ITD mutation burden for the prognosis in FLT3-ITD positive acute myeloid leukemia patients / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the impact of ITD mutation characteristics on the overall survival (OS) and complete remission duration (CRD) in FLT3-ITD positive non-M3 acute myeloid leukemia (AML).</p><p><b>METHODS</b>Capillary electrophoresis was used to detect the FLT3-ITD characteristics after PCR amplication. Single or multiple mutations were identified by the numbers of peak. FLT3-ITD mutation burden was calculated by the peak area of mutant divided by the wild-type and mutant peak areas. Clinical data was collected and followed up in the FLT3-ITD mutation patients.</p><p><b>RESULTS</b>Multiple ITD mutations were common in patients aged 60 and above. Patients with single ITD mutation had higher percentage of blasts in bone marrow than multiple ITD mutations (0.758 vs 0.638, P=0.028). The numbers and length of FLT3-ITD mutation had no impact on prognosis. Patients with less than 10% of ITD mutation burden showed no difference with the intermediate-risk c-kit group in OS and CRD, but the two groups had longer OS and CRD than ITD mutation burden above 10% (OS: undefined, undefined, 9.9 months, P<0.05; CRD: undefined, undefined, 6.7 months, P<0.05). In patients with ITD mutation burden above 10%, cases with NPM1 or CEBPA mutation alone had markedly longer CRD than ITD mutation alone (25.0 vs 5.1 months, P=0.003), while OS were similar (11.4 vs 8.0 months, P>0.05).</p><p><b>CONCLUSION</b>Non-M3 AML patients with less than 10% FLT3-ITD mutation burden had a better prognosis than those above 10%.</p>

The effect of up-regulated expression of Rap1GAP on the invasion ability of HL-60 cells in vitro and in vivo / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of up-regulation of Rap1GAP on the invasion ability of leukemic HL-60 cells in vitro, and to establish leukemia mouse model to verify the effects in vivo.</p><p><b>METHODS</b>Quantitative RT-PCR and Western blot methods were used to detect the expression of Rap1GAP in Venus/HL-60 (vehicle control) and Rap1GAP/HL-60 cells (R1 andR2). Transwell method was used to examine the invasion ability in vitro. Quantitative RT-PCR and gelatin zymograph were used to study the expression of MMP-2 and MMP-9. Four-week-old BALB/c nu/nu mice were pre-treated and inoculated with leukemic cells from different groups, several index including survival time were then monitored.</p><p><b>RESULTS</b>Rap1GAP mRNA level of R1 and R2 increased about 16-17 folds as compared to the control cells. The invasion rate of R1 and R2 are (55 ± 5)% and (59 ± 4)%, which are significantly higher than (14 ± 4)% of the control cells. The mRNA level of MMP-9 was up-regulated about 12.0 folds in R1 and R2 cells compared to the corresponding control cells. The median survival times of R1 and R2 mice are (32.00 ± 1.85) d and (33.37 ± 2.50) d, respectively, which are shorter than (43.62 ± 2.32) d of the control group. Three mice of R1 and R2 groups showed leukemic cells infiltration in meninges tissue, and the genes of Rap1GAP and MMP-9 were amplified by PCR method.</p><p><b>CONCLUSION</b>Up-regulated expression of Rap1GAP increased the invasion ability of HL-60 cells accompanied with enhancement of MMP-9 expression in vitro, and the experiment in mouse model also confirmed that Rap1GAP enhanced the invasion of HL-60 cells in vivo.</p>

