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Objective To investigate the CD47 expression in de novo acute myelogenous leukemia (AML) patients with normal karyotype and its clinical significance. Methods One hundred thirty-seven cases of de novo AML with normal karyotype and 3 healthy volunteers were selected. Relative CD47 expressions in normal bone marrow hematopoietic stem cells (HSC) and multipotent progenitor (MPP) from healthy volunteers, as well as bone marrow mononuclear cells (MNC) and leukemia stem cells (LSC, Lin-CD34+CD38-CD90-) from AML patients were determined by flow cytometry. CD47 expression on the Lin-CD34+CD38-LSC-enriched fraction of specimen was determined by flow cytometry. The FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) was detected by using the Genome Analyzer platform. CD34+CD38-CD47hi and CD34+CD38-CD47lo expressing cells were identified and purified using FACS. Two groups of cells were inoculated with MethoCult H4445 medium on agarose-containing methylcellulose plates. After 12 days, MPP colony forming units (CFU) were counted, and 1×105 CD34+ CD38- CD47lo and CD34+ CD38-CD47hi cells were transplanted into NSG (NOD-SCID IL-2R γ null) mice irradiated by 280 cGy, and mice were sacrificed after 8 weeks. The ratio of human CD45+cells was detected by flow cytometry. Results The expression of CD47 in AML patients was higher than that in the healthy control. CD47 was expressed in all FAB (French-American-British) subtypes of AML. No significant difference in CD47 expression among different FAB subtypes was found (F=0.545, P>0.05). Among the 37 patients with CD34+CD38-CD47hi, 17 (46 %) were FLT3-ITD negative, and 20 (54 %) were FLT3-ITD positive. Among the 100 patients with CD34+CD38-CD47hi, 63 (63%) cases were FLT3-ITD negative, 37 (37%) cases were FLT3-ITD positive. The rate of FLT3-ITD positive in patients with CD34+ CD38- CD47lo had no statistical difference compared with patients with CD34+CD38-CD47hi (χ2= 3.79, P> 3.79). The CD34+CD38-CD47lo or CD34+CD38-CD47hi which was selected by FACS, was inoculated with the methylcellulose plate containing agarose for 12 days, and CD34+CD38-CD47lo cells could form CFU. The NSG mouse transplantation experiment showed that CD34+CD38-CD47lo cells could be reconstructed hematopoiesis, and CD34+CD38-CD47hi implantation failed. Conclusion CD34+CD38-CD47hi could enrich LSC, which may be a potential marker to detect minimal residual disease.
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Objective To investigate the inhibiting effects of alcohol extract from Dioscore bulbifera on proliferation, colony formation and migration of cancer cell lines. Methods Alcohol extract from Dioscore bulbifera was prepared using Soxhlet extraction. Human gastric cancer cell line MGC803 was treated with different concentrations(0, 60, 120 mg/L)of al?cohol extract from Dioscore bulbifera. In vitro, proliferation, colony formation and migration of gastric cancer cells were detect?ed by MTT, colony formation experiments and Transwell assay respectively. Results The proliferation(day2-day 6, F=29.130, 21.864, 67.826, 36.015, 43.656, P<0.01)and colony formation(F=11.918,P<0.01)of gastric cancer cells were significantly inhibited by administration of alcohol extract from Dioscore bulbifera at both 60 mg/L and 120 mg/L . The migra?tion(F=4.258,P<0.05)of gastric cancer cells were significantly suppressed after cells were treated with120 mg/L alcohol ex?tract from Dioscore bulbifera. Conclusion Alcohol extract from Dioscore bulbifera significantly inhibit proliferation, colony formation and migration of gastric cancer cells.
