Objective To establish the Flow-FISH method for simultaneous detection of telornere length and cell differentiation antigen. Methods HL60, Raji, Molt4 cells were cultivated. Each step and the conditions of the Flow-FISH procedure were optimized, standardized and validated, then 14 acute leukemia patients were observed for the changes of telomere length combined with differentiation antigen after complete remission by the method. Results Cells were stained with Alexa Fluor(R) 647-labeled antibody. Anti-gen-anfibedy complexes were covalenfly cross-linked onto the cell membrane before telomere staining. Cells were hybridized with telomere-specific fluorescein isothiocyanate (FITC)-conjugated peptide nucleic acid (PNA) probes followed by being counterstained with propidium iodide(PI). Multicolor Flow-FISH was performed to analyze telomere length and differentiation antigen simultaneously. The patients showed longer telomere and lower antigen expression after complete remission. Conclusion The Flow-FISH method for simultaneous detection of telomere length and differentiation antigen was successfully established which might prove to be a promising means for leukemia research especially in those without special molecular markers.

BACKGROUND: Recombinant adeno-associated virus 2(rAAV-2) has attracted considerable attention due to its nonpathogenic nature in contrast to other viral vectors such as adenoviral and retroviral vectors in gene therapy attempts.OBJECTIVE: To explore rAAV-2 transduction to bone marrow mesenchymalstem cell(BMSC) in vitro and evaluate the possibility of using rAAV-2 as a vector for gene therapy of acute myelogenous leukemia(AML).DESIGN: An open experiment with cells as the observational subjects.SETTING: Department of Hematology, Nanfang Hospital, Southern Medical University.MATERIALS: The experiment was conducted in the Department of Hematology, Nanfang Hospital, Southern Medical University from February to July 2004. We used passages 3 to 5 BMSCs derived from six de novo AML patients and four healthy volunteers in this study.METHODS: BMSC was isolated from 6 to 10 mL of bone marrow aspirates obtained from the iliac crests of the patients who had been diagnosed as having de novo AML and from those of healthy volunteers. The acquired BMSC was infected by rAAV-2 which contained enhanced green fluorescent protein (rAAV-2-eGFP) at different multiplicity of infection(MOI) (MOI = 1 × 102,1 × 103, 1 × 104, 1 × 105, 1 × 106, 1 × 107) . Then we observed through phase contrast fluorescent microscope and flow cytometer to evaluate green fluorescent protein(GFP) expression 10 to 14 days after transduction. GFP expression was observed as the rAAV-2-eGFP transduced BMSC cultured in vitro. We also observed the in vitro gene expression profile of GFP in rAAV-2-eGFP transduced BMSC which was selected by neomycin ( G418). First, we confirmed GFP expression in BMSC through phase contrast fluorescent microscope, then on flow cytometer to detect the percentage of GFP expression.MAIN OUTCOME MEASURES: The efficiency of rAAV-2-eGFP transduction to BMSC. GFP expression was observed through phase contrast fluorescent microscope and flow cytometer at different time points after transduction.rAAV-2-eGFP to BMSC derived from normal volunteers and AML patients had no significant differences. GFP began to express 10 to 14 days after transduction, and the transduction efficiency ranged from 0. 3% to 1.4%. By changing infection condition, we could not make a higher transduction efficiency( P ＞ 0.05) . One round infection of BMSC by rAAV-2-eGFP at a MOI of 1 × 105 was ( 1. 030 ± 0. 034) %, 3 rounds of infection of BMSC by rAAV-2-eGFP at a MOI of 1 × 105 was (1. 140 ±0. 036)%, and coinfected by LipofectAMINE was (1. 380 ± 0. 054)%. However, 293 cell line which was the package cell of rAAV-2 could be efficiently transduced by AML patients transduced by rAAV-2-eGFP at MOI = 1 × 105: The percentage of GFP expression cell gradually decreased from 1.14% at day 12 after transduction to 0. 6% as cell passaged from 2 to 3, and maintained at a level of 0. 5% to 0. 6% later on till 61 days after transduction. After selected by neomycin(G418) 1 month later, rAAV-2-eGFP transduced BMSCs could maintain a long-term GFP expression at a level of 6.0% in vitro without significant decay within 100 days of observation period after transduction.CONCLUSION: The advantages of rAAV-2 mediated gene transduction lie in safety, no immune response to the host, and long-term expression maintained by the target gene. rAAV-2 and BMSC can be used for in vitro gene therapy, and as a systemic gene delivery system, it might be an alternative for systemic gene therapy in the future.

