Effects of DNMT1 Gene Silencing on Methylation of SOCS-1 Gene in Myeloma Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of DNA methyhransferase l (DNMT1) gene silencing on methylation of suppressor of cytokine signaling (SOCS-1) in multiple myeloma RPMI 8226 cells.</p><p><b>METHODS</b>Recombinant plasmid pshRNA-DNMTl was transfected into multiple myeloma RPMI 8226 cells by lipofectamine 2000. RT-PCR and Western blot were used to detect the mRNA and protein expression of DNMTl in RPMI 8226 cells respectively before and after transfection. Methylation-specific polymerase chain reaction was used to detect the methylation level change of SOCS-1 gene in RPMI8226 cells transfected.</p><p><b>RESULTS</b>DNMTl targeted short hairpin RNA(shRNA) was successfully inserted into the plasmid vector pshRNA. RT-PCR results showed that the relative mRNA expression level of DNMTI gene in RPMI 8226 cells transfected with pshRNA was 0.176±0.004 which was significantly lower than that in cells transfected by empty vector (0.956±0.033, P<0.01). Western blot analysis showed that the relative expression level of DNMT1 protein of RPMI 8226 cells transfected by pshRNA was 0.065±0.014, which was significantly lower than that in transfected cells by empty vector(0.415±0.027) (P<0.05). These results indicated that the recombinant plasmid pshRNA could effectively knock down the expression of DNMT1 gene in RPMI 8226 cells. Methylation analysis showed that the methylation level of SOCS-1 gene was obviously reduced after transfection.</p><p><b>CONCLUSION</b>DNMT1 gene in RPMI 8226 cell can be silenced by shRNA. DNMT1 gene silencing can significantly induce SOCS-1 gene hypomethylation, which indicates that DNMT1 may play an important role in the process of SOCS-1 hypermethylation.</p>

Expression of pituitary tumor transforming gene in patients with acute lymphoblastic leukemia / 中国实验血液学杂志

The aim of this study was to explore the expression of pituitary tumor-transforming gene (PTTG) in acute lymphoblastic leukemia (ALL) and its relationship with the pathogenesis of ALL, as well as study the difference of the PTTG expression in ALL patients with Ph1 chromosome and without Ph1 chromosome. The mRNA expressions of PTTG in bone marrow from 28 patients with ALL and 28 normal controls were quantitatively detected by real-time quantitative polymerase chain reaction (real-time PCR). The results indicated that the expression of PTTG mRNA was significantly higher in ALL patients (1.9428E5 ± 1.8372E5) than that in normal controls (4.5766E3 ± 1.1817E3) (P < 0.05). The expression of PTTG mRNA was higher in Ph1 chromosome positive patients. The initial expression of PTTG mRNA was lower in patients achieved complete remission than that in patients with non-complete remission. It is concluded that the overexpression of PTTG may be related to the progression and genesis of ALL. Overexpression of PTTG may be intimately related to the progression and genesis of Ph1 chromosome positive ALL. It provides a new ideas to research the pathogenesis and genic target treatment of ALL.

Expression of FoxO3a in patients with acute myeloid leukemia and its clinical significance / 中国实验血液学杂志

This study was aimed to investigate the expression and clinical significance of forkhead box protein O3a (FoxO3a) in the patients with acute myeloid leukemia (AML). Western blot was used to detect the FoxO3a protein expression in bone marrow samples from 44 newly diagnosed AML patients and 5 healthy donors. Additionally, 14 patients' samples were reevaluated when they got complete remission (CR). The results showed that FoxO3a expression (FoxO3a/β-actin 0.43 ± 0.19) in newly diagnosed AML patients was much higher than that in healthy donors (FoxO3a/β-actin 0.19 ± 0.06) (P < 0.001). The FoxO3a level was down-regulated when CR was got and there was not significant difference between patients in CR and healthy donors (P > 0.10). The correlation analysis showed that the level of FoxO3a expression positively correlated with the white blood cell count of AML patients at the time of diagnosis. Although FoxO3a expression did not positively correlate with the CR rate, the higher FoxO3a expression in AML patients showed a shorter remission duration. It is concluded that FoxO3a may be a oncoprotein in AML, and the high FoxO3a expression is associated with poor prognosis.

