Mobilized peripheral blood stem cells (PBSC) have largely replaced the use of bone marrow as a source of stem cells for both allogeneic and autologous stem cell transplantation.G-CSF with or without chemotherapy is the most commonly used regimen for stem cell mobilization.Some donors or patients,especially the heavily pretreated patients,fail to mobilize the targeted number of stem cells with this regimen.The generation of new mobilization agents and new therapies can improve the mobilization of hematopoietic stem cells.

Objective To observe the efficacy of imatinib on the treatment of bcr-abl positive essential thrombocythemia (ET). Methods A case of bcr-abl positive ET resistant to hydroxyurea (HU) treating with imatinib (200～400 mg/d) was reported and related literatures were reviewed. Results A case of bcr-abl positive ET was initially treated with 1.5～2.0 g/d HU, the platelet count decreased to 562x109/L after 4 weeks; however, the platelet count increased to (1020～1330)×109/L treating with same dose of HU 16 months later. With the elevation of HU to 3.0 g/d, platelet count was still high(1290～1780)x109/L companied with the very low white blood cell count(0.3～0.9)×109/L. While treating with imatinib (400 mg/d) for 1 month,the platelet count decreased to 390×109/L and white blood cell count was 0.5×109/L; Furthermore, treating with 200×300 mg/d of imatinib, the platelet and white blood cell count recovered in normal after 1 month,and bcr-abl fusion gene negative 2 months later. Conclusion Imatinib may be the effective targeting drug for the bcr-abl positive ET, and the bcr-abl positive ET is sensitive to low dose imatinib.

Objective To investigate the efficacy of imatinib combining with allogeneic hematopioetic stem cell transplantation or chemotherapy for bcr-abl positive acute lymphoblastic leukemia (ALL). Methods 12 cases were diagnosed on morphology, cytochemistry, immunophenotype and bcr-abl fusion gene. The induction is imatinib (400 mg/d) combining chemotherapy. 8 cases accepted allogeneic hematopoietic stem cell transplantation after complete remission (CR). If bcr-abl became positive, the patient was treated with imatinib (400～600 mg/d). 3 cases were tested with imatinib alternating chemotherapy after cr. Results 11 patients gained CR, CR rate 91.7 %; 5 patients (41.7 %) became bcr-abl negative through 2 courses induction. 3 cases relapsed after transplantation. 2 cases relapsed in imatinib combining chemotherapy group. The median remission interval is 16 months (imatinib combining transplantation group) and 10 months (imatinib combining chemotherapy group) (P <0.01) respectively. The median survival time is 18 months (imatinib combining transplantation group), and the other group (imatinib combining chemotherapy) is 12 months (P <0.01). Conclusion Imatinib combining chemotherapy achieved high CR rate for the bcr-abl positive ALL. Imatinib combining allogeneic hematopoietic stem cell transplantation is superior to imatinib combining chemotherapy for CR patients.

AIM: To explore the characters of fibroblast-like (F-L) cells cultured from granunocyte clony stimunating factor (G-CSF)-mobilized peripheral blood cell (PBC) harvests. METHODS: The adherent cells in the PBC harvests were cultured for 2 week in the mediums of RPMI-1640/L-DMEM/G-CSF or interleukin-3 (IL-3) plus RPMI-1640, the cultured F-L cells were analyzed by flow cytometry (FC). RESULTS: The adherent non-confluent F-L cells obtained from the four groups were similar in their phenotypes: CD33+, CD11c+, CD64+, CD14+, CD45+, HLA-DR+, CD86+, CD34-, CD38-, CD3-, CD19-, CD56-, CD29-, CD44-, CD105-. The F-L cells are similar to monocytes except CD38-and were distinct from dendritic cells (DC) or mesenchymal stem cells (MSC). CONCLUSION: The cultured F-L cells are macrophages rather than DC or MSC. G-CSF, rhIL-3 enhances their numbers.

