Effect of heat stress on expression of gp96 in K562 cell line of the chronic myeloid leukemia and its significance / 中国实验血液学杂志

This study was purposed to investigate the effect of different heat stress conditions on expression level of heat shock protein gp96 in K562 cell line of chronic myeloid leukemia in order to provide experiment basis for seeking optimal heat stress condition increasing extraction amount of gp96 from K562 cells. The expression changes of gp96 in K562 cell line was detected by immunocytochemistry under 38, 40, 42, 44, 46 and 48 degrees C for 30 minutes in water, by flow cytometry under 40, 44, 48 and 52 degrees C for 30 minutes in water, by Western blot under 40, 44, 48 and 52 degrees C for 30 minutes in water. Immunocytochemistry assay showed that gp96 existed mainly in cytoplasm. The peak of gp96 expression was at 30 minutes after 48 degrees C in water. The result of flow cytometry was consistent to immunocytochemistry detection results under temperatures 40, 44, 48 and 52 degrees C (P < 0.01). Western blot showed that detection result was the same as the immunocytochemistry and flow cytometry detections. In conclusion, the expression of gp96 in K562 cell line reached peak at 30 minutes after 48 degrees C in water. This condition may be an effective preparative condition for extraction of gp96 from K562 cells.

Differentiation and function of human dendritic cells influenced by heat shock protein gp96 purified from K562 cells / 中国实验血液学杂志

This study was to establish the method of purifying heat shock protein GP96 from K562 cells and explore the differentiation and function of human DC influenced by heat shock prolein (HSP). Using ammonium sulfate precipitation, conA-sepharose affinity chromatography and DEAE-sephacel anion exchange chromatography GP96 from K562 cells lysate was isolated and purified. The identification of the purified protein was controlled by Western blot with anti-human GP96 IgG. Human dendritic cell derived from peripheral blood mononuclear cell were cultured with purified GP96. The phenotype changes of DC were analyzed by flow cytometry and MLR was detected by MTT. The results showed that 60-80 microg GP96 was purified successfully from 1 x 10(10) K562 cells. DC stimulated with HSP-GP96 had higher expression rates of CD83, CD86, HLA-DR and lower expression rates of CD1a and had stronger ability to induce T cells proliferation. It is concluded that heat shock protein GP96 can be isolated and purified from K562 cells and could induce maturation of dendritic cell. HSP-DC vaccine show stronger ability to induce T cell proliferation than DC.

Effect of transforming growth factor-beta1 on proliferation and apoptosis of mononuclear cells of cord blood / 中国实验血液学杂志

To explore the mechanism of transforming growth factor-beta1 (TGF-beta1) effect on umbilical cord blood mononuclear cells proliferation and apoptosis, 5-bromo-2'-deoxyurine (BrdU) incorporation assay was adopted to detect effect of TGF-beta1 on synthesis of DNA in cells. Western blot method was used to examine effect of TGF-beta1 on expression of cyclin A, Cyclin D1, CDK2 and CDK4 in G1 phase of cell cycle. Giemsa staining and flow cytometry (FCM) were performed to detected effect of TGF-beta1 on cell apoptosis. The results showed that (1) after culture of cells with IMDM containing 10% FBS, 10% FBS + 1 ng/ml TGF-beta1, 10% FBS + 2 ng/ml TGF-beta1 or 10% FBS + 5 ng/ml TGF-beta1 for 12 hours the OD values of TGF-beta1 group were significantly lower than control group (P <0.01); after culture for 24 hours the OD values of 1 ng/ml TGF-beta1 group had no significant difference compared with control group (P >0.05), but the OD values of 2 ng/ml and 5 ng/ml TGF-beta1 groups were significantly lower than control group (P <0.05). (2) 2 ng/ml TGF-beta1 could significantly inhibit the production of cyclin A, cyclin D1, CDK2 and CDK4, the protein levels were significantly lower than control group. (3) when the cells were co-cultured with 2 ng/ml TGF-beta1 for 12 and 24 hours, Giemsa staining and FCM detection could display typical apoptosis, the apoptosis rates were 14.42% and 31.98%, while apoptosis rate in control were 4.71% and 5.76%. It is concluded that TGF-beta1 can inhibit production of G1 cyclins and CDKs of umbilical cord blood mononuclear cells, arrest cells in the G1 phase of cell cycle and induce cell apoptosis. Thus, TGF-beta1 may be an important negative modulator in hematopoiesis.

The heat shock protein 70 antisense oligomers enhance the sensitivity of bladder cancer cell EJ to mitomycin C / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate whether the heat shock protein (HSP) 70 antisense oligomers can enhance the sensitivity of bladder cancer cell EJ to mitomycin C.</p><p><b>METHODS</b>The HSP70 mRNA of EJ cells was blocked by the 10 micromol/L HSP70 antisense oligomers, while its effect on cell growth was evaluated by methyl thiazolyl tetrazolium (MTT) and colony forming ability test.</p><p><b>RESULTS</b>The HSP70 expressions in HSP70 antisense treated group were lower than the corresponding sense and nonsense treated groups (P < 0.01). While, the increased sensitivity of EJ to mitomycin C was found in antisense treated group, compared with the corresponding sense and nonsense treated groups (P < 0.01).</p><p><b>CONCLUSION</b>The sensitivity of bladder cancer cell EJ to mitomycin C was enhanced by the blockage of the HSP70 expression.</p>

A quantitative assay for telomerase activity in peripheral blood mononuclear cells from patients with acute leukemia / 中国实验血液学杂志

To establish a quantitative assay for telomerase activity and analyze the telomerase activity in peripheral blood mononuclear cells (PBMNC) from patients with acute leukemia, a fluorescent dye, PicoGreen, was added to the products after telomere repeat amplification protocol. The samples were excited at 480 nm and the fluorescence emission intensity was measured at 520 nm using a spectrofluorometer. Telomerase activity was detected in PBMNCs from 20 cases of normal individuals and 25 patients with acute leukemia. The results showed that the fluorescence of PicoGreen binding to double-stranded DNA specifically was enhanced with increase of DNA quantities. In conclusion, the met hod is rapid, simple and quantitative, the telomerase activities of PBMNCs from acute leukemia patients are significantly higher than that of the normal controls.