A preliminary study on efficacy of allogeneic hematopoietic stem cell transplantation by myeloablative conditioning regimen with fludarabine for high risk leukemia patients / 中国实验血液学杂志

The aim of this study was to explore the efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) by myeloablative conditioning regimen with fludarabine for high risk leukemia patients. 25 refractory and relapsed leukemia patients underwent allo-HSCT with new conditioning regimen consisted of fludarabine, busulfan and cyclophosphamide. Donors for 15 patients were sibling, but donors for the rest 10 patients were all unrelated. HLA matched and mismatched donors were for 15 and 10 patients respectively. The graft versus host disease (GVHD) prophylaxis included cyclosporine A and methotrexate, while mycophenolate mofetil and rabbit anti-T-lymphocyte globulin (ATG) were used in case of unrelated and HLA mismatched HSCT. The results showed that unrelated donor HSCT in 10 cases was successful (100%), 14 out of 15 patients with donors of sibling or parent also reconstructed their haematopoietic system. One mismatched patient (4/6) died of graft failure. The time from transplantation to ANC > 0.5 × 10(9)/L and Plt > 20 × 10(9)/L were 13 (11 - 19) days and 13 (12-20) days after transplantation respectively. The cumulative incidence of grade II-IV acute GVHD and chronic GVHD was 12.5% (3/24) and 47.4% (9/19), respectively. In a follow-up duration of 6-84 months, 12 patients were dead, out of which 8 died of relapse; 1 cases died of regimen-associated side effect. 3 cases died of serious infection. The other 13 patients remained alive and disease-free survival probability was 48.7%. It is concluded that allo-HSCT by myeloablative conditioning regimen with fludarabine is a safe and effective option for high risk leukemia patients, which reduces aGVHD incidence and regimen-associated side effect, but it should be modified for higher rate of relapse.

Expression of pituitary tumor transforming gene in patients with AML / 中国实验血液学杂志

The aim of study was to explore the expression of pituitary tumor-transforming gene (pttg) in acute myeloid leukemia (AML) and its relationship with the pathogenesis of AML, simultaneously to investigate the difference of the pttg expression among AML different subtypes. The expressions of pttg mRNA were quantitatively detected by real-time fluorescence quantitative polymerase chain reaction (real-time PCR) in bone marrow from 47 patients with AML and 28 normal controls. The results indicated that the expression of pttg mRNA was significantly higher in AML patients [(1.1323 ± 1.3934) × 10(5)] than that in normal controls [(4.5766 ± 1.1817) × 10(3)] (p < 0.05). The expression of pttg mRNA was higher in M(3) patients than that in other AML subtypes, such as M(1), M(2), M(4), M(5). It is concluded that the overexpression of pttg may be related to the pathogenesis and progression of AML, in which the overexpression of pttg may be more intimately related to the pathogenesis and progression of M(3). This study provides a new idea to research the pathogenesis and targeted gene therapy of AML.

Significance of platelet parameters and lactate dehydrogenase level in differential diagnosis for thrombocytosis / 中国实验血液学杂志

This study was purposed to explore the clinical application of mean platelet volume (MPV), platelet distribution width (PDW), platelet-large cell ratio (P-LCR), lactate dehydrogenase (LDH) level in the differential diagnosis of thrombocytosis. The clinical applications of 3 platelet routine laboratory parameters (MPV, PDW, P-LCR) and LDH were examined in 1048 patients with thrombocytosis-related diseases: reactive thrombocytosis (RT), chronic myeloproliferative disease (CMPD) including chronic myeloid leukemia (CML), essential thrombocythemia (ET) and polycythemia vera (PV). Receiver operating characteristic (ROC) curve was used to predict the cause of thrombocytosis. The results indicated that there were significant differences in MCV, PDW, P-LCR and LDH level between RT and CMPD groups (p < 0.05). The area under ROC curve of PDW and P-LCR for prediction of CMPD were 0.96 (95% CI: 0.93 - 0.99) and 0.89 (95% CI: 0.84 - 0.95) respectively, and whose optimal cut-off value was 11.95%and 23.05% respectively. Three types of CMPD were characterized as follows: high P-LCR and high LDH level in chronic myeloid leukemia, whose optimal cut-off value was 424 U/L and 26.10% respectively; slightly high LDH level and high Plt count in ET, the optimal cut-off value of Plt was 939 x 10⁹/L. In conclusion, these characteristics of MPV, PDW, P-LCR and LDH levels may be useful for simple and primary differential diagnosis of the thrombocytosis-related disease mentioned above.