Up-regulation of TIMP-2 expression promotes SHI-1 leukemic cells proliferation and infiltration in immunodeficiency mice / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>MMPs and TIMPs play important roles in tumor angiogenesis and invasion. Studies have shown that TIMP-2 has two roles in tumor invasion. However, its role in leukemic infiltration has not been well investigated. This study explored the roles of TIMP-2 in extramedullary infiltration of acute monocytic leukemic SHI-1 cells both in vitro and in vitro.</p><p><b>METHODS</b>A retroviral vector carrying the human TIMP-2 cDNA was constructed and transfected into the monocytic leukemic cell line SHI-1. The expression of TIMP-2 in the positive clones was determined. The proliferation of SHI-1 cells was examined by MTT assay. Trans-Matrigel invasion assays were used to investigate the infiltration ability in vitro. SHI-1 cells were intravenously injected into pre-treated nu/nu mice to investigate the infiltration ability feature in vitro.</p><p><b>RESULTS</b>The expression of TIMP-2 on the cell membrane was significantly elevated in SHI-1/TIMP-2 cells. Over-expression of TIMP-2 promoted the cells proliferation and the invasions in vitro. The SHI-1/TIMP-2 cells demonstrated higher infiltration ability when intravenously injected into nu/nu mice.</p><p><b>CONCLUSION</b>Over-expression of TIMP-2, especially on the cell membrane, may play important roles in promoting the proliferation and infiltration of SHI-1 leukemic cells.</p>

Influence of bone marrow mesenchymal stem cells transplantation on the expression of AMPA receptor protein in rats with spinal cord injury / 中华创伤杂志

Objective To observe the influence of bone marrow mesenchymal stem cells (BMSCs) transplantation on the expression of alpha-amino hydroxymethyl-oxazole propionic acid (AMPA) receptors GluR1 and GluR2 in rats with spinal cord injury (SCI) so as to investigate the potential anti- chronic stress mechanism of BMSCs transplantation in treatment of rats with spinal cord injury.Methods A total of 48 adult male SD rats were equally divided into three groups:control group,treatment group and model group.The rats in the model and treatment group underwent lower thoracic SCI with the modified Allen' s method,and the rats in control group received only laminectomy.At day 7 after thoracic SCI,100 μl of Hank's buffered saline solution containing 1.0 × 106 BMSCs was injected into the subarachnoid space from L4-L5 intervertebral space in the treatment group and control group,and the same amount of Hank' s buffered saline solution was injected in the model group.The motor function of the rat posterior limbs was assessed by Basso-Beattie-Bresnahan (BBB) scale before and after operation.Half of the rats were anesthetized at days 14 and 28 postoperatively to harvest brains which were frozen and cut in a cryostat to detect the expressions of GluR1 and GluR2 proteins by immunohistochemistry.Results After BMSCs transplantation,the motor function of the posterior limbs in the treatment group was improved progressively.At day 14 after transplantation,the number of GluR1-positive cells of the model and treatment group was higher than that of the control group in the hippocampus CA1 region (P ＜0.05,P ＜0.01 respectively) ; GluR2-positive cells had the similar tendency,without significant difference(P ＞ 0.05 ).At the 28th day after transplantation,GluR1 positive cells of the model group were higher than those of the control group in CA1,CA3,DG regions and those of the treatment group in CA1,CA3 regions (P ＜0.05,P ＜0.01,respectively) ; GluR1 positive cells of the model and treatment group were higher than their counterpart at day 14 after grafting procedure,with significant difference (P ＜0.05,P ＜0.01,respectively).GluR2 positive cells of the treatment group were higher than those of the control group in the basolateral amygdale (BLA) (P ＜0.05 ) and had similar tendency with GluR1 expression in other regions ( P ＞ 0.05 ).Conclusion BMSCs transplantation implies a potential antichronic stress mechanism of SCI rats,since it can improve the motor function of posterior limbs in rats with lower thoracic SCI and regulate the expressions of AMPA receptor GluR1 and GluR2.