Objective To study the relationship between the MDR1 gene expressions and CD56 antigen expression in patients with de novo acute myeloid leukemia(AML) and to explore the role of this two factors in clinical drug resistance and their correlation. Methods A real-time quantitative RT-PCR method was established for detecting MDR1 expression levels and three-color flow cytometry analysis using CD34/ SSC gating was used to examined CD56 antigen expression in 79 de novo AML patients. Results CD56 an-tigen was recorded in 19 out of 79 cases (24.1%) and particularly in those with M5 cytotypes. Moreover, CD56 expression was significantly associated with unfavorable cytogenetic abnormalities (P<0.05), Patients with t(8:21)had a significantly higher incidence (57.1%, 4/7) of CD56 expression than those with favora-ble karyotype(P<0.05). CD56~+ AML patients had a higher incidence of splenohepatomegalia and lactate dehydrogenase level than CD56~- patients(P<0.05). The median expression levels of MDR1 was statistical-ly higher in CD56~+ AML patients than that in CD56 patients(P<0.001). Patients with both high levels of MDR1 and CD56~+ had a significantly lower CR(complete remission) rate than those with both low MDR1 level and CD56 (58.8% vs 89.2%, P<0.01). Conclusion There is a linear correlation between MDR1 gene expression and CD56 expression in AML. Quantification of the MDR1 gene expression together with CD56 antigen expression is more effective to the judgement of prognosis in AML.

Objective To investigate the variation of immune index in patients with systemic lupus erythematosus (SLE) treated with autologous purified CD+34 cells transplantation and to clarify the relationship with pathogenesis and prognosis. Methods Flow cytometry (FCM) and enzyme linked immunosorbent assay(ELISA) were used to test lymphocyte subsets, C3, C4, CH50, autoantibodies and immunoglobulin for 18 cases of SLE before and after transplantation. Results The results showed that the ratio of all the T cell subsets reduced obviously in early postgraft and recovered gradually in 1 to 3 months after transplantation except CD45 RO+CD+4 cells. The levels of serum C3, C4, CH50 increased significantly after transplantation. No case relapsed within one year after transplantation, but 2 patients relapsed one year after transplantation. The levels of the indexes in the patients with relapse were significantly lower than those in the patients with persistent remission, including C4 in the entire course, CH50 in the 3rd and 12th month after transplantation and CD45 RA+ CD+8 cells in the 6th month after transplantation. However, the ratio of CD45 RO+ CD+4 cells in the first month after transplantation in the patients with relapse was higher than that in the patients with persistent remission. Conclusion Autologous purified CD+34 cells transplantation is effective for treating SLE. Survey of immune indexes before and after transplantation is important to investigate the pathogenesis of SLE. Moreover, these immune indexes can be used to predict therapeutic efficacy of SLE.

ObjectiveTo investigate the therapeutic effects of the conditioning regimen with or without total body irradiation on allogeneic hematopoietic stem cell transplantation in acute leukemia.Methods We retrospectively evaluated clinical outcomes in 287 allo-HSCT recipients with acute leukemia (ALL- 105,AML-129,and AUL-53) who received myeloablative conditioning regimen with or without total body irradiation from January 2002 to August 2011.Two hundred and six patients obtained complete remission (CR) and 81 non-remission (NR) before transplantation.One hundred and ninety-nine patients received conditioning with total body irradiation (TBI+ Cy group,9 Gy given over 2 days),and 88 patients received busulfan (BuCy group,3.2 mg·kg-1 ·d-1 ),both followed by cyclophosphamide.ResultsThere were no statistically significant differences in hematopoietic reconstitution,regimen-related toxicity (RRT),graft-versus-host disease (GVHD) and relapse between two groups.For patients with AML and AUL,there was no significant difference in the 5-year survival between the two regimens (P＞ 0.05),while for ALL-CR patients,the TBI + Cy regimen had a higher over survival rate (52.0％ vs.31.3％,LogRank=4.249,P＜0.05) and DFS (50.4％ vs.27.8％,LogRank =4.445,P＜0.05) than BuCy.In TBI + Cy group and BuCy group,the proportion of CD19+ B cells at the first month after HSCT was (4.04 ± 1.86)％ and (1.47 ±0.99) ％ (P＜0.05),that of NK cells at 12th month after HSCT was (23.38 ± 12.19) ％ and (13.11± 7.99) ％ (P＜0.05),and that of CD4+ CD45RO+ cells at 9th month after HSCT was (14.63 ±6.17)％ and (9.07 ± 3.12)％ (P＜0.01),respectively.ConclusionUsing TBI-containing regimen was more effective for treating ALL-CR patients than busulphan-containing regimen,but no difference was found for long-term outcomes in patients with AML and AUL between the two regimens.The 9 Gy TBI-based regimens may not affect recipients' thymic function,T-cells reconstitution and immune tolerance,coming out a non-increase of GVHD.

Tetrapyrrole compounds are a class of compounds with important functions. They exist in living organisms and have been widely used in agriculture, food, medicine, and other fields. The cumbersome process and high cost of chemical synthesis, as well as the shortcomings of unstable quality of animal and plant extraction methods, greatly hampered the industrial production and applications of tetrapyrrole compounds. In recent years, the rapid development of synthetic biology has provided new tools for microorganisms to efficiently synthesize tetrapyrrole compounds from renewable biomass resources. This article summarizes various strategies for the biosynthesis of tetrapyrrole compounds, discusses methods to improve its biosynthesis efficiency and future prospects, with the aim to facilitate the research on biosynthesis of tetrapyrrole compounds.
Biomass ; Plants/genetics* ; Synthetic Biology ; Tetrapyrroles