Child, Preschool ; Female ; Humans ; Sarcoma ; diagnosis ; surgery ; Stomach Neoplasms ; diagnosis ; surgery ; Stromal Cells ; pathology

This study was aimed to detect and identify the promoter CpG island methylation of γ-globin gene in peripheral blood mononuclear cells from patients with β-thalassemia major and healthy adult in Guangxi province, as well as to analyze the difference of promoter methylation rate of each CpG sites between them, and then to screen the promoter CpG island main methylation sites which maybe influence γ-globin expression. The template DNA was modified by bisulfite genomic sequencing PCR; the promoter sequences of γ-globin gene were amplified by technique Touchdown PCR, and then the PCR products were cloned and sequenced for obtaining methylation status of each CpG sites in target fragments, and then the accurate methylation sites and levels were detected quantitatively. The results indicated that the 4 CpG methylation sites locating at 28, 122, 231 and 234 bp in sequences were hypermethylated. As compared with healthy adults, the DNA methylation rate of 122 and 231 bp CpG sites in patients with β-thalassemia major was obviously lower, however, methylation rates of 28 and 234 bp sites were not significantly different between patients and healthy adults. It is concluded that the methylation sites 28, 122, 231 and 234 bp of γ-globin gene promoter are found both in patients with β-thalassemia major and healthy adults. The 122 and 231 bp sites are identified preliminarily to be involved in the regulation of γ-globin expression. This study provides the experimental evidence for alleviating the clinical symptoms of β-thalassemia major and targeting gene treatment through the regulation of γ-globin.
Adolescent ; Adult ; Case-Control Studies ; Child ; CpG Islands ; DNA Methylation ; Female ; Humans ; Male ; Promoter Regions, Genetic ; Young Adult ; beta-Thalassemia ; genetics ; gamma-Globins ; genetics

Combined Modality Therapy ; Humans ; Medicine, Chinese Traditional ; Practice Guidelines as Topic

Objective To investigate the correlation between the CT perfusion and microvessel density (MVD),expression of vascular endothelial growth factor(VEGF)in neoplasm of head and neck.Methods Eighty-eight lesions of head and neck were scanned by spiral CT.The largest axial surface of the mass was searched on unenhanced imaging,and at this level the dynamic contrast enhanced scan series was acquired.Time-density curves (TDC)were created from circular or oval regions of the interest drawn over the mass,target artery by Toshiba Xpress/SX spiral CT with perfusion functional software.The parameters were measured including:peak height (PH ),peak time (PT ),mean transit time (MTT), contrast enhancement ratio(RPH),and perfusion flow (PF).Histopathological slides of 35 masses were carefully prepared for the anti-CD34 and VEGF immunohistochemical staining and tumor microvessel density and calculation of VEGF expression scores.The parameters of CT perfusion were correlatively study with MVD and VEGF.Results(1)The TDC of CT perfusion imaging could be classified into 3 types.The TDC of 53/77 (68.9% )malignant tumors presented the type with rapid ascending and rapid descending after injecting contrast.The TDC of 6/9 malignant lymphomas showed low platform curve。(2)The PF median of thyroid carcinoma was 82.2(41.0,183.4)ml?min~(-1)?100 g~(-1).There was significantly difference in the parameters of CT perfusion among thyroid carcinoma and squamaous cell cancer (Median 23.8 (7.0, 108.4)ml?min~(-1)?100 g~(-1))and lymphomas (Median 24.5(13.2,78.6)ml?min~(-1)?100 g~(-1)).(3) MVD in benign tumors was (44.7?3.4),and in malignant tumors,it is (49.6?14.8 ).There was no significantly difference in MVD between benign and malignant tumors.High VEGF expression was found in 15 malignant tumors and 1 benign tumors,low VEGF expression was found in 9 malignant tumors and 10 benign tumors.(4)There were no significantly difference in VEGF expression and MVD.There was good correlation between MVD (M 40.0 )and PH (M 26.9 ),RPH (M 14.5 ),PF (M 46.8 )(r = 0.35,45.49, 0.41 ).There was correlation between VEGF(M 4.0)and MTT(M 16.7 )(r = -0.41 ).Conclusion The TDC and CT perfusion could be helpful to differentiate benign from malignant tumors. CT peffusion in neoplasm of head and neck is correlated with MVD and VEGF,and may reflect MVD and expression of VEGF.