Effect of low dose radiation on expression of p16 gene in chronic myelogenous leukemia cells / 中华放射医学与防护杂志

Objective To investigate the effect of low dose radiation on the expression of p16 gene in chronic myelogenous leukemia.Methods Leukemic stem cells(LSCs)which expressed CD34+,CD38- and CD123+ were isolated from bone marrow cells obtained from twenty patients newly-diagnosedas chronic myeloid leukemia with EasySepTM magnet beads.Hematopoietie stem cells(HSCs) which expressed CD34+ and CD38- were isolated from human cord blood cells obtained from twenty full-term deliveries with EasySepTM magnet beads as control.HSCs vs LSCs samples were further divided into three dose groups,including 0,12.5 and 50 cGy,respectively.RT-PCR and real-time quantitative reverse transcription-polymerase chain reaction methods were used to detect mRNA expression of p16 gene in HSCs and LSCs after irradiation.Cells were harvested at different time for detection of cell cycle and apoptosis by flow cytometer.Results p16 mRNA level in CML-LSCs was increased slightly at 12.5 cGy,and significantly increased at 50 cGy(Z=-3.39,P<0.01),but ho significant change was found in HSCs.The percentage of CML-LSCs cell in G0/G1 stagewas increased 48 h after 12.5 cGy irradiation,and 72 h post-irradiation with 50 cGy.The apoptosis rate of CML-LSCs was gradually raised after LDR,especially at 72 h post-irradiation of 50 cGy[(17.75±11.76)%vs(6.13±4.71)%,Z=-2.37,P<0.01 ].Conclusions p16 gene transcription could be up-regulated by low dose radiation,which might provide a theoretical evidence for CML therapy and LDR in leukemic clinical application.

Characteristics of TCR β gene rearrangements in adult patients with T-lineage acute lymphoblastic leukemia and its significance in quantitation of minimal residual disease / 中华检验医学杂志

Objective To develop allele specific oligonucleotide(ASO) -PCR assay based on TCR βgene rearrangements and provide a screening method for minimal residual disease (MRD) in adult patients with T-lineage acute lymphoblastic leukemia (T-ALL).Methods DNA samples from newly diagnosed 20 adult T-ALL patients were obtained.The TCR β gene rearrangements were detected by multiplex PCR,which included 38 paired of primers in 3 reaction tubes.Gel electrophoresis and two-color Gene Scanning was also applied for clonality analysis of TCR β followed by sequencing and subsequent blasting for monoclonal PCR products in four patients.ASO primers were designed based on the sequence of junction regions.MRD were detected in the bone marrow by RQ-PCR with ASO upstream primers, consensus Jβprobes and downstream primers.Results The detection rate of the clonal TCR β rearrangements was 85.0% (17/20).At least one complete Vβ-Jβ rearrangement could be detected at the time of diagnosis in 16 out of 17 patients(94.1%, 16/17).Incomplete Dβ-Jβ rearrangement could be detected in 7 patients (41.2% ,7/17).The positivitity rate of Vβ-Jβ to Dβ-Jβ was 2∶1 (94.1% versus 41.2% ).Two-color Gene Scanning analysis showed the Jβ2 family was used more frequently than the Jβ1 family (73% versus 27% ).The slopes of the standard curves ranged from - 3.60 to - 3.27.The correlation coefficients of all four standard curves were more than 0.99.The detection sensitivity of ASO-PCR was 4 × 10 -5 μg/μl.The fluorescence background were detected at a low level.Quantitative MRD values of TCR β rearrangement in sequential BM specimens of 4 adult T-ALL patients were monitored during the treatment, including complete remission after induction and after consolidation therapy. RQ-PCR showed the MRD values of TCR β rearrangement were gradually decreased in response to the treatment.Conclusions The quantification of TCR β rearrangement by ASO-PCR approach is sensitive, specific and reliable for the accurate evaluation of malignant clones.It is suitable for the monitoring of minimal residual disease of adult T-ALL patients.

The expression of insulin-like growth factor-binding protein related protein 1 (IGFBP-rP1) in children with acute leukemia and its clinical significance / 肿瘤

