Apoptosis of U937 cell line promoted by matrine through MAPK signal transduction pathway / 中国中药杂志

<p><b>OBJECTIVE</b>To study the anti-cancer effect of matrine (Mat) on U937 cell line and its possible molecular mechanism.</p><p><b>METHOD</b>The cells were cultured in medium containing either 0.1, 0.2, 0.3, 0.4, or 0.5 g x L(-1) of Mat. The morphological alteration was observed by inverted microscopy and electron microscopy. Cell proliferation was analyzed by Try pan blue staining and MTT. The method of Western Blot was used to detect phosphorylation activity of MAPK.</p><p><b>RESULT</b>Matrine had a significant inhibitory effect on proliferation of U937 cell line at the concentration of 0.2 g x L(-1). Treated with matrine of 0.2 g x L(-1) for 48 h, U937 cells became smaller and appeared more round than previously. The number of U937 cells showing apoptosis increased with elevation of the concentration of the matrine. Matrine had an ability of inhibiting the activity of ERK and increasing the activities of p38 and JNK to some degree in U937 cells.</p><p><b>CONCLUSION</b>Matrine can inhibit the proliferation of U937 cell line in vitro and induce its apoptosis possibly through inhibiting the activity of ERK and increasing the activities of p38 and JNK in U937 cells.</p>

The influence of the tyrosine Kinase inhibitor imatinib mesylate on the cell repair function after DNA damage in K562 cells / 中华检验医学杂志

Objeetive To construct the cell DNA damage models for the human CML K562 cell line before or after imarlnib mesylate treatment and observe the repairing process dynamically for investigating the iniluence of imatinib mesylate on the repair function of K562 cells after cell DNA danlage.Methods The MTT assay was used to estimate the optimal pretreatment concentration of imatinib mesylate in K562 cells and Western blot was employed to evaluate the phosphorylation status in K562 cells after imatinib mesylate treatment to estimate BCR/ABL tyrosine kinase inhibition by imatinib mesylate.The comet assay was used to detect the DNA damage induced by hydrogen peroxide at various concentrations in K562 cells with or without the pretreatment of imatinib mesylate.A dynamic observation on the repairing process after cell DNA damage was made by the comet assay.Results The pretreatment by imatinib mesylate for K562 cells was optimized to be at a final concentration of 1 μmol/L for 24 h as revealed by the MTT assay additionly imatinib mesylate treatment at this concentration could effectively inhibit the phosphorylation of the BCR-ABL fusion protein at Tyr177(Deusityrate 0.100±0.018).When compared with the control group(Deusityrate 0.425±0.039),the BCR/ABL phosphorylation at Tyr177 was significantly decreased by (77. 11±5.59) % (t=4. 57,P＜0. 05). The cell DNA damage models for both imatinib mesylate-nontreated and imatinib mesylate-pretreated K562 cell groups were constructed with hydrogen peroxide treatment at a final concentration of 10 μmol/L for 10 min at 4℃ as confirmed by the comet assay. When compared with the control imatinib mesylatenontreatod K562 cell group,the time duration required for the DNA repair in imatinib mesylate-pretreated K562 cell group was significantly prolonged (F= 97.79,P＜0. 05 ). Conclusions The cell DNA damage models for the leukemic K562 cell groups before or after imatinib mesylate treatment were successfully constructed and the tyrosine kinase inhibitor imatinib mesylate for BCR/ABL fusion protein was revealed to attenuate the DNA repair capacity of the K562 cells after DNA damage.