OBJECTIVETo explore the effect of liposomal transfection of cyclin A antisense oligodeoxynucleotide (ASON) on HL-60 cell proliferation and apoptosis.

METHODSBy liposomal transfection, cyclin A ASON was co-cultured with HL-60 cells, the cell growth curve was determined by MTT assay and cell apoptosis electron-microscopy in situ cell apoptosis detection kit (POD), the protein and mRNA of cyclin A and bcl-2 were measured by FACS and RT-PCR, the role of cyclin A ASON in the development of leukemia was tested by the tumor formation in nude mice.

RESULTS(1) In the cyclin A ASON liposomal transfection group (group A), the proliferation of HL-60 cell was significantly inhibited as compared to those in cyclin A ASON group (group B) (68.9% vs 24.8%) (P < 0.01). (2) The expressions of cyclin A and bcl-2 of group A were significantly lower than those in the control group (1.1% vs 38.8%, P < 0.01; 21.9% vs 65.0%, P < 0.01, respectively), and the DNA ladder and apoptosis body was displayed. (3) In group A, the rate of tumor formation in nude mice was lower, the time for tumor formation was longer and the volume of tumor was smaller than those in control group.

CONCLUSIONLiposomal transfection of cyclin A ASON can inhibit in vitro proliferation of leukemia cells and induce in vivo apoptosis of the tumor cell, which might provide a new target for gene therapy.

Animals ; Apoptosis ; drug effects ; Cell Division ; drug effects ; Cyclin A ; genetics ; physiology ; Genetic Therapy ; HL-60 Cells ; Humans ; Leukemia ; therapy ; Liposomes ; Mice ; Mice, Inbred BALB C ; Oligonucleotides, Antisense ; pharmacology ; Transfection

OBJECTIVETo investigate the expression and regulation mechanism of mismatch repair (MMR) genes in chronic myeloid leukemia (CML).

METHODSExpression of MMR genes hMSH2, hMSH3, hMSH6, hMLH1 and hPMS2 mRNAs in 62 CML patients and K562 cell line were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Expression of bcr-abl mRNA and MMR genes mRNA were detected by RT-PCR in 26 CML patients with allogeneic peripheral blood stem cell transplantation (allo-PBSCT) and 4 CML patients on imatinib treatment. Expression of bcr-abl mRNA was detected by RT-PCR and tyrosine phosphorylation of BCR-ABL fusion protein by Western blot.

RESULTSExpression of hMSH2, hMSH3 and hMLH1 mRNA was significantly lower in CML and K562 cells than in normal control (P < 0.05). In 26 CML with allo-PBSCT and 4 CML patients on imatinib treatment, expressions of hMSH2, hMSH3 and hMLH1 mRNA was enhanced while expression of bcr-abl mRNA decreased. In CML MNC after imatinib treatment and in K562 cells, expression of hMSH2, hMSH3 and hMLH1 mRNA was enhanced while tyrosine phosphorylation of BCR-ABL fusion protein decreased.

CONCLUSIONExpressions of hMSH2, hMSH3 and hMLH1 mRNA were down-regulated by bcr-abl fusion gene.