Objective:To explore the expression of insulin-like growth factor-binding protein related protein 1(IGFBP-rP1) gene in children with acute leukemia and its potential significance. Methods:Real-time fluorescence quantitative PCR (RFQ-PCR) method was used for detecting IGFBP-rP1 mRNA expression in bone marrow (BM) cells of 168 children with acute leukemia. The results were compared with those of 30 non-leukemia children in control group. Meanwhile the relationship between IGFBP-rP1 expression level and clinical prognosis was analyzed according to clinical prognostic factors of children acute leukemia. Results:Expression level of IGFBP-rP1 in initial acute leukemia children was significantly higher than that of non leukemia children (P<0.01). It was higher in acute myeloid leukemia (AML) than in acute lymphoblastic leukemia (ALL)(P =0.013). The transcription level of IGFBP-rP1 mRNA in patients who had complete remission (CR) were lowest, which was nearly the same as non-leukemia childish patients. It increased again when leukemia relapsed, which was significantly higher than that in CR. However, as far as ALL was concerned, IGFBP-rP1 expression levels had no significant difference between newly-diagnosed, complete remission, and recurrent groups.Conclusion:IGFBP-rP1 may be involved in the initiation and development of childish leukemia. It has the potential to become a new target for AML treatment.

Study on the KIR receptor-ligand model in treatment of acute lymphoblastic leukemia / 中华微生物学和免疫学杂志

Objective To investigate the effect of KIR-HLA receptor-ligand model on the unrelated allo-hematopoietic stem cell transplantation (Allo-HSCT) of acute lymphoblastic leukemia (ALL). Methods The KIR genotype of 23 pairs of ALL patients and their HLA-matched unrelated donors obtained from the Database of China Marrow Donor Program. KIR genotype was determined using PCR-SSP. The expression of inhibitory KIR(iKIR) was determined by flow cytometry analysis on recipients after HSCT. Results Among all 23 donor/recipient pairs, 17 donors with KIR2DL2/L3 could find corresponding HLA-Cw1, 3, 7, 8, 12, 14 ligands in their recipients. Six donors with KIR2DL1 could match with HLA-Cw6, 15 in recipients. Sixteen donors with KIR3DL1 could recognize HLA-Bw4 and 12 donors with 3DL2 could find HLA-AI1 in their corresponding recipients, respectively. Ninteen patients were successfully transplanted, and the death rate of transplantation were 33.3% (2/6)and 40.0% (2/5) in KIR receptor-ligand matched model and the graft versus leukemia(HVG) KIR ligand-mismatching pattern. The frequency of acute graft versus host disease(GVHD) was 50.0% and death rate was 12.5% (1/8) in GVH KIR ligand-mismatching. The incidence rate of activated GVHD(aGVHD) was 20.0% in the HVG KIR ligand-mismatching. Five donor/recipient pairs of KIR gene typing were the KIR-haplotype A, 2 donor/recipient pairs with KIR2DS4 * 001/002 were died, 3 donor/recipient pairs with KIR2DS4 * 003-007 were obtained the disease free survival. The expression of CD158a/2DL1 was low when the patient had no aGVHD, but became much higher when aGVHD occurred. The percentage of NK cell of the patients was decreasing since transplantation, but still higher than normal after HSCT[ (23.4 ± 3.8 ) % vs (2.04 ± 0.58) %, P < 0.05 ]. Conclusion Analysis on KIR-HLA gene loci pattern may provide a useful parameter in predicting the clinical outcome of HLA-matched unrelated allogeneic hematopoietic stem cell transplantation for leukemia patients. Moreover, it may help to increase overall survival and disease free survival after HSCT by preventing the development of GVHD.

THINKING THE WORK MODE OF MEDICAL RECORD AND STATISTIC IN VIEW OF CONSTANTLY IMPROVING / 现代医院

Applying the value, cost and delivery of Lean Management, with the point of view of hospital president, health bureau and personnel of the medical record(TMR) and statistics, to analyze the problems existed in present work mode of TMR and statistics, and help the personnel of TMR and statistics to change thinking style, to find a new way to embody oneself value, at the same time solve the orientation of the TMR and statistics in hospital administration.

