AIM: To study the preventive effect of fibronectin on hepatic failure induced by endotoxin in mice.METHODS: The survival rate was observed in endotoxemia mice injected with fibronectin from human plasma. The tissue damage and expression of TNF?, IL-1?, IL-6 mRNA in hepatocyte were detected by the methods of histology, ultrastructure, DNA fragementation and RT-PCR. RESULTS: ①Fibronectin obviously reduced the mortality of endotoxemia mice sensitized by D-galactosamine(GalN). ②Histopathology showed that less necrosis occurred on the hepatocyte of endotoxemia mice injected with fibronectin, compared with saline control. ③Ultrastructure and DNA fragmentation showed fibronectin suppressed hepatocyte apoptosis induced by LPS. ④Fibronectin down-regulated the overexpression of TNF?, IL-1?, IL-6 mRNA on hepatocyte induced by LPS. CONCLUSION: Fibronectin supports endotoxemia mice surrival by down-regulating the expression of TNF?, IL-?, IL-6, it may be a potent therapy for endotoxemia.

Aim To study the effects of Triptolide (TPL) on HL-60 cells in vitro and in vivo.Methods MTT was used to examine the effects of TPL on proliferation of HL-60 cells;TUNEL assay was used to detect apoptotic cells.The effect of TPL on xenograft growth of HL-60 cells was evaluated by tumor inhibition rate.Results In vitro TPL inhibited the proliferation and induced apoptosis of HL-60 cells in a dose-dependent manner.In vivo TPL inhibited xenograft growth of HL-60 cells with the highest inhibition rate of 53.5%.Conclusion TPL is able to inhibit the proliferation of HL-60 cells and induce apoptosis of HL-60 cells in vitro and in vivo.

AIM: To study the expression of cytokines and their receptors in leukemia cell lines and normal blood cells. METHODS: RT-PCR was used to detect expression of mRNA for cytokines in leukemia cell lines(HL-60,U937,K562,HEL,DAMI,MEG-01,HUT78 and CA) and normal blood cells, including CD34 + cells, megakaryocytes,platelets, peripheral mononucleates cells and granulocytes. RESULTS: ①CD34 + cells simultaneously expressed mRNA for IL-1(?,?),IL-3, IL-6 , G-CSF, GM-CSF and their receptors and SCFR,MPL as well. The granulocytes only expressed IL-6,IL-6R,G-CSFR,GM-CSF. Megakaryocytes and platelets only expressed IL-3R,IL-6,IL-6R,MPL.Interestingly, TGF? 1 ,TNF? and their receptors sustained to express in normal cells.②Most leukemia cell lines were found to simultaneously express at least two or more stimulating cytokines and receptors ,while TGF? 1 , TNF? and their receptors were expressed in all the leukemia cell lines we observed. CONCLUSIONS: ①Multi-autocrine loops exist in leukemia cells;②Imbalance of autocrine loops of positive and negative cytokines may be related to leukemia.

AIM: To study the effect of TGF-? 1 and TN F-? antisense PS-ODNS on ex vivo expansion of hematopoietic stem/progenitor cell s (HSPC). METHODS: CD 34 + cells were purified from fres h umbilical cord blood by immunomagnetic beads, and mononuclear cells were purifi ed from bone marrow by Ficoll-hypaque . The effects of TGF-? 1 and /or TNF-? an tisense PS-ODNS on ex vivo expansion of CD 34 + cells、CFU-GEMM、CFU-G M、CFU-E and BFU-E were detected by using liquid and semi-solid culture systems . RESULTS: TGF-? 1 antisense PS-ODNS cooperated with cytokines increased the number of CD 34 + cells,CFU-GEMM,CFU-GM,CFU-E and BFU-E , which was as 4,2.6,2.7,1.8,2.1 times as that of the control (the cytokine s combination), respectively. TNF-? antisense PS-ODNS cooperated with cytokines respectively increased the number of CD 34 + cells, CFU-GEMM, CFU-GM, CF U-E and BFU-E by 4, 2 9, 2 6, 1 7, 1 8 times as that of the control. The ab ove two antisense PS-ODNS cooperated with cytokines could respectively increased the number of CD 34 + cells, CFU-GEMM, CFU-GM, CFU-E and BFU-E by 5 3,2 1, 2 7, 1 9, 1 8 times as that of the control. CONCLUSION: I nhibition of endogenous TGF-? 1 and TNF-? by antisense PS-ODNS will be one of the effective methods to expand HSPC ex vivo.

AIM: To explore the existence of deficiency of TGF-?_1 in leukemia cells and its possible mechanism in the pathogenesis of leukemia. METHODS: The levels of TGF-?_1 were detected by ELISA in the cultured supernatant of leukemia cell lines and primary cells from patients with acute leