Effect of lentinan against immunosuppression of lymphocytes cultured in simulated microgravity environment / 中国实验血液学杂志

This study was aimed to evaluate the effect of lentinan on the immune function of splenic lymphocytes in rotary cell culture system (RCCS) microgravity environment. The splenic lymphocytes from mice were separated and cultured in the normal gravity and the microgravity environments. The cells were treated with lentinan solution (0, 10, 20 and 40 µg/ml). After incubated with lentinan for indicated times (24, 48 and 72 h), the cell proliferation, secretion of cytokine and the expression of cell surface markers were detected by MTT method, ELISA and flow cytometry respectively. The results indicated that lentinan of above mentioned concentrations did not obviously promote the lymphocyte proliferation, but increased the secretion of IL-2 and IFN-γ and enhanced the expression of lymphocyte surface markers CD4 and CD8 in microgravity environment. It is concluded that lentinan has the ability to enhance the lymphocyte immune function in microgravity environment.

Cordyceps sinensis enhances lymphocyte proliferation and CD markers expression in simulated microgravity environment / 中国实验血液学杂志

This study was aimed to explore the effect of cordyceps sinensis enhancing lymphocyte proliferation and surface CD marker expression in simulated microgravity environment. The splenic lymphocytes were separated from mice and cultured in the rotary cell culture system simulated microgravity environment. The cells were treated with different concentration of cordyceps sinensis solution (0, 6.25, 12.5, 25 and 50 µg/ml) for 24, 48 and 72 h respectively, then the cells were harvested, and analyzed for cell proliferation and the expression of cell surface markers (CD4 and CD8). The results showed that under simulated microgravity environment, the lymphocyte proliferation was inhibited. When the concentration of cordyceps sinensis was 25 or 50 µg/ml, the lymphocyte proliferation, CD4 and CD8 expressions all increased, but 50 µg/ml cordyceps sinensis could inhibit the proliferation ability with the time prolonging. It is concluded that the suitable concentration of cordyceps sinensis displayed the ability to enhance the lymphocyte proliferation and CD marker expression in simulated microgravity environment. These results may be valuable for screening drugs which can be potentially against immunosuppression under simulated microgravity.

Overexpression of CHIP in chronic myeloid leukemia K562 cells induces mitotic abnormality / 中国实验血液学杂志

This study was aimed to investigate the possible influence of a novel E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70/Hsp70-interacting protein) on biological characteristics of cancer cells. Stable overexpression models in CML K562 cells were established via lipofectamine-mediated wild type CHIP and its TPR or U-box deletion mutants gene transfection. Followed G418 pressure selection, K562-CHIP stable transfected cell clones were obtained by limited dilution. The proliferation status and cell cycle were observed by MTT assay and FACS. The expression of related proteins and morphological changes were detected by Western blot and Wright-Giemsa staining. The results showed that overexpression of wild type CHIP did not inhibit cell proliferation, but slightly increased cell ratio of G(2)/M phase. CHIP gene had no effect on the stability of BCR-ABL kinase protein. HDAC inhibitor FK228-induced BCR-ABL degradation did not enhanced by CHIP. Notably the enlarged cells and abnormal mitotic cells remarkably increased in K562 WT-CHIP cells, indicating that CHIP may involve in the regulation of mitotic process. It is concluded that wild type CHIP induces mitotic abnormity in K562 cells.

Construction and selection of RNAi plasmid vector for suppressing GATA-3 gene expression / 中国实验血液学杂志

GATA-3 plays a central role in the Th2-mediated immunoreaction. This study was aimed to construct and select plasmid vectors of siRNA which can effectively and specifically suppress the gene expression of GATA-3. Plasmid including PSi338, PSi717 and PSi1232 were designed and constructed for GATA-3 regarded as target gene and transfected into murine P815 cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot were used to detect the inhibition of GATA-3 mRNA as well as its protein in P815 cells. The results demonstrated that the expressions of mRNA and protein of GATA-3 in P815 cells were inhibited significantly by both of PSi338 and PSi717. It is concluded that PSi338 and PSi717 siRNA plasmid vectors have been successfully constructed, and both vectors are effective and specific siRNA plasmids for suppressing GATA-3 gene expression.

Histone deacetylase inhibitor SAHA induces inactivation of MAPK signaling and apoptosis in HL-60 cells / 中国实验血液学杂志

The study was aimed to investigate the molecular mechanisms of histone deacetylase inhibitor SAHA-induced apoptosis of acute myeloid leukemia cell line HL-60. The effect of SAHA on HL-60 cell proliferation was detected by MTT assay and the cell morphological changes were observed with Wright-Giemsa and Hoechst33342 staining. The cell cycle distribution was determined by flow cytometry and the expression of cell signaling proteins were detected by Western-blot analysis. The results showed that SAHA inhibited the proliferation of HL-60 cells in dose- and time-dependent manners, after 2 micromol/L SAHA exposure for 12 - 48 hours, the cell cycle was arrested at G(0)/G(1) phase and apoptotic cell death was confirmed by either defined apoptotic bodies stained by Hoechst33342, Western blot showed cleaved-PARP, which represents the activation of caspase 3. The Western blot analysis indicated the activation of two important survival signal pathways after SAHA treatment, the phosphorylation of Raf and its downstream ERK kinases were remarkable downregulated, whereas the phosphorylation of AKT and its downstream molecular mTOR were not changed. It is concluded that SAHA-induced apoptosis of HL-60 cells is mediated by inactivation of p44/42 MAPK signaling pathway.

