A novel exonuclease protection mediated PCR assay (EPM-PCR) to detect the interaction of protein and DNA at a dioxin-responsive enhancer (DRE) upstream of the CYP1A1 gene in rat hepatic cytosol was established. A double-stranded DNA fragment containing two binding sites was designed and incubated with the aryl hydrocarbon receptor (AhR) transformed by 2,3,7,8-tetrachlorodibenzo-p dioxin (TCDD) to generate TCDD: AhR: DNA complex which could protect receptor-binding DNA against exonuclease II (Exo III) digestion. With Exo III treatment, free DNAs were digested and receptor-bound DNAs remained that could be amplified by PCR. By agarose gel electrophoreses a clear band (285bp) was detected using TCDD-treated sample, while nothing with control samples. To detect transformed AhR-DRE complex, 2 fmol DNAs and 3 ug cytosol proteins were found to be sufficient in the experiment. Compared with gel retardation assay, this new method is more sensitive for monitoring the Ah receptor-enhancer interaction without radioactive pollution.
Binding Sites ; Cytochrome P-450 CYP1A1/*genetics ; Cytosol/metabolism ; DNA-Binding Proteins/chemistry ; Exodeoxyribonucleases/chemistry ; Liver/*metabolism ; Polymerase Chain Reaction ; Rats, Sprague-Dawley ; Receptors, Aryl Hydrocarbon/*chemistry ; Tetrachlorodibenzodioxin/*analogs & derivatives ; Tetrachlorodibenzodioxin/chemistry

A novel exonuclease protection mediated PCR assay (EPM-PCR) to detect the interaction of protein and DNA at a dioxin-responsive enhancer (DRE) upstream of the CYP1A1 gene in rat hepatic cytosol was established. A double-stranded DNA fragment containing two binding sites was designed and incubated with the aryl hydrocarbon receptor (AhR) transformed by 2,3,7,8-tet rachlorodibenzo p dioxin (TCDD) to generate TCDD: AhR: DNA complex which could protect receptor-binding DNA against exonuclease Ⅲ (Exo Ⅲ) digestion. With ExoⅢ treatment, free DNAs were digested and receptor-bound DNAs remained that could be amplified by PCR. By agarose gel electrophoreses a clear band (285bp) was detected using TCDD-treated sample, while nothing with control samples. To detect transformed AhR-DRE complex, 2 fmol DNAs and 3 ug cytosol proteins were found to be sufficient in the experiment. Compared with gel retardation assay, this new method is more sensitive for monitoring the Ah receptor-enhancer interaction without radioactive pollution.

AIM: To find new methods for heavy metals pollution in traditional Chinese medicine(TCM) with macroporous chelating resins. METHODS: We emulsificated crude extracts of TCM and adjusted its pH to 7 first and the pretreated crude extracts was treated by three kinds of macroporous resins(D401,D402 and D001) respectively,then we detected the concentrations of 5 heavy metals(Cu,Pb,Cd,Hg,As) and flavonoids in the crude extracts with or without treatment. RESULTS: The concentrations of all heavy metals decreased significantly after treatment of D401 and D402.The concentrations(mg/L) of Cu,Pb,Cd changed from 0.500,0.521,0.078 to 0.117 and 0.236,0.174 and 0.165,0.024 and 0.045,respectively,and Hg and As became beyond the detection limit after the treatment,but the use of D001 affected a bit quantities of 5 heavy metals.Moreover,the concentration of flavonoids kept almost the same after treatment of such 3 resins.On the condition of room temperature,we found that a fluent velocity of 30 m/s and pH of 7 would bring the superior treatment of D401 to excessive heavy metals in TCM. CONCLUSION: Macroporous chelating resins(D401 and D402) can be employed in the treatment of excessive heavy metals in crude extracts of TCM.

Eight patients with severe aplastic anemia received unrelated cord blood transplantation at the Guangzhou First People’s Hospital from June 1998 to December 2007. The patients were conditioned with decreased dosage of immunosuppressive agents of cyclophosphamide and antilymphocytic globulin. The median infused donor total nucleated cell were 5.69?10 7/kg of recipient weight, and the CD34+ cell was 4.10?105/kg of recipient weight. Methotrexate and corticoid methylprednisolone were used for prophylaxis of graft versus host disease (GVHD). The time to reach an absolute neutrophil count of 0.5?109/L ranged from 7 to 25 days (median: 17 days) and the time to reach a platelet count of 20?109/L ranged from 13 to 102 days (median: 35 days) after transplantation. DNA finger print map of 7 patients showed donor and recipient chimera. Two patients developed grade I to II acute GVHD, which was controlled. One patients developed grade II chronic GVHD, which was controlled by using methylprednisolone. Five patients had lived for 10-108 months, with no diseases. Taken together, unrelated umbilical cord blood transplantation is effective for adult patients. Partial conditioning regiment could ensure engraft of unrelated umbilical cord blood transplantation.