Expression of FLT3 internal tandem duplication in pediatric patients with acute myeloid leukemia and its correlation with multidrug resistance / 中国实验血液学杂志

This study was aimed to investigate the expression of FLT3 internal tandem duplication (FLT3-ITD) in pediatric patients with acute myeloid leukemia (AML) and to analyse the clinical features of patients with mutations and the relation of FLT3-ITD with multidrug resistance gene 1 (mdr1). RT-PCR was used to determine the expressions of FIT3-ITD and mdr1 gene in bone marrow samples from 81 new diagnosed pediatric patients with AML, the cytogenetics and immunophenotypes of bone marrow cells were routinely examined. The results indicated that the FLT3-ITDs were detected in 8 out of 81 pediatric patients (9.88%) and all mutations detected were hybrid, while less frequently this mutation was detected in adult patients. Although they were irrelevant with sex and immunophenotypes, the mutations seemed predominant in older pediatric patients. The leukocyte counts and bone marrow blast cell counts in pediatric patients with FLT3-ITD at diagnosis were higher than those in pediatric patients without FLT3-ITD (p = 0.001 and p = 0.041 respectively), but the normal chromosomes were found in most pediatric patients with FLT-ITD. The patients with FLT3-ITD had lower induction remission rate (only 25%), but the patients without FLT3-ITD had higher remission rate (76.1%). According results detected by RT-PCR, the mdr1 gene was found in 27 pediatric patients, but only 3 out of 8 pediatric patients with FLT3-ITD were detected to express both FLT3-ITD and mdr1, which suggests unrelation between FLT3-ITD occurrence and mdr1 expression. It is concluded that the FLT3-ITD is frequent mutation in pediatric patients with AML, the prognosis is worse and the induction remission rate is lower in these patients, but the FLT3-ITD not relates with the mdr1, which suggests that the common MDR modulators may be un effective for therapy of the patients with FLT3-ITD.

Treatment of stage IV neuroblastoma with allogeneic hematopoietic stem cell transplantation in children / 中国当代儿科杂志

<p><b>OBJECTIVE</b>At present there is no effective therapeutic approach for stage IV neuroblastoma. We report our experience with allogenic hematopoietic stem cell transplantation as a means of treating this disorder in one child.</p><p><b>METHODS</b>A 7-year-old boy with stage IV neuroblastoma received allogenetic hematopoietic stem cell transplantation. The donor was his mother who was haploid HLA-matched to the patient. Conditioning regimen consisted of fludarabin and melphalan. Stem cells were collected from peripheral blood and bone marrow of the donor.</p><p><b>RESULTS</b>The patient achieved hematopoietic reconstruction and was converted to full donor chimerism according to short tandem repeat sequence-polymerase chain reaction detection. The patient's neutrophil count recovered to more than 0.5 x 10(9)/L 10 days after transplantation. The patient's platelet count recovered to more than 20 x 10(9)/L 11 days after transplantation. Acute graft versus host disease occurred 8 days after transplantation and was improved after treatment. The patient survived in a 210-day-follow-up.</p><p><b>CONCLUSIONS</b>Haploid HLA-matched allogeneic hematopoietic stem cell transplantation from parent donor was an alternative, safe and effective treatment for children with stage IV neuroblastoma.</p>

Clinical study on transplantation of G-CSF-mobilized allo-PBSC and allo-bone marrow for leukemia patients / 中国实验血液学杂志

To study clinical outcome of G-CSF-mobilized allo-peripheral blood stem cell (PBSC) and allo-bone marrow (BM) transplantation for patients with leukemia, donors were injected G-CSF 8-10 microg/(kg.d) for 5 days, PBSC were collected on day 4-5 and G-CSF mobilized BM was extracted on day 7. Conditioning regimen consisting of fludarabine, busulfan and cyclophosphamide. The results showed that transplanted cells in all patients were engrafted, the median days of neutrophil exceeding 0.5 x 10(9)/L and platelet exceeding 20 x 10(9)/L were 10.2 days (range 9 - 12 days) and 12.5 days (range 12 - 14 days), respectively. Patients were monitored up to 100 days, 4 of 12 patients (33.3%) developed II aGVHD, 10 of 12 patients (83.3%) developed limited cGVHD. The median follow-up duration was 5 months. Two patients died, the others were alive in disease-free situation. In conclusion, allogeneic transplantation of G-CSF mobilized PBSC plus BM was safe and effective treatment for patients with leukemia, the therapy provides rapid and sustained engraftment without increase in incidence of aGVHt and cGVHD.