Differentially expressed genes in human giant-cell lung cancer lines with different metastatic potentials / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To screen genes differentially expressed in two human giant-cell lung cancer lines of same origin but with different metastasis potentials.</p><p><b>METHODS</b>Suppression subtractive hybridization (SSH) was done twice on two giant-cell lung cancer lines, PLA-801C and PLA-801D (hereafter abbreviated as C and D), of same origin but with low (C) and high (D) metastatic potentials. In the first round, SSH C was used as tester and D as driver, while in the second round, the tester and driver were interchanged. The sequences acquired from both rounds of SSH were spotted on glass slides respectively and screened by hybridizing with two-color fluorescence probes. Clones that had different expression levels on chips were also confirmed by RNA dot blot or Northern blot.</p><p><b>RESULTS</b>There were 16 sequences with high expression in C as compared to those in D, and 79 sequences with high expression in D compared to those in C. After sequencing, most of them were found to be highly homologous to those encoding the following proteins: (1) cytokines and their receptors; (2) kinases and related proteins; (3) other proteins including enzymes, heat shock proteins, receptors, proteins of cell skeleton and mitochondria, products of oncogenes, etc; (4) some proteins deduced from gene sequences with yet unknown functions.</p><p><b>CONCLUSION</b>The alterations in expression of some known genes, including HSP70, AXL receptor tyrosine kinase and 14-3-3zeta, might have impact on metastasis of giant-cell lung cancer. Whether some differentially expressed genes newly revealed are metastasis-related needs further study.</p>

The specific killing of human melanoma cells by replication selective adenovirus / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To construct replication selective adenovirus AdhepE1 targeting human melanoma and observe its specific killing of human melanoma cells in vitro.</p><p><b>METHODS</b>Adenovirus E1 region, the murine tyrosinase promoter and enhancer DNA sequences were acquired respectively by PCR cloning. The shuttle plasmid of replication-selective adenovirus targeting human melanoma was constructed by DNA recombination. Replication-selective adenovirus AdhepE1 was generated by homologous recombination. The human melanoma cell line SK-Mel-1 and hepatocellular carcinoma cell line HepG2 were attacked separately by lower dose of AdhepE1. Change of cell morphology was observed and the surviving cells were calculated. The expression of E1A was assayed by RT-PCR to verify the specific-replication of AdhepE1.</p><p><b>RESULTS</b>Replication selective adenovirus AdhepE1 targeting human melanoma was acquired by PCR. Human melanoma cell line SK-Mel-1 was sensitive to oncolytic killing of AdhepE1 whereas HepG2 was little responsive. The results of RT-PCR suggested that AdhepE1 replicated specifically in human melanoma cells.</p><p><b>CONCLUSION</b>AdhepE1 can selectively kill human melanoma cells.</p>

Purification of HLA-DR molecules / 军事医学科学院院刊

Objective:To purify HLA-DR molecules. Methods: Anti-HLA-DR antibody L243 was purified and coupled with CNBr activated Sepharose 4B gel. Immunoaffinity column was used to purify HLA-DR molecules. Results:Twenty micrograms of HLA-DR molecules were isolated from about 5 g Epstein-Barr virus-transformed human B lymphoblastoid cell line RAJI lysates by affinity chromatography. The purified HLA-DR molecules existed in α/β heterodimers form and could bind to conformation-dependent antibody L243. These HLA-DR molecules were separated into two strands,α and β,by boiling denaturation. These results are the basis for studying MHC Ⅱ binding peptide motif and CD4＋ T cell epitopes of antigens in future.

FK228 blocks cells survival signal pathways and induces apoptosis of prostate cancer DU145 cells / 中国肿瘤生物治疗杂志

Objective:To investigate the underlying mechanism of histone deacetylase (HDAC) inhibitor FK228-in- duced apoptosis of the prostate cancer cell line DU145.Methods:The inhibitory effect of FK228 on DU145 cell growth and its cytotoxicity were determined by MTT assay;cell cycle arrest was detected by flow cytometry assay;morphological change was observed by Giemsa staining;and defined kinase protein levels were determined by Western blot analysis.Re- suits:FK228 obviously inhibited DU145 cells growth,arrested cell cycle at G_0/G_1 phase,induced cells morphological changes and degraded several kinase proteins,including EGFR,Her2,Raf-1,Src,Cdk4 and IAP member Survivin.The degradation of these kinases blocked Raf-Mek-Erk and PI3K/Akt survival signal pathways,inducing apoptosis.Condu- sion:FK228 may induce DU145 cell apoptosis through depletion of multiple kinase proteins and blockade of survival sig- nal pathways of DU145 cells.