The clinical and laboratory features of acute promyelocytic leukemia: an analysis of 513 cases / 中华内科杂志

Objective To investigate the clinical and laboratory features of acute promyelocytic leukemia (APL).Methotis 513 APL patients in the last two decades were retrospectively analyzed in this research.We investigated the clinical features including age,sex,abnormality of peripheral hemogram before treatment.therapeutic effect and follow-up and laboratory data such as morphology,immunology,cytogenetics and molecular biology(MICM).Results The median age of the APL patients was 33 years old and the ratio of male and female was 1.21:1.Before treatment,the median level of WBC was 4.3×109/L and the deteetion rate of abnormal promyelocyte on blood film was 85.8%;with immunophenotypie detection,the expression levels of CD117、CD34、HLA-DR、CD7、CD14 and CD19 in APL were found to be lower and the expression 1evels of CD2、CD33 and MPO higher than those in other subtypes of acute myelocytie leukemia(AML)(beth P<0.01).Specific abnormal chromosome t(15;17)was detected in 91.7%of the patients,of whom 75.9%had standard translocation of t(15;17),being the most common one and 15.8% of the patients had t(15;17)with additional abnormal chromosome.There was only 7.5%of the patients with nolnlal karyotype.However,the presence of both simple translocation and complex translocation was seldom seen.With molecular biological detection.PML/RARα fusion gene positive rate was 99.6%.In a relativelv long clinical follow-up,we found that the complete remission(CR)rate in APL patients was 84.7%.incidence of DIC was 13.4%and five-year survival rate was 30.7%.111e median count of WBC in CR group was lower than that non-remission group(P<0.01).There were no significant differences on expressions of CD34 and CD2 and changes of cytogenetics between the two groups(P>0.05).Conclusions Comprehensive evaluation of MICM could be of important significance in the diagnosis and prognosis iudgrnent for APL patients.The CR rate in these patients with high WBC eount was considerable low.

All-trans retinoic acid as a single agent induces complete remission in a patient with acute leukemia of M2a subtype / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To present a special case with the karyotype and molecular marker of acute myeloid leukemia (AML)-M2 who was induced to complete remission by all-trans retinoic acid (ATRA) alone.</p><p><b>METHODS</b>A recently hospitalized young female patient with acute leukemia was initially diagnosed as M3 subtype based on morphological French-American-British (FAB) classification. Karyotype analysis using standard G and R banding techniques and RT-PCR were applied to further define the diagnosis. After primarily cultured bone marrow cells from the iliac aspiration were tested for in vitro induced differentiation, the patient was treated with oral all-trans retinoic acid alone, 60 mg per day until complete remission was achieved. Peripheral blood and bone marrow changes were monitored over the whole treatment course.</p><p><b>RESULTS</b>The characteristic chromosomal aberration for M3, the t(15;17) reciprocal translocation, was not found while a t(8;21) translocation was verified. Furthermore, an amplified product of the AML-1/ETO fusion gene instead of the PML/RAR alpha fusion gene was detected by RT-PCR and the diagnosis was corrected from M3 to M2. Primary cultured bone marrow cells can be fully induced to terminal differentiation after 4 days exposure to ATRA. A hematological complete remission was achieved after 40 days treatment with ATRA as a single therapeutic agent, suggesting an alternative pathway mediating ATRA-induced myeloid differentiation.</p><p><b>CONCLUSION</b>A leukemia patient with a subtype other than M3, such as M2 in this case, may also be induced to complete remission by the mechanism of ATRA-induced terminal differentiation. This implies that there may be a pathway other than PML/RAR alpha fusion gene product which mediates ATRA-induced myeloid maturation in leukemia cells.</p>

