Construction and expression of fusion protein TRX-hJagged1 in E.coli BL21 / 中国实验血液学杂志

This study was purposed to construct prokaryotic expression vector and to investigate the expression of Notch ligand Jagged1 in E.coli. An expression vector pET-hJagged1 was constructed, which can be inserted in Jagged1 with different lengths, but the DSL domain of human Jagged1 should be contained. Then the recombinant plasmids were transformed into the competent cell of E.coli BL21, and the expression of the fusion protein was induced by IPTG. Fusion protein was purified from the supernatant of cell lysates via the Nickel affinity chromatography. The results showed that prokaryotic expression vectors pET-hJagged1 (Bgl II), pET-hJagged1 (Hind I) and pET-hJagged1 (Stu I) were successfully constructed, but only pET-hJagged1 (Stu I) could express the soluble TRX-hJagged1. The purified TRX-Jagged1 protein could be obtained via the Nickel affinity chromatography, and then confirmed by Western Blot. It is concluded that prokaryotic expression vector pET-hJagged1 is successfully constructed, but only pET-hJagged1 (Stu I) can express the soluble TRX-hJagged1 and the TRX-Jagged1 fusion protein is obtained through the prokaryotic expression system, which laid a solid foundation for further to explore the effects of Jagged1 in hematopoietic and lymphoid system.

Clinical analysis of peripheral blood stem cell mobilization regimens in autologous transplantation for treating non-Hodgkin's lymphoma / 中国实验血液学杂志

The purpose of this study was to compare the efficacy of CEP plus G-CSF and CVP plus G-CSF regimens in the mobilization and collection of peripheral blood hematopoietic stem cells (PBHSC), and in the hematopoietic recovery. 57 patients with non-Hodgkin's lymphoma (NHL) underwent autologous PBHSC transplantation were analyzed retrospectively. The PBHSC were mobilized and collected by using CEP plus G-CSF and CVP plus G-CSF respectively, and were retransfused into these NHL patients after preconditioning, then the mobilization efficacy, adverse reactions and hematopoietic recovery were analyzed. The results showed that the WBC count decreased to ≤ 1.0 × 10(9)/L, platelet amount dropped to ≤ 40 × 10(9)/L during peripheral blood stem cell mobilization of all patients, which indicated successful collection of PBHSC. The mean value of (4.38 ± 3.40) × 10(8)/kg mononuclear cells (MNC) containing (2.79 ± 2.53) × 10(6)/kg CD34(+) cells were collected in CEP plus G-CSF group, while the mean value of (3.31 ± 1.23) × 10(8)/kg MNC containing (2.02 ± 0.87) × 10(6)/kg CD34(+) cells were collected in CVP plus G-CSF group. The efficacy of mobilization in CEP plus G-CSF group was significantly higher than that in CVP plus G-CSF group (p < 0.05). After preconditioning, bone marrow was suppressed in all patients. The average time of WBC count recovery to ≥ 1.0 × 10(9)/L was 11.4 days in CEP plus G-CSF group and 12.3 days in CVP plus G-CSF group; the average time of platelet amount recovery to ≥ 50 × 10(9)/L was 18.6 days in CEP plus G-CSF group and 19.3 days in CVP plus G-CSF group. The statistical analysis showed no significant difference in the average time of hematopoietic recovery between 2 groups. It is concluded that autologous PBHSC transplantation shows significant effect for treatment of patients with NHL. Either modified CEP or CVP plus G-CSF regimen is safe and effective in PBHSC mobilization. The CEP plus G-CSF regimen is better than CVP plus G-CSF regimen.

Activating effects of protein transduction domain mediated BCR/ABL protein on CML T cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the activating effect of protein transduction domain (PTD) mediated BCR/ABL protein on T cells from CML patients.</p><p><b>METHODS</b>The plasmid containing PTD and b3a2 bcr/abl of CML was constructed by genetic engineering and expressed in E. coli. The peripheral blood mononuclear cells from CML patients were stimulated in vitro with purified PTD-BCR/ABL protein and the expression of the early activation antigen CD(69) on CD(8)(+) and CD(4)(+) T cells was detected by flow cytometry (FCM).</p><p><b>RESULTS</b>The optimal concentration of PTD-BCR/ABL protein for activating CD(8)(+) T cells in vitro was 100 micro g/ml, CD(69) expression peaked in three days stimulation. CD(8)(+) T cells were activated in 10 of 15 CML patients, the expression rate of CD(69) was (15.01 +/- 3.75)%. CD(4)(+) T cells were activated in 4 of 15 patients, the expression rate of CD(69) was (10.32 +/- 3.08)%. Both CD(8)(+) and CD(4)(+) T cells were activated simultaneously in 3 of them. However, neither CD(4)(+) nor CD(8)(+) T cells was activated by stimulation with BCR/ABL protein in all 15 specimens, the expression rate of CD(69) on CD(8)(+) and CD(4)(+) T cells was (1.36 +/- 0.31)% and (1.41 +/- 0.43)%, respectively. There was no difference compared with that of PBS control group (P > 0.05).</p><p><b>CONCLUSION</b>By using a PTD-mediated antigen delivering system, exogenous BCR/ABL protein can be delivered into APC, processed and presented onto surface of APC to activate Ag-specific CD(8)(+) and CD(4)(+) T cells in vitro.</p>

Clinical analysis of retinoic acid syndrome developed in 11 patients with acute promyelocytic leukemia / 中国实验血液学杂志

To explore the clinical features, risk factors an d treatment of retinoic acid syndrome (RAS) in patients with acute promyelocytic leukemia (APL) treated with retinoic acid, the clinical and laboratory data of 11 APL patients with RAS were retrospectively analysed. The results showed that earlier and more common symptoms of RAS were successively dyspnea (11/11), fever (10/11) and hydrothorax (6/11). Higher WBC count (> or = 15.0 x 10(9)/L) in the course of treatment of all-trans retinoic acid susceptible to develop RAS (9/11). The RAS patients were treated with dexamethasone without discontinuing the treatment of retinoic acid, complete remission was achieved in 10 cases and one patient died from disseminated intravascular coagulation. It is concluded that the identification and dexamethasone treatment of RAS in earlier period are extremely important for obtaining better clinical curative effect, and it does not influence therapeutic effect of continuing application of retinoic acid.

Alteration of the Cell Cycle during the Differentiation of HL-60 Cells by Induction of Retinoic Acid / 中国实验血液学杂志

To study the alteration of the cell cycle during the differentiation of human myeloid leukemia cell line HL-60 induced with all trans-retinoic acid (RA), the flow cytometry was used to assay the various phases of cell cycle in HL-60 cells treated with RA. The results showed: (1) S + G(2)/M phase proportion kept relative invariability during the 48 hours incubation of HL-60 cells with RA, however, the proportion alteration of S-phase cells was associated with the RA concentrations. At 10(-6) mol/L RA, the proportion of S-phase cells appeared a temporarily increasing peak followed by persistent decrease of S-phase proportion. At 10(-5) mol/L RA, S-phase cell proportion only appeared the persistent decreasing tendency. (2) Re-culture of HL-60 cells without RA showed the decrease of S + G(2)/M and S-phase cells was associated with the increase of differentiated cells, but not all HL-60 cells were triggered into differentiation at the same time. Once the cells start to differentiate, even if there is no RA presence, the HL-60 cells were still differentiated until maturation. In conclusion, HL-60 cells are able to differentate and maturate after exposure to RA for a period of time. S-phase proportion is related to the concentrations of RA. Once the cells start to differentiate, even if there is no RA presence, the HL-60 cells still differentiatiated until maturation.