantation].

Objective: To investigate the value of sequential and quantitative analysis of donor chimerism (DC) to predict the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT), to determine the optimal time point of adoptive immunotherapy and to estimate the efficacy of adoptive immunotherapy.

Methods: Quantitative analysis of DC was performed with multiplex PCR amplification of short tandem repeats markers (STR-PCR) and capillary electrophoresis with fluorescence detection in 84 patients who received allo-HSCT. Sequential chimerism both prior to and following adoptive immunotherapy were evaluated in 16 patients.

[Mobilization of peripheral blood stem cells with mitoxantrone and high-dose cytarabine chemotherapy and rhG-CSF in patients with hematopoietic malignancies].

Objective: To evaluate the efficacy of mitoxantrone combined high dose of cytarabine and recombinant human granulocyte colony-stimulating factor (MAG) regimen for mobilizing autologous peripheral blood stem cells (APBSC) in patients with hematopoietic malignancies.

Methods: From December 1995 to April 2003, 14 lymphoma and 29 acute leukemia patients were treated with high-dose cytarabine (2 g/m2 every 12 h, days 1 and 2) and mitoxantrone (10 mg/m2, days 2 and 3), followed by 300 microgram recombinant human granulocyte colony-stimulating factor per day (rhG-CSF 300 microg/d) i.e, the MAG regimen as mobilization regimen of peripheral blood stem cells.

[Application of sequential and quantitative analysis of donor chimerism in donor lymphocyte infusion].

In order to study the value of sequential and quantitative analysis of chimerism in determination of optional time of donor lymphocyte infusion (DLI) and prediction of efficacy of DLI, six patients with leukemias who relapsed or failed of engraftment were treated with DLI. Serial and quantitative analyses of donor chimerism (DC) both prior to and following DLI were performed by multiplex PCR amplification of STR markers (STR-PCR) and capillary electrophoresis with fluorescence detection. The results showed that at the time of relapse or graft rejection, STR-PCR indicated the decreasing donor chimerism in all six patients, at levels ranging from 27.

[Application of sequential and quantitative monitoring of chimerism in allogeneic hematopoietic stem cell transplantation].

Objective: To establish multiple short tandem repeat (STR) amplification by fluorescence labeling polymerase chain reaction (PCR) combined with capillary electrophoresis for quantitative determination of chimerism, and to evaluate the status of engraftment and predict the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT).

Methods: Thirty-one patients received bone marrow transplantation (BMT) or nonmyeloablative allogeneic stem cell transplantation (NST) were evaluated. Peripheral blood and bone marrow were co-llected before and after transplantation in different period.

[Dynamic changes of cytokines in G-CSF mobilized peripheral blood].

Objective: To investigate the level and significance of interleukin-8 (IL-8), soluble intercellular adhesion molecule (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) in patients peripheral blood (PB) during mobilization for peripheral blood stem cells harvesting.

Methods: The levels of IL-8, sICAM-1 and sVCAM-1 in patients were dynamically assayed by ELISA during the mobilization procedure and the number of CD(34)(+) cell, white blood cell (WBC) and platelet (BPC) by flow cytometric analysis and hematometry respectively. Colony formation was assayed by using semisolid methycellulose culture.