Objective To study the distributive characteristics of HLA-A,B,DRBI alleles and haplotypes patients with ALL in southern Chinese Han.Methods The frequencies of HLA-A,B,DRB1alleles and haplotypes were estimated by Expectation-Maximization method based on the genotypes of 572patients with ALL and 5645 unrelated health donors,and then compared by chi-square test.Results The frequencies of HLA-A33(7.15%vs 9.3%,OR=0.73,P＜0.05),B58(5.93%vs 8.75%,OR=0.64,P＜0.05),DRB1*17(5.15%vs 6.30%,OR=0.82,P＜0.05)alleles and HLA-A33-B58-DRB1*17(2.46%vs 4.14%,OB=0.35,P＜0.05)haplotype were significantly lower in ALL patient groups than that in controls.The frequencies of HLA-A3(2.1%vs 1.26%,OR=1.7,P＜0.05),B51(7.25%vs 5.78%,OR=1.3,P＜0.05)and DRB*12 (16.13%vs 12.99%,OR=1.35,P＜0.05)alleles and A2-B51-DRB1*12(1.24%vs.0.89%,OR=1.66,P＜0.05)haplotype were significantly higher in ALL patient groups than that in controls.Conclusion These results indieated that HLA-A33-B58-DRB1*17 haplotype was a associated with a diminished incidence of ALL.and HLA-A3 auele or A2-B51-DRB1*12 haplotype was weakly associated with ALL.

AIM: To establish a arsenic trioxide (As 2O 3 )-resistant leukemic cell line to explore the mechanism of resistance to As 2O 3, and the relationship between the resistant cell line and the multidrug resistance was also investigated. METHODS: The arsenic trioxide (As 2O 3 )-resistant leukemic cell line was established by exposing the cells to the increasing concentration of As 2O 3. MTT assay was used to detect the cytotoxicity. Cell cycle was detected by PI assay. Flow Cytometry was used to detect the P-glycoprotein on the surface of the cells, the intracellular concentration of DNR, and the immuetype of the cells. RESULTS: The cell doublings time and the cell cycle of the arsenic trioxide (As 2O 3 )-resistant leukemic cell line, K562/AS2, is similar to that of K562. The relative resistant fold of K562/AS2 to As 2O 3, DNR, VP16 and Ara-C was 7.4, 2.9, 3.8 and 1.1, respectively. The relative resistant fold of multidrug resistant cell line, K562/ A02, to As 2O 3, DNR, VP16 and Ara-C was 0.8?94?2.5 and 0.9, respectively. The fluorescence of the P-glycoprotein on the surface or of the DNR inside the cells detected was not significantly different between the K562 and the K562/AS2 cell lines. CONCLUSIONS: A cell line, K562/AS2, resistant to clinical achieving level (2 ?mol/L) of As 2O 3 has been established. The relative resistant fold of K562/ AS2 to As 2O 3 is about 7.4 fold to the parent K562 line sensitive to As 2O 3. Partial resistance of K562/AS2 to DNR and VP16 is observed , the mechanism of which is unrelated to the P-gp, the expression product of multidrug resistance gene 1 (mdr1).

Objective To explore the relationship between intracellular concentration of arsenic trioxide (ATO) in ATO-resistant K562 cells (K562/AS2) and ATO resistance level.Methods The K562/AS2 cells were established by gradually increasing the concentration of ATO from the parental cell line,K562.Arsenic concentration was measured with atomic fluorescence photometry.Cell viability was assessed using MTT assay.Results At exposure to 1 μg/ml ATO for 24 h,48 h and 72 h,the arsenic concentration in the K562/S cells were all significantly higher than that in the K562/AS2 cells,(15.63± 0.42) μg/L vs 0 μg/L,(22.27±0.15) μg/L vs (3.51±0.12) μg/L and (24.31±0.21) μg/L vs (3.61±0.11) μg/L (P ＜ 0.05).With increasing concentration and the extension of incubation time,concentration of arsenic in cells was gradually increased (P ＜ 0.05),which increase quickly between 1 μg/ml and 2 μg/ml.The growth inhibition rate of K562/AS2 cells was also gradually increased (P ＜ 0.05),which increased quickly between 1 μg/ml and 2 μg/ml.Linear correlation analysis showed that when the K562/AS2 cells was exposed to ATO for 24 h,48 h and 72 h,respectively,the cell growth inhibition rates were positively correlated with the intracellular concentration of ATO.Conclusions Either increasing concentrations of ATO or prolonging the exposure time to ATO can increase intracellular concentration of ATO in ATO-resistant cells,and intracellular arsenic concentration is positively related to the cytotoxicity of ATO to K562/AS2 cells.Therefore,the sensitivity to ATO of ATOresistant K562 cells could be restored by increasing the intracellular concentration of ATO.