Adult ; Aged ; Antineoplastic Agents ; pharmacology ; Benzamides ; DNA Mismatch Repair ; Female ; Fusion Proteins, bcr-abl ; genetics ; metabolism ; Gene Expression ; Gene Expression Regulation, Neoplastic ; Humans ; Imatinib Mesylate ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Male ; Middle Aged ; Piperazines ; pharmacology ; Pyrimidines ; pharmacology ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

This study was aimed to investigate the expression and regulation mechanism of DNA-dependent protein kinase catalylic subunit (DNA-PKcs) in chronic myeloid leukemia (CML) and its role in blast crisis of CML. Expression of DNA-PKcs mRNA was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and DNA-PKcs protein by Western blot in 62 CML patients and K562, as compared to those of 23 normal individual controls. In 26 CML patients received allogeneic peripheral blood stem cell transplantation (allo-PBSCT) and 4 CML patients treated with imatinib, the expression of bcr-abl mRNA and DNA-PKcs protein was detected by RT-PCR and Western blot, respectively. After treatment with imatinib in mononuclear cell (MNC) of CML patients and K562 in vitro, expression of DNA-PKcs mRNA was detected by RT-PCR and DNA-PKcs protein level, tyrosine phosphorylation of bcr-abl fusion protein were detected by Western blot. The results showed that the expression of DNA-PKcs protein was significantly lower in CML and K562 than those in normal control (P<0.05). In 26 CML patients received allo-PBSCT and 4 CML patients treated with imatinib, the expression of DNA-PKcs protein was enhanced while the expression of bcr-abl mRNA decreased. After treatment of MNC of CML and K562 with imatinib in vitro, the expression of DNA-PKcs protein was enhanced while tyrosine phosphorylation of bcr-abl fusion protein decreased. It is concluded that the expression of DNA-PKcs protein is down-regulate by bcr-abl fusion gene, and the bcr-abl fusion gene down-regulate the expression of DNA-PKcs protein by post-transcriptional mechanism; the decrease of DNA-PKcs protein expression may be one of mechanisms underlying the acute transformation of CML.
Adult ; Aged ; Benzamides ; Bone Marrow Cells ; metabolism ; DNA-Activated Protein Kinase ; biosynthesis ; genetics ; Female ; Fusion Proteins, bcr-abl ; biosynthesis ; genetics ; Humans ; Imatinib Mesylate ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; therapy ; Male ; Middle Aged ; Peripheral Blood Stem Cell Transplantation ; Piperazines ; therapeutic use ; Pyrimidines ; therapeutic use ; RNA, Messenger ; biosynthesis ; genetics

To evaluate the separation of T lymphocyte subsets by immunomagnetic beads and to find optimization of strategy for specific binding of antibody-coated beads to cells, two strategies to isolate enriched T lymphocyte subpopulation CD4+ T cells and CD8+ T cells from small volumes (< 5 ml) of peripheral blood by using immunomagnetic beads or complement cytotoxicity method were compared. The purity and activity of CD4+ T cells and CD8+ T cells were measured by using flow cytometry, trypan-blue dye exclusion test, etc. The results showed that the yields of CD4+ T lymphocytes and CD8+ T lymphocytes by using immunomagnetic beads were (94.2 +/- 1.4)% and (93.8 +/- 3.0)% respectively, higher than those of control group and the group of using completement cytotoxicity method (P < 0.05). At the same time, the yields of CD4+ T lymphocytes and CD8+ T lymphocytes by using complement cytotoxicity method were (76.0 +/- 2.8)% and (77.0 +/- 3.0)% respectively, higher than those of unenriched group (P < 0.05). The trypan-blue dye exclusion test confirmed that there were no influences on activity of CD4+ T cells and CD8+ T cells when immunomagnetic beads were used for separation of these cells from peripheral blood. It is concluded that the immunomagnetic bead method has a higher efficiency for separation of CD4+ T cells and CD8+ T cells from peripheral blood than complement cytotoxic method, especially for small sample. This method has no influence on activity and proliferation of T lymphocyte subpopulations, and would be expected to establish conditions for research of biological characteristics of CD4+ T cells and CD8+ T cells in future.
CD4-Positive T-Lymphocytes ; cytology ; CD8-Positive T-Lymphocytes ; cytology ; Flow Cytometry ; Humans ; Immunomagnetic Separation ; methods ; Leukocytes, Mononuclear ; cytology ; immunology

OBJECTIVETo investigate the subcellular expression of mammary serine proteinase inhibitor (Maspin) in oral squamous cell carcinoma and its relationship to the clinicopathological features.

