Biological Effects of the SARI Over-expression on K562 Cell Line / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To construct a lentivirus vector carrying SARI gene and to investigate its biological effects on K562 cells.</p><p><b>METHODS</b>SARI was amplified from the plasmid containing SARI cDNA and subcloned into pLOV.CMV.eGFP virus vector. After sequencing, lentivirus packaging, titering, the viruses of SARI-pLOV.CMV.eGFP were harvested and tansfected into the K562 cells. Real-time quantitive PCR and Western blot were performed to validate the SARI expression at the level of mRNA and protein respectively. Simultaneously, the proliferation, apoptosis and cell cycle of K562 cells were detected by CCK-8 and flow cytometry respectively.</p><p><b>RESULTS</b>The SARI overexpressed lentivirus vector was successfully constructed. The mRNA and protein levels of SARI increased significantly in the pLOV.CMV.eGFP-SARI group, which was confirmed by Q-PCR and Western blot; as compared with blank and mock groups, SARI over-expression leaded to significant proliferation inhibition and increased apoptosis of K562 cells, without visible effects on cell cycle.</p><p><b>CONCLUSION</b>the over-expression of SARI gene obviously suppresses the cell proliferation of the K562 cells as well as promotes the apoptosis. The results implied that the induction of the SARI gene expression may be an important candidate therapeutic method for the CML.</p>

Cross-lineage expression in 505 patients with acute lymphoblastic leukemia by multiparametric flow cytometry analysis / 中国实验血液学杂志

The expression of immunological markers of one hematopoietic lineage on the abnormal cells of another lineage (cross-lineage expression) is a known feature of leukemia. The present study was aimed to investigate the cross-lineage expression in ALL cells. The cross-lineage expression in ALL cells from 505 patients was detected by flow cytometry using 23 monoclonal antibodies (McAbs) in triple staining combinations. The results showed that in whole ALL, the expression of myeloid antigens occurred in 56.4% of the cases, and CD13 was the most frequently expressed myeloid marker (32.7%) followed by CD33 (29.5%), CD15 (19.2%) and CD11b (7.7%). CD13/CD33 expressions were more frequent in CD34(+) cases than in CD34(-) cases. In B-ALL, T-cell antigen CD4, CD5, CD7 and CD2 were found in 27 (6.3%), 12 (2.8%), 8 (1.9%), and 6 (1.4%) cases respectively, and CD7(+), CD2(+) and CD4(+) cases commonly expressed CD13/CD33. In T-ALL, B-cell antigen cCD79a, CD19 and CD22 were found in 6 (8.1%), 5 (6.8%), and 2 (2.8%) cases respectively, and all of CD19(+) and CD22(+) cases were all accompanied with CD13/CD33. It is concluded that cross-lineage expression in ALL mostly exists in the immature stages, ALL cells more frequently express phenotypes B(+)M(+), T(+)M(+) and occasionally B(+)T(+)M(+), but B(+)T(+)M(-) phenotype is extremely rare.

Effects of acute myeloid leukemia cell supernatant on the proliferation and apoptosis of CD4+ and CD8+ T cell subsets / 中国实验血液学杂志

To study the effects of supernatant derived from acute myeloid leukemia (AML) cell lines on proliferation and apoptosis of CD4(+) and CD8(+) T cell subsets and to investigate the mechanism by which AML escapes from immune recognition, lymphocytes were labeled with CFSE and were stimulated with anti-CD3 and anti-CD28 in presence or absence of supernatants from three AML cell lines (HL-60, NB4, U937). After culture, cell suspensions were labeled with 7AAD and CD4 PE (or CD8 PE). Cells were then detected by flow cytometry and their proliferation and apoptosis were analyzed. The results showed that supernatants from two of three cell lines (HL-60 and NB4) inhibited the proliferation of CD4(+) and CD8(+) T cells, and the degree of inhibition showed a dose-dependent way. Similarly, the apoptosis of stimulated CD4(+) T cells was inhibited, but stimulated CD8(+) T cells remained unaffected by supernatant from HL-60 and NB4. In contrary, the apoptosis of proliferative CD8(+) T cells were increased significantly by HL-60 and NB4 supernatant. It is concluded that soluble factors derived from AML cell lines inhibit the proliferation of CD4(+) and CD8(+) T cells and induce the apoptosis of proliferative CD8(+) T cells, that may be one of the mechanisms by which the immunity was suppressed.

Proliferation and apoptosis in vitro of umbilical cord blood CD34+CD38- hematopoietic early progenitor cells / 中国实验血液学杂志

To cultivate CD34(+)CD38(-) cells isolated from umbilical cord blood of healthy puerperal women over a longer-period of time for observation of cell division, proliferation, apoptosis, and effects of stem cell factor on the growth of CD34(+)CD38(-) cells, with flow cytometry, CD34(+)CD38(-) cells were isolated from umbilical cord blood of 10 healthy puerperal women and cultivated in stem cell media with supplement of IL-3, IL-6, GM-CSF, EPO, IGF-1 and SCF 6 kinds cell growth stimulating factors for six months. The cell growth curves were established. The effects of stem cell factor on the growth of CD34(+)CD38(-) cells and cell apoptosis were investigated with the single cell gel electrophoresis technique and flow cytometry method, respectively. The results showed that CD34(+)CD38(-) cells isolated from umbilical cord blood were capable of proliferating after being cultivated in vitro over a longer-period of time with no evidence of the presence of excessive apoptosis. In conclusion, under appropriate culture conditions, CD34(+)CD38(-) hematopoietic early progenitor cells from umbilical cord blood can serve as a resource providing a large amount of primitive cells for transplantation therapy after a longer period of cultivation and proliferation in vitro.