Objective To investigate sequence variations of 12 miRNA genes in multiple myeloma(MM) in order to find whether sequence variations in miRNA genes are associated with tumorigenesis and discuss the clinical significance of MM associated with miRNA genes mutations. Methods The miRNA gene mutations in 20 cases of MM, 4 MM-derived cell lines and 20 controls were detected by the methods of polymerase chain reaction single stranded conformation polymorphism (PCR-SSCP) and silver staining technique. Both clinical features and laboratory results were analyzed simultaneously. Results The electrophoretic patterns showed a total of three variations in miR-19a, miR-19b and miRNA-335,which were observed in 3 MM cells (15 %, 3/20). We also found variations of miRNA-335 in MM-derived cell lines KM-3and RPMI8226. However, no sequence alteration in the miRNA genes was observed in our set of controls. One of the three MM patients died, and two of them were detected mutations at the terminal stage of the disease.Conclusion A relative high frequency of miRNA gene mutation was found in MM and MM derived cell lines, which suggests possibility of a main mechanism underlying tumorigenesis. And, detecting miRNA gene mutations in MM might be benefit to evaluate the progression and prognosis of disease.

Objective To construct a recombinant adenovirus vector of Hairpin RNA specific for MRP1 gene and study its inhibition of MRP1 gene expression in K562/AS2 cell line resistant to AS_2O_3 (ATO). Methods A MRP1-specific hairpin RNA recombinant adenovirus vector was constructed and used to infected K562/AS2 cells. Expression level of MRP1 mRNA detected by real-time fluorescent quantitative PCR. MRP1 protein detected by flow cytometry. MTT method was used to detected the cytotoxicity of ATO and etoposide. Results MRP1 mRNA and protein expression level in K562/AS2 cells before and after the pAd-MRPl-shRNA adenovirus infection was (34.70±0.28 vs 4.19±0.03, P <0.05) and (26.40±0.16 vs 10.85±0.37, P<0.05), respectively. RR of K562/AS2 to arsenic trioxide and etoposide was (11.4078±0.3183 fold vs 1.6126±0.3015 fold, P<0.05) and (5.9141 ±0.0149 fold vs 1.7664±0.1038 fold, P <0.05), respectively. The reversal fold of ATO and etoposide was (7.2409±1.3668) and (3.3555±0.1886), respectively. Conclusion Successfully constructed pAd-EGFP-U6-shRNA-MRPl adenovirus vector, the vector of infection K562/SA2 cells can inhibit MRP1 gene expression and reverse the resistance of the ATO and etoposide.

Objective To investigate the reversal effect of Topo Ⅱα-shRNA and Topo Ⅱβ-shRNA on Topo Ⅱ gene in K562/AS2 cells. Methods Three pieces of Topo Ⅱα-shRNA and three pieces of Topo Ⅱβ-shRNA were designed,synthesized and transfected into K562/AS2 cells by liposome. Expression level of Topo Ⅱα and Topo Ⅱβ mRNA were determined by real time fluorescence quantitative PCR. The expressions of Topo Ⅱα and Topo Ⅱβ protein were assayed with flow cytometer. Results After treated with Topo Ⅱα-shRNA or Topo Ⅱβ-shRN A for 24 hours,the expression level of Topo Ⅱα mRNA and Topo Ⅱβ mRNA protein in K562/AS2 cells decreased at most (78.22±0.01) %,(31.17±1.27) % (P <0.05),and (57.36±0.01)%,(23.98±1.22) % (P <0.05) respectively. Conclusion The expression of Topo Ⅱ gene can be down-regulated after infected with Topo Ⅱ-shRNA in K562/ AS2 cell line.