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Objective: To construct cytokine-induced killer (CIK) cell vehicles carrying recombinant adenovirus carrying tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene, and preliminarily observe its anti-hepatoma ability. Methods: Lymphocytes were isolated from peripheral blood to culture CIK cells. The phenotypic identification of CIK cells was performed by flow cytometry (FCM). Then, the lentiviral pLenti-hCD40L-E1AB containing CD40L promoter and the recombined adenovirus vector pAd5/35-TRAIL were constructed, respectively. The two viruses were infected into CIK cells by two-step method. After that, the secretory function and proliferative capacity of CIK cells as well as the effect on angiogenesis and the ablility of colony-formation of hepatoma cells were assessed by ELISA, MTT method, Tubule formation assay and soft agarose assay, respectively. Results: The CIK lymphocytes grew vigorously, in which the expressions of CD3, CD56, CD11a and CD226 were positive, while the expressions of CD8 and CD305 were negative. The lentiviral pLenti-hCD40L-E1AB containing active hCD40L promoter and adenovirus E1 gene was successfully constructed, and the recombined adenovirus vector pAd5/35-TRAIL containing human TRAIL gene was also constructed. In CIK cells infected with Ad5/F35-TRAIL and pLenti-hCD40L-E1AB, the expression level of interferon-? was significanly increased (P < 0.05), and the angiogenesis and the colony-formation rate of hepatoma cells were inhibited, but the proliferative capacity of CIK cells was less affected. Conclusion: CIK cell vehicles carrying adenovirus and expressing TRAIL gene are successfully constructed. The growth inhibition of hepatoma cells may be induced by CIK cells. Copyright © 2013 by TUMOR.
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BACKGROUND AND PURPOSE: Endothelial impairment is a linking mechanism between obstructive sleep apnea (OSA) and cardiovascular diseases. Profiles of endothelial microparticles (EMPs) and endothelial progenitor cells (EPCs) reflect the degree of endothelial impairment. The aims of this study were to measure the levels of EMPs and progenitor cells in OSA, determine the correlations between these factors and OSA severity and the degree of atherosclerosis, and document any changes in these factors after therapy. METHODS: Subjects with (n=82) and without (n=22) OSA were recruited prospectively. We measured the number of colony-forming units (CFU) in cell culture as the endothelial progenitor cell index, and the number of EMPs using flow cytometry with CD31 [platelet endothelial cell adhesion molecule (PECAM)], CD42 (platelet glycoprotein), annexin V, and CD62E (E-selectin) antibodies at baseline and after 4-6 weeks of continuous positive airway pressure (CPAP) therapy. Carotid intima-media thickness (IMT) was regarded as a marker of atherosclerosis. RESULTS: The levels of PECAM+CD42- (p<0.001), PECAM+annexin V+ (p<0.001), and E-selectin+ microparticles (p=0.001) were higher in OSA subjects than in non-OSA subjects. The number of CFU did not differ between the two groups. OSA severity independently predicted the levels of PECAM+CD42- (p=0.02) and PECAM+annexin V+ (p=0.004). Carotid IMT was correlated with OSA severity (p<0.001), PECAM+CD42- (p=0.03), and PECAM+annexin V+ (p=0.01). Neither OSA severity nor carotid IMT was correlated with either the number of CFU or E-selectin+. CPAP therapy decreased the occurrence of E-selectin+ (p<0.001) in 21 of the OSA subjects, but had no effect on the other microparticles of the number of CFU. CONCLUSIONS: OSA led to the overproduction of EMPs, which moderately correlated with OSA severity and the degree of atherosclerosis, and partly responded to therapy. The endothelial impairment might contribute to future cardiovascular events.
Subject(s)
Annexin A5 , Antibodies , Atherosclerosis , Cardiovascular Diseases , Carotid Artery Diseases , Carotid Intima-Media Thickness , Cell Culture Techniques , Colony-Forming Units Assay , Continuous Positive Airway Pressure , Endothelial Cells , Endothelium , Flow Cytometry , Prospective Studies , Sleep Apnea, Obstructive , Stem CellsABSTRACT
pcDNA3.1/Hygro(-)+pCMV?. Conclusions Transactivation competence of genotype B HBx was higher than that of genotype C HBx, while the antiproliferative and apoptosis effects of genotype B HBx was lower than of genotype C HBx. B and C genotype-specific functional differences of HBx may closely co-related with the pathogenicity of HBV.