METHODSThe Maspin protein subcellular expression was detected in 45 patients with oral squamous cell carcinoma by immunohistochemical staining. The relationship between the Maspin protein subcellular expression and the clinicopathological parameters was analyzed.

RESULTSThe negative rate of nuclear maspin expression was 64% (29/45), and the weakly positive rate was 11% (5/45), and the strong positive rate was 24% (11/45). Nuclear maspin expression was negatively correlated with T stage (P = 0.019), lymph node metastasis (P = 0.038) and postoperative metastasis (P = 0.004), but positively correlated with the patients' survival rate (P = 0.014). The negative rate of cytoplasmatic maspin expression was 38% (17/45), and the weakly positive rate was 31% (14/45), and the strong positive rate was 31% (14/45). Cytoplasmatic maspin expression was negatively correlated with lymph node metastasis (P = 0.038) and postoperative metastasis (P = 0.004), but positively correlated with the patients' survival rate (P = 0.014).

CONCLUSIONSMaspin expression may be a significant marker in predicting prognosis of oral squamous cell carcinoma.

Adult ; Aged ; Aged, 80 and over ; Biomarkers, Tumor ; metabolism ; Brain Neoplasms ; secondary ; Carcinoma, Squamous Cell ; metabolism ; pathology ; secondary ; Cytoplasm ; metabolism ; Female ; Humans ; Lung Neoplasms ; secondary ; Lymphatic Metastasis ; Male ; Middle Aged ; Mouth Neoplasms ; metabolism ; pathology ; Neoplasm Staging ; Serine Proteinase Inhibitors ; metabolism ; Serpins ; metabolism ; Survival Rate

OBJECTIVETo investigate the changes of open probability (Po) of large conductance Ca(2+)-activated K(+) channel (BK channel) in diabetic coronary smooth muscle cells and elucidate the underlying cellular electrophysiology mechanisms of coronary dysfunction.

METHODSRat coronary smooth muscle cells were isolated from control group and diabetic group. BK single channel currents were recorded by patch clamp technique in inside-out configuration. Open probabilities were calculated and compared between two groups. After exposure to DHS-1, a specific BK channel activator, Po at 0.2 and 1 µmol/L free Ca(2+) were compared between control and diabetic groups.

RESULTSIn the presence of 0.2 µmol/L free Ca(2+), the Po at baseline was significantly lower in diabetic rats than in control rats (0.0032 ± 0.0012 vs. 0.095 ± 0.036, P < 0.05). Cytoplasmic application of DSH-1 significantly increased the Po to 0.335 ± 0.096 (P < 0.05 vs. baseline) in control rats, whereas DSH-1 had no effect in diabetic rats (Po = 0.022 ± 0.018, P > 0.05 vs. baseline). In the presence of 1 µmol/L free Ca(2+), the Po at baseline was also significantly lower in diabetic rats than in control rats (0.210 ± 0.055 vs. 0.458 ± 0.077, P < 0.05). Cytoplasmic application of DHS-1 further robustly enhanced Po to 0.823 ± 0.019 (P < 0.05 vs. baseline) in control rats and to 0.446 ± 0.098 in diabetic rats (P < 0.05 vs. baseline of diabetic rats; P < 0.05 vs. control rats with DHS-1).

CONCLUSIONThe decrease of Po of BK single channel in coronary smooth muscle cells may be a potential cause for coronary dysfunction in diabetic rats.

Animals ; Coronary Vessels ; metabolism ; physiopathology ; Diabetes Mellitus, Experimental ; metabolism ; physiopathology ; Large-Conductance Calcium-Activated Potassium Channels ; metabolism ; Male ; Muscle, Smooth, Vascular ; cytology ; physiopathology ; Myocytes, Smooth Muscle ; metabolism ; Patch-Clamp Techniques ; Rats ; Rats, Sprague-Dawley