Telomerase activity in myelodysplastic syndrome / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To study telomerase activity (TA) and its variation in bone marrow mononuclear cells from patients with myelodysplastic syndrome (MDS) at different stages in comparison with normal bone marrow cells and leukemic cells.</p><p><b>METHODS</b>The TA was semi-quantitatively determined in mononuclear cells from 20 normal bone marrow samples, 21 patients with MDS at different stages and 32 cases of acute leukemia by using a polymerase chain reaction-enzyme linked immuno-sorben assay (PCR-ELISA) kit.</p><p><b>RESULTS</b>The TA in normal bone marrow cells was in the range of 0 to 0.3 units (U) with a mean of 0.11 +/- 0.08 U. Among them, 3 samples were considered positive in accordance with the standard recommended by the kit's pamphlet. In bone marrow cells from patients with acute leukemia, the TA was ranging from 0 to 0.96 U with a mean value of 0.42 +/- 0.26 U. The positive rate was 78.1% which was significantly different from that in normal bone marrow (BM) (P < 0.01). In case of myelodysplastic syndrome, the average level of TA was 0.27 +/- 0.19 U (ranging from 0 to 0.97 U) with a positive rate of 66.7%. In comparison with normal BM cells, the difference was significant (P < 0.05). Particularly, the MDS high-risk subgroup exhibited a significantly higher activity of telomerase (P < 0.05). In comparison with INT-1 and INT-2 subgroups in MDS patients based on international prognostic scoring system (IPPS), the difference in TA was also significant (P < 0.05). The abnormality in cell karyotype was not correlated with TA.</p><p><b>CONCLUSION</b>The normal bone marrow cells demonstrate TA at a marginal level while a remarkably increasing level may be seen in acute leukemia patients. The BM cells from MDS patients display a moderate TA among which the high risk MDS subgroup with a poor prognostic IPPS score exhibited markedly higher TA.</p>

Construction and Expression of Retroviral Vector Encoding Enhanced Green Fluorescent Protein / 中国实验血液学杂志

Retroviral vectors are wildly used as vehicles for gene transfer into hematopoietic cells based on its potency for efficient gene delivery and integration of transgene in host genome. The development of better transduction protocols depends on gene markers that allow a rapid detection and effective selection of genetically transduced cells. In this study, the enhanced green fluorescent protein (EGFP), a gene that is optimized for detection and expression in mammalian cells, was firstly amplified and cloned by high-fidelity PCR. The vector LGSN carrying EGFP gene was then constructed and the retroviral producer cell lines that yield high titers of LGSN vector in supernatants were developed by liposome-mediated transfection in combination with cross infection. Both GP + envAm12 murine fibroblasts and K562 leukemic cells transduced with EGFP virus demonstrated a stable green fluorescence signal readily detectable by flow cytometry or fluorescence microscopy in up to 97% and 86% of examined cells, respectively. The integration of LGSN provirus in transduced cells was confirmed by PCR analysis. These results indicate EGFP is a suitable reporter molecule for gene transfer and expression in hematopoietic cells. Therefore, the bright and long-term expression of EGFP in living cells will advance the study of gene therapy in vitro and in vivo, particularly for human applications.

Expression of the Wilms' Tumor Gene WT1 and Detection of Minimal Residual Disease in Acute Leukemia / 中国实验血液学杂志

The WT1 gene encoding a zinc finger DNA-binding protein was identified as a tumor suppressor gene being responsible for Wilms' tumor. Recently, aberrant expression of WT1 gene and an inverse correlation between its expression levels and prognosis have been demonstrated in acute myeloid leukemia (AML), suggesting it is a novel tumor marker for leukemic blast cells. To explore whether the WT1 may be used as a marker for detection of minimal residual disease (MRD) in acute leukemia, we examined the sensitivity of the nested reverse transcriptase-polymerase chain reaction (RT-PCR) by using WT1 gene primers in comparison with tumor-specific marker genes, such as PML/RARalpha gene in NB4 cells or bcr-abl gene in K562 cells. In all samples, the integrity of RNA was confirmed by amplification of the c-abl gene as an internal control. The limits in amount of leukemic cells detected by two-step RT-PCR with primers for WT1 or tumor specific fusion gene were 10(-4) and 10(-5) in NB4 cells and 10(-3) to 10(-4) and higher than 10(-6) for K562 cells, respectively. None was WT1 positive in peripheral blood mononuclear cells (MNC) from 29 blood donors, while bone marrow MNCs from eight of 21 cases (38.1%) of nonmalignant patient WT1 gene expression were found. Our results suggested that monitoring of WT1 expression makes it possible to rapidly assess the effectiveness of treatment and follow up MRD in AML cases regardless of the presence or absence of tumor-specific markers.