Construction of BaF3-P210 cell line stably expressing BCR/ABL protein and its biological activity / 第三军医大学学报

Objective To construct the transformed mouse BaF3-P210 cell line stably expressing BCR/ABL and to initially investigate the influence of BCR/ABL on the cell biological characteristics of BaF3 cell line. Methods The retroviral vector with bcr/abl gene was transfected into the packaging cell line. The BaF3 cells were infected with the collected viral supernatant. The transgenic BaF3-P210 cell line stably expressing BCR/ABL were screened and subcloned. The integration of the bcr/abl gene in the genome of the target cell was determined by PCR and DNA sequencing,trypan blue staining assay,flow cytometry and MTT assay. Results The bcr/abl gene was integrated into the BaF3 cell genome; RT-PCR and Western blot verified the stable expression of the bcr/abl gene and protein in the screened subclone cell line BaF3-P210. Compared with the control group,the cell proliferation rate was promoted (P

Construction of GPI-anchored bcr/abl and its expression on COS-7 cells membrane / 第三军医大学学报

Objective To construct a recombinant eukaryotic expression plasmid of glycosylphosphatidyl inositol(GPI)-anchored bcr/abl and explore its expression at mRNA and protein level.Methods The gene fragment encoding bcr/abl was amplified by PCR using the plasmid containing the cDNA sequence of P210 as template and then inserted into a eukaryotic expression vector pBudCE4.1.The constructed recombinant plasmid pBudCE4.1-bcr/abl was identified by restriction analysis and DNA sequencing.Lymphocytes were isolated from human peripheral blood and their total RNA was extracted.The gene fragment encoding GPI was amplified by RT-PCR using the obtained RNA as template and was inserted into the constructed recombinant plasmid pBudCE4.1-bcr/abl in order to anchor GPI and bcr/abl.The constructed recombinant plasmid pBudCE4.1-bcr/abl-GPI was transfected into COS-7 cells,and the expressions of objective fragment were detected by RT-PCR and Western blotting.Results The results of restriction analysis,PCR and DNA sequencing proved that GPI-anchored bcr/abl fusion fragment was correctly inserted into vector pBudCE4.1.The expression of bcr/abl fusion gene and fusion protein were identified in transfected COS-7 cells and on their membrane.Conclusion The recombinant plasmid pBudCE4.1-bcr/abl-GPI was successfully constructed and expressed on the membrane of COS-7 cells,which found a basis of cell immunity with GPI-anchored bcr/abl fusion gene.

In vitro study of the apoptosis effect of DNR on HL-60 cells and its relationship with ROS, CER and NF-kappaB / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the effect of DNR on HL-60 cells apoptosis in vitro and the related mechanism.</p><p><b>METHODS</b>The apoptosis of HL-60 was observed by microscope, flow cytometry (FCM) and DNA electrophoresis and various apoptosis-associated proteins expression by immunocytochemistry (IC) and FCM assays; the changes of apoptosis in HL-60 cells treated with DNR or suppressors PDTC or FB1 were also observed.</p><p><b>RESULTS</b>When treated with 0.2 approximately 2.0 micro mol/L DNR, the percentage of apoptotic HL-60 cells increased with the dose increasing and the time extending, and the typical apoptotic cells and the appearance of apoptotic DNA ladder were observed. It was shown that after treatment with 1 micro mol/L DNR, the fluorescence intensity index (FI) of both bcl-2 and c-myc in HL-60 cells decreased, the FI of Bax, caspase-3 increased at 2 h, but decreased at 5 h, the FI of NF-kappaB increased. After adding PDTC, the apoptosis percentage of HL-60 cells decreased, but FB1 didn't present these effect.</p><p><b>CONCLUSION</b>It suggested from the results that at certain concentration, DNR can induce the apoptosis of HL-60 cells in vitro. The mechanism was supposed by suppressing the expression of bcl-2 and c-myc and activating the expression of Bax and caspase-3, NF-kappaB and ROS had the marked correlation with the apoptosis process, but the ceramide synthase wasn't associated with it.</p>

Committed differentiation of hematopoietic cells from human umbilical cord blood in vitro / 第三军医大学学报