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Objective To identify the surface marker of bone marrow-derived liver stem cells and to isolate the stem cells, to investigate the differentiation of the stem cells. Methods The quantitative variations of the cells with stem cell surface markers, including ? 2-microglobulin negative (? 2m -), Thy-1 +,CD34 +,Flt-3 +,IL-3R +,and c-kit + markers, were detected by using flow cytometry in the bone marrow of several rat models with liver injury. Each stem cell population was then isolated using a magnetic bead cell-sorting procedure. The isolated cells were cultured in a system containing cholestatic serum and hepatocyte growth factor (HGF). The morphology of the cells was observed, and the expressions of albumin, AFP, and CK8/18 were detected with immunohistochemistry technique. Results ? 2m - cells elevated significantly in each of the rat models.After being co-cultured with cholestatic serum and HGF, ? 2m - cells showed multilateral transformation and resembled hepatcytes morphologically. The differentiated cells expressed albumin, AFP, and CK8/18, all known to be the characteristic markers of hepatocyte. The other cell population showed little quantitative changes, and did not express the same proteins. Conclusions The quantitative variation of ? 2m - cells corresponds to the severity of liver injury. ? 2m - cells have the ability to trans-differentiated into hepatocytes in vitro. They might be the marker of liver stem cells.
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Objective To study the influence of culture supernatant of psoriatic peripheral blood mononuclear cells (PBMCs) on the colony-forming of bone marrow-derived hematopoietic stem cells and progenitor cells. Methods Bone marrow-derived mononuclear cells were separated by density gradient centrifugation. Methylcellulose semi-solid culture medium was used to culture the bone marrow mononuclear cells in culture systems. The PBMC culture supernatant from psoriatic patients or normal controls were added to the culture, to observe their influence on the marrow-derived high proliferative potential colony forming cells (HPP-CFCs), erythroid and granulocyte-macrophage colony forming units (CFUs-E and CFUs-GM) of bone marrow hematopoietic stem/progenitor cells from healthy individuals. Results The values of HPP-CFCs, CFUs-E and CFUs-GM were significantly lower in the bone marrow cells stimulated by the supernatant of cultured psoriatic PBMCs than those stimulated by the supernatant of cultured normal PBMCs and those in the spontaneous proliferation group (P 0.05). Conclusions Psoriatic PBMCs have specific biological activity and can inhibit the colony forming of bone marrow hematopoietic cells from healthy individuals.
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Objective To explore the mechanism of hematolo gic abnormality in patients with systemic lupus erythematosus(SLE).Methods Suppression of granulocytic and ery throid colony formation of bone marrow cells fromhealthy individuals was examined in vitro by using methylcell ulose culture with sera or IgGdelete d sera from patients with SLE.Results①Both colony-forming units of erythr ocytes(CFU-E)(in 66.7%of samples tested)and granulocytes(CFU-GM)(in 70%of samples tested)of normal bone marrowcells were sign ificantly inhibited by sera frompatients with SLE(P
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40 human fetuses were used in this study. The range of fetal age varied from 3 months to full terms. All fetuses used were normal and their parents were healthy. The CFU-C from bone marrow, liver, spleen and peripheral blood were studied in vitro with agar culture technique modified by Metcalf.Experiments proved that the CFU-C of bone marrow may be observed in the fetuses in 3rd month, however, it increased rapidly in 4th month and maintained higher level until the end of fetal life. The progenitor cell population was detected at very high level in liver from 4th month to 6th month fetuses. The number of CFU-C of fetal liver were low in 7—10th month. The circulating progenitor cells in peripheral blood of 4, 5 and 6-month fetuses Were on high level but it was lower than that of liver and bone marrow.