Protective effect of the bone marrow cells transfected with multidrug resistance gene on the reconstruction of murine hematopoietic function / 中国病理生理杂志

AIM: To investigate the protective effect of the bone marrow cells transfected with human multidrug resistance gene (MDR1) on the reconstruction of murine hematopoietic function.METHODS: The mononuclear cells of the bone marrow from donors, BALB/C mice, treated with 5-Fu previously, were isolated and transfected with human multidrug resistance gene in vitro , then transplanted to the tertiary recipients. After lethal irradiation(8.5 Gy) and bone marrow transplantation, the recipients were selected with Taxol 7 mg/kg intraperitoneal injection, VCR 5 mg/kg or DNA 5 mg/kg intravenous injection. The survival rate and blood pictures of mice as well as the integration and expression of target gene MDR1 were studied. RESULTS: The lethal irradiated murine hematopoietic function could be reconstructed and protected from toxicity of high doses Taxol, VCR and DNR selection after reinfusing the hematopoietic progenitor cells containing human multidrug resistance gene (MDR1). The survival rate and survival time of experimental mice were higher than that in the control group. The integration and expression of MDR1 gene in recipients were confirmed by PCR, RT-PCR and FCM. CONCLUSION: The integration and expression of human multidrug resistance gene in recipients may play an important role in the reconstruction and protection of murine hematopoietic function.

Transfection of WT1 gene isoforms and establishment of leukemia cell lines stably overexpressing WT1 gene / 中国病理生理杂志

AIM: To transfer 4 full-length WT1 isoforms cDNA into the leukemia cell line NB_4 so as to provide a cell model for studying the WT-1 gene function. METHODS: The eukaryotic expression recombinant vectors for WT1 isoforms (pCB6+/WT1) were introduced into the leukemia cell line NB_4 by electroporation. The positive cell clones were screened by G418 culture. The integration of WT1 gene isoforms in NB_4 cells as confirmed by PCR. The mRNA and protein of WT1 were detected by RT-PCR and Western blotting. RESULTS: WT1 gene isoforms were successfully transferred into NB_4 cells. WT1 mRNA and protein expression in the G418-selected cells increased remarkably compared with the control. CONCLUSION: WT1 gene isoforms were effectively transferred into NB_4 cells by electroporation and stably expressed in the transfected cells.

The abnormalities of microenvironment in myelodysplastic syndrome / 中国病理生理杂志

Myelodysplastic syndrome (MDS) is considered as a preleukemic course, characteristic of hypercellular marrow and pancytopenia. Many studies have demonstrated that defects occur in the heamtopoietic cells from patients with MDS. Recently, many abnormal changes in apoptosis, proliferation, ability of hematopoietic support, cytokine secretion, clonal origin of stromal cells and angiogenesis have also been revealed in the bone marrow microenvironment of MDS patients.

Effects of lovastatin on ras expression and p21~(Ras) membrane localization in human promyelocytic leukemia NB4 cells in vitro / 中国病理生理杂志

AIM: To explore the effects of cholestoral and mevalonate synthesis inhibitor lovastatin (LOV) on the proliferation of NB4 cells and elucidate the mechanisms. METHODS: Cell proliferation was analyzed by MTT assay;the expression of H, K, N- ras oncogenes in NB4 cells at different time point after LOV treatment were determined by semi-quantitative RT-PCR. Both total p21 Ras protein and p21 Ras protein on the cellular membrane were examined by flow cytometry. RESULTS:①LOV inhibited the proliferation of NB4 cells. ②All three kinds of ras were expressed in NB4 cells. ③LOV caused no increase in H, K, N- ras mRNA level. Amount of total p21 Ras protein did not change as the time varied. Concomitantly,p21 Ras protein localized on the cellular membrane decreased. CONCLUSION:LOV inhibits the proliferation of NB4 cells. Targeting HMG-CoA reductase, LOV blocks the isoprenylation of p21 Ras protein which affects its anchorage on the cellular membrane. No change in the H, K, N- ras mRNA and total p21 Ras protein expression is detected.