BACKGROUND: At present, non-viral vectors have become a hotspot in gene transfection research because of their strong points of lower toxicity,low immunologic reaction, target orientation, and easy assembly.OBJECTIVE: To evaluate the role of chitosan-DNA nanoparticles in transfection efficiency and cellular toxicity of tumor cells.DESIGN: Observational control trial. SETTING: Institute of Environmental Medical Sciences, Tongji Medical College of Huazhong University of Science and Technology.MATERIALS: Zeta potential/ particle size analyzer; spectrofluorometer;Hoechst 33258; PLPS-3'EGFP plasmid; lung cancer cell A549 and human hepatocarcinoma cell line HepG2.METHODS: This experiment was conducted in the Institute of Environmental Medical Sciences, Tongji Medical College of Huazhong University of Science and Technology, from December 2004 to December 2005. The green fluorescent protein gene was used as report gene; chitosan green fluorescent protein plasmid nanoparticles were prepared with re-coherence gy of prepared nanoparticles; Zeta potential/ particle size analyzer was used to measure the diameter and superficial potential of the nanoparticles;enzyme-protection test was used to measure anti DNA enzyme degradation and lung cancer cell A549 were transfected in vitro. Transfection of the cells was observed under the inverted fluorescence microscope after 24, 48nanoparticles.characteristics: Nucleic acid encapsulation efficiency of chitosan nanoparticles was 91.7%, and the nanoparticles presented globular shape with the mean diameter of 149nm and superficial potential of +20.5 mV.Transfection rate of nanoparticles: It reached the peak 48 hours later; the transfection rate of A549 was 95% while that of HepG2 was only about chitosan could inhibit the growth of HepG2 and A549, and the inhibitory effect of chitosan on cellular growth was stronger than that of the nanoparticles.CONCLUSION: Nanoparticles of chitosan plasmid can transfect HepG2and A549, two kinds of tumor cells, and have inhibitory effects on their growth, suggesting that nanoparticles, as the carrier of DNA, can be used in the transfection of tumor cells.

Objective To investigate the effect of down-regulation of lysine specific demethylase 1 (LSD1) by shRNA on the apoptosis and cell cycle of human acute myelogenous leukemia cells.Methods The lentiviral vector-mediated LSD1-shRNA was transfected into human acute promyelocytic leukemia HL-60 cells and acute monocytic leukemia SHI-1 cells.The expressions of LSD1 mRNA and protein were examined by real time quantitative PCR and Western blot,respectively.The flow cytometry was applied to detect the apoptosis and cell cycle distribution after AnnexinV-PE/7-AAD and PI dying,respectively.Results The expressions of LSD1 mRNA and protein in HL-60 and SHI-1 LSD1-shRNA group were significantly decreased compared with the blank control group and the negative shRNA group (P ＜ 0.01,respectively).The apoptosis levels of HL-60 and SHI-1 cells were significantly increased after knockdown of LSD1 (P ＜ 0.01).Moreover,the cell cycle distribution in the G0/G1 phases was also significantly increased(P ＜ 0.01).Conclusion LSDI-shRNA promotes cell apoptosis and increases the percentage of cells in the G0/G1 phases of human acute myelogenous leukemia cells.

Objective To discriminate morphology and immunophenotype differences between hematogones and lymphoblast to provide some references for the correct identification of hematogones and minimal residual leukemia cells.Methods Immunophenotypes were detected by flow cytometry in a total of 132 bone marrow from 58 patients with acute B lymphoblastic leukemia during diagnosis,remission and relapse.Hematogones were identified based on combination of CD34/CD10/CD19/CD45 or CD34/CD10/CD45/CD19/CD20/CD38.Results Among 132 specimens,45 (34 ％) were identified hematogones,the detection range was 0-36 ％.Three specimens appeared in diagnosis patients,one in relapse,and the remaining 41 cases in remission.The detection rate of hematogones was 62 ％ (41/66) in the remission cases.More than 5 ％ leukemia cells of nucleated cells were detected in diagnosis and relapse,and less than 5 ％ residual leukemia cells was in 24 specimens from remission patients.In 28 specimens,the co-existence of hematogones and leukemia cells was found,including three in diagnosis,one in relapse and the remaining 24 in remission.Hematogones were characterized in term of variable expression of CD45 and very low side scatter.The early hematogones expressed CD34.With maturation increasing,hematogones gradually lacked CD34.CD19 and CD10 were presented in whole hematogones stage.Early hematogones had expression of CD10.Lymphoblasts showed maturation arrest and more homogeneous populations.SSC values of hematogones were higher than that of normal B cell progenitors.Antigen overexpression or underexpression was not found in normal hematopoietic progenitor B cells,and hematopoietic progenitor B cells did not appear cross-lineage markers,CD20+ cells exhibited continuous distribution from negative to weak positive for normal hematogones.Conclusions Hematogones were present in diagnosis,remission and relapse cases with acute B lymphoblastic leukemia,especially abundant in bone marrow after chemotherapy.It should be careful to diagnose and discriminate the malignant cells from benign cells.By comprehending continuous and complete maturation spectrum of antigen expression for normal hematogones,knowing phenotype of leukemia cells drift change patterns and using multiparameter flow cytometry and optimal antibody combination,it is significant in identifying residual lymphoblasts from hematogones and improving the detection accuracy in minimal residual disease.