Objective To explore the capacity of hematopoietic cells from human umbilical cord blood (UCB) induced by hematopoietic growth fators (HGFS) to differentiate committedly to granulocyte in vitro . Methods Suspension culture of isolated mononuclear cells from fresh UCB samples were conducted for 5 weeks in IMDM medium containing UCB serum and HGFS. The expansion folds of nucleated cells, the changes of CD34+antigen by immunofluorometry, and the colony forming unit granulocyte macrophage(CFU GM) by progenitor clonogenic assay were observed. Results Culture of mononuclear cells with the combination of granulocyte colony stimulating factor(G CSF), granulocyte macrophage colony stimulating factor (GM CSF), stem cell factor(SCF), interluekin 3(IL 3) and interleukin 6(IL 6) for 3 weeks markedly increased the total count of nucleated cells and CFU GM, being 58.3 and 12.2 folds of the pre cultured, and the cultured cells were mainly neutrophils. However, CD34+ cells in nucleated cells declined from 0.97% to 0.05%. According to the culture condition, a single portion of fresh UCB could increase nucleated cells by (5.1?2.3) fold and CFU GM by (2.2?0.95) fold. Conclusion Hematopoietic cells of UCB may be induced to differentiate committedely to granulocyte lineage by this culture system in vitro to result in the increase of granulocytes and CFU GM in single portion of cord blood.

ANALYSIS OF THERAPEUTIC EFFECTS OF HOAP OR HOCP REGIMEN IN ACUTE NONLYMPHOCYTIC LEUKEMIA / 重庆医科大学学报

Sixty-seven patients with acute nonlymphocytic leukemia were treated with HOAP or HQCP regimen. The complete remission (CR) was 41.8%, partial remission(PR) 22.4%, non remission(NR)35.8%. The CR rate was of no significant difference between HOAP and HOCP regimens, neither betoeen the leukemia cell types. The remission time was significantly longer in patients with CR than that in patients with PR and NR

DIAGNOSTIC SIGNIFICANCE OF PLASMA ENDOTOXIN AND SERUM C-REACTIVE PROTEIN IN PATIENTS WITH LEUKEMIA COMPLICATED BY INFECTION / 重庆医科大学学报

The concentration of plasma eadotoxin and serum C-reactive protein(CRP) were measured in 48 cases with leukemia.Plasma endotoxin was apparently higher in 9 cases with gram-negative bacterial infection than those with other organisms infection.The serum CRP concentration was strongly positive in patients with leukemia complicated by bacterial infection.It is suggested that measurement of plasma endotoxin and serum CRP would aid the diagnosis of G- bacterial infection in patients with leukemia

Mechanism research and effects of clock gene Per2 in K562 leukemia cells on their proliferation,differentiation and apoptosis / 第三军医大学学报

Objective To investigate the effects of circadian clock gene Period2(Per2)on the proliferation,differentiation and apoptosis of K562 cells and its probable molecular mechanism.Methods The Per2 expression plasmid pcDNA3.1-Per2 and empty control plasmid were respectively transfected into K562 cells with cationic liposome,and the resistant cells stably expressing Per2 gene were obtained by G418 selection.Their morphological changes were observed under light microscope following Wright-Giemsa staining.Trypan blue excluding staining and MTT assay were employed to evaluate cell proliferation.Flow cytometry was performed to analyze cell cycle distribution and cell apoptosis,and electron microscopy was used to detect cell apoptosis.Meanwhile,the expressions of proliferation and apoptosis associated proteins,such as P53,Cyclin B1 and C-Myc,were respectively detected by RT-PCR and Western blot analysis at mRNA and protein level.Results The K562/Per2 cell line stably expressing Per2 gene was screened out.As compared with either the empty plasmid transfected group(K562/empty)or the untreated group(K562/untreated),K562/Per2 cells was smaller in volume and showed no obvious cellular differentiation.Circadian clock gene Per2 could significantly inhibit both growth and proliferation of K562 cells.The percentage of K562 cells in G2/M phase increased [K562/Per2 group(36.1?5.5)%,K562/empty group(12.5?2.9)%,untreated group(9.7?2.3)%,P