Immunophenotypes in 115 patients with acute myeloid leukemia by multi-color flow cytometry / 中国实验血液学杂志

Immunophenotyping has become common in the diagnosis and classification of leukemia. To evaluate the immunophenotype of acute myeloid leukemia (AML), multiparameter flow cytometry and CD45/SSC gating were used to analyze the surface and cytoplasmic antigen expressions in 115 cases of AML. The results were compared with the French-American-British (FAB) Cooperative Group classification to help define the best use and role of multiparameter flow cytometry in the diagnosis and proper classification of AML. The results showed that CD38, CD38 and CD13 were the most commonly expressed antigen (94.8%, 91.3% and 89.6%, respectively). CD7 was the most commonly expressed lymphoid antigen (20.2%), followed by CD19 (16.5%) and CD2 (15%). Some immunophenotypes correlated with FAB type, including increased frequency of CD2 in M(3); lack of HLA-DR, CD34 and CD56 expression in M(3); increased frequency of CD19 in M(2), CD14 and CD56 in M(5) and lack of MPO in M(0). In conclusion, multiparameter flow cytometry is a reliable technique in the diagnosis of AML, and some immunophenotypes correlate with FAB type.

Effect of peroxisome proliferator-activated receptor activators on tumor necrosis factor-alpha expression in neonatal rat cardiac myocytes / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the effect of peroxisome proliferator-activated receptor-alpha (PPAR alpha) and PPAR gamma activators on tumor necrosis factor-alpha (TNFalpha) expression in neonatal rat cardiac myocytes.</p><p><b>METHODS</b>Primary cultures of cardiac myocytes from 1- to 3-day-old Wistar rats were prepared, and myocytes were exposed to lipopolysaccharide (LPS) and varying concentrations of PPAR alpha or PPAR gamma activator (fenofibrate or pioglitazone). RT-PCR and ELISA were used to measure TNFalpha, PPAR alpha, and PPAR gamma expression in cultured cardiac myocytes. Transient transfection of TNFalpha promoter with or without nuclear factor-kappaB (NF-kappaB) binding site to cardiac myocytes was performed.</p><p><b>RESULTS</b>Pretreatment of cardiac myocytes with fenofibrate or pioglitazone inhibited LPS-induced TNFalpha mRNA and protein expression in a dose-dependent manner. However, no significant changes were observed on PPAR alpha or PPAR gamma mRNA expression when cardiac myocytes were pretreated with fenofibrate or pioglitazone. Proportional suppression of TNFalpha promoter activity was observed when myocytes was transiently transfected with whole length of TNFalpha promoter (-721/+17) after being stimulated with LPS and fenofibrate or pioglitazone, whereas no change of promoter activity was observed with transfection of TNFalpha reporter construct in deletion of NF-kappaB binding site (-182/+17).</p><p><b>CONCLUSIONS</b>PPAR alpha and PPAR gamma activators may inhibit cardiac TNFalpha expression but not accompanied by change of PPAR alpha or PPAR gamma mRNA expression. Therefore PPAR alpha and PPAR gamma activators appear to play a role in anti-inflammation. The mechanism may partly be involved in suppression of the NF-kappaB pathway.</p>

Effect of peroxisome proliferator-activated receptors activators on plasminogen activator inhibitor-1 expression in HepG-2 cells / 中国应用生理学杂志

<p><b>AIM</b>To investigate the effect of different peroxisome proliferator-activated receptors (PPARs) activators on plasminogen activator inhibitor-1 in HepG-2 cell line and explore the effect of PPARs on PAL-1 gene expression.</p><p><b>METHODS</b>Stearic acid, oleic acid, linoleic acid, fenofibrate, pioglitazone were used in the treatment of HepG-2 cell culture. The level of PAI-1 and PPARs mRNA was measured by reverse transcription-polymerase chain reaction (RT-PCR) and the level of PAI-1 activity and PPARs protein was determined by colorimetric assay and western blotting respectively.</p><p><b>RESULTS</b>The mRNA and activity of PAI-1 significantly increased in the groups of oleic acid and linoleic acid compared with the control, but decreased in the group of fenofibrate. There were no significant changes in both groups of stearic acid and pioglitazone. The alterations in the level of PPARs mRNA and protein were not detected in all the treated groups compared with the control.</p><p><b>CONCLUSION</b>Peroxisome proliferator-activated receptors activators play important roles in the PAI-1 gene expression and regulation. It is likely mediated by the activation of PPARalpha, but there might be other mechanisms.</p>