Dendritic cells cultured with human umbilical cord serum instead of fetal calf serum / 中国实验血液学杂志

To investigate whether the dendritic cells (DC) could grow up in cultural system with umbilical cord serum (UCS), the UCS was used in the culture instead of fetal calf serum. The phenotype of dendritic cells was detected by flow cytometry and the antigen presenting ability of DC in allo-MLR was measured by MTT assay. The results showed that DC grown in UCS (UCS-DC) had higher expression rate of CD86, CD83 and HLA-DR than that in grown in FCS (FCS-DC). (P < 0.05), and their expression of CD1a was lower than that of FCS-DC. The ability to induce T cell proliferation had no difference between UCS-DC and FCS-DC. It is suggested that dendritic cells with more mature phenotype had been produced in the medium containing UCS than those in the medium containing FCS, and UCS-DC possessed potent ability to stimulate proliferation of allogeneic T cells.

p21(WAF1) transfection decreases sensitivity of K562 cells to VP-16 / 中国实验血液学杂志

The objective of this study was to explore the effect of p21(WAF1) on the sensitivity to chemotherapeutic agent VP-16, etoposide, in leukemia cell line K562. A p21(WAF1) retroviral expression vector was constructed, and mediated by FuGENE(TM)6, it was transfected into K562 cells, which without p21(WAF1) expression. The ecotropic expression of p21(WAF1) in K562 cells was identified by RT-PCR and Western blot, and named K562-p21(WAF1) cell. The K562-p21(WAF1) cells were exposed to VP-16 at different concentrations and different times, then, the sensitivity to VP-16 was examined by cell viability and MTT assay, the apoptosis induced by VP-16 was examined by DNA fragments electrophoresis and flow cytometric Annexin V-PI dual labeling technique. The results showed that the ecotropic expression of p21(WAF1) decreased the sensitivity of K562 cells to VP-16. After treatment of VP-16, the DNA ladder was examined in control K562-neo cells at 20 microgram/ml, but in K562-p21(WAF1) cells at 80 microgram/ml. With FCM, the number of apoptotic K562-neo cells was 14.9% and 25.4% respectively after treated with 20 microgram/ml VP-16 for 12 and 24 hours, and it was 6.94% and 10.96% in K562-p21(WAF1) cells. Our results suggest that the expression of p21(WAF1) could reduce the sensitivity of K562 cells to VP-16 and inhibit the apoptosis of K562 cells

Transfer and Expression of Recombinant Human Thrombopoietin Gene in COS-7 Cells and Mice In Vivo / 中国实验血液学杂志

In order to investigate the expression of recombinant TPO gene in COS-7 cells and in vivo of the mouse model, eukaryotic expressing plasmid pcd2/TPO with human TPO cDNA was constructed with DNA recombinant techniques. The plasmid pcd2/TPO was transiently transfected into the COS-7 cells by means of lipofection, the naked pcd2/TPO plasmid was injected into the skeletal muscle of mice with electric pulses. RT-PCR and ELISA methods were used to detect the TPO expression of the transfected COS-7 cells, both showed high level expression. The MTT test showed the expressed TPO had proliferative activity to TPO-dependent cell line. High efficiency of gene transfer in transgenic mice was also observed by RT-PCR and immunohistochemical methods. The serum TPO level [(1 185 +/- 264) ng/L] in transgenic mice was quite different compared with the normal mice [(250 +/- 76) ng/L]. All these results provided solid foundations for the research of TPO gene therapy in the future.

p21(WAF1) Gene Transfection Inhibits Proliferation of Leukemia Cell Line K562 / 中国实验血液学杂志

To explore the functional role of p21(WAF1) gene on the proliferation of leukemia cell line K562, a p21(WAF1) retroviral expression vector was constructed. Mediated by FuGENE trade mark 6, p21(WAF1) was transfected into leukemia cell line K562, which was without p21(WAF1) expression. After selected in G418, K562 cell clones that expressed p21(WAF1) stabaly were isolated and named K562-p21(WAF1). The ectopic expression of p21(WAF1) mRNA and protein in K562 cells was identified by RT-PCR and Western Blot. The cell proliferaton was tested in liquid and soft agar culture after transfection. The cell cycle was tested by FCM. The expression of p21(WAF1) protein and mRNA could be detected in K562-p21(WAF1) cells. A strong inhibition of cell proliferation was observed in K562-p21(WAF1) cell clones cultured in liquid media as well as soft agar (P < 0.01). The cell number in G(0)/G(1) phase was remarkably increased. The findings showed that p21(WAF1) can inhibit the proliferation of leukemia cells, and it could be a potential target gene for leukemia gene therapy.