Objective To investigate the detectable significance of multiparameter flow cytometry (MFC) for the first visiting and minimal residual disease (MRD) in the patients with multiple myeloma .Methods MFC was used to identify the plasma cells by the expression of CD138 or CD38 antigen in 74 patients with multiple myeloma .By combining surface antigens like CD45 ,CD56 , CD19 ,CD20 ,CD117 and the cytoplasm Kappa and Lambda light chain ,the aberrant myeloma cells were differentiated from normal plasma cells .Results In the 44 first visiting cases ,the positive expression of CD138 can be detected in all cases ,while the expres‐sion of CD19 was negative and 42 cases (95% ) were negative or weak positive expression for CD45 .The detection rates of CD38 , CD56 ,CD20 and CD117 were 98% ,93% ,45% and 41% ,respectively .The cytoplasm Kappa and Lambda light chains were showed the limited expression .Of the patients with MM ,14 cases were used for evaluating the change of immunophenotype at first visiting and during the treatment process ,among them ,11 cases(79% ) appeared the changes in at least one of aberrant phenotypes .4 cases (29% ) had the significant enhancement of antigen marker fluorescence intensities after chemotherapy and 7 cases (50% ) had sig‐nificant decrease of antigen marker fluorescence intensities after chemotherapy .CD45 ,CD19 and cytoplasm immunoglobulin light chains were the most stable marker ,no obvious antigen marker changes were found during the treatment ,while there was a signifi‐cant antigen density change in 2 cases of CD38 (14% ) ,7 cases of CD56 (50% ) ,4 cases of CD20 (29% ) and 2 cases of CD117 (14% ) .Of the 30 cases for evaluating MRD immunophenotype ,the abnormal myeloma cells were detected in 25 cases .In 5 cases ,no expression of limited Kappa and Lambda light chains was found and the ratio of Kappa and Lambda was 0 .5 - 2 ,which were identi‐fied as negative for MRD .Conclusion The multiparameter flow cytometry has important significance in evaluating the diagnosis , therapeutical effect and prognosis .The detection by adopting cytoplasm immunoglobulin light chains can improve the accuracy in MRD detection .

Objective To evaluate the effectiveness and side effects in sibling and unrelated HLA identical allogeneic hematopoietic stem cell transplantation for multiple myeloma patients under 45-year-old at early stage. Methods Three patients with multiple myeloma ranged from 38 to 44-year-old received two courses of chemotherapies and achieved partial remission. Sibling HLA identical allogeneic hematopoietic stem cell transplantations were underwent in case 1 and 2, and unrelated were in case 3. The conditioning regimens for case 1 and 2 included fludarabine, busulfan plus cyclophosphamide, and of case 3 included modified busulfan, cyclophosphamide plus antithymocyte globulin. Cycloporine A combined with methotrexate were used to prevent GVHD in the case 1 and 2, and methotrexate, mycophenolate and cycloporine A were used in case 3. Results All patients achieved full donor chimerism without graft failure. Grade Ⅱ acute GVHD and extensive chronic GVHD were found in case 1, but not in case 2 and 3. The period of follow-up of case 1, 2 and 3 were 48, 27 and 6 months, respectively, and all of them were alive with no signs of relapse. Conclusion The multiple myeloma patients under 45-year-old underwent sibling and unrelated HLA identical allogeneic hematopoietic stem cell transplantation at early stage after chemotherapy remission have the low treatment-related mortality, high complete remission rate and may prolong long-term survival.

To investigate the enhancement of immunological activity of CpG ODN by chitosan gene carrier in mice, the effect of lymphocyte proliferation was detected in mice by using MTT, the levels of IgG and cytokines (IL-2 and IL-12) in serum were measured by ELISA and peripheral blood T lymphocyte subsets CD4(+), CD8(+) were analyzed by flow cytometry. Our results showed that spleen lymphocytes isolated from the CS-CpG ODN group of mice showed the strongest proliferation (SI=1.551), and the levels of IgG, IL-2 and IL-12 in serum were higher than those of other groups. Compared with the immunization with CpG ODN, the immunization with CS-CpG ODN gene carrier was more efficient in up-regulating the percentage of CD4(+)T cells and the ratio of CD4(+)/CD8(+) of mice. It was concluded that CS gene carrier of CpG ODN was much more effective in improving immunity of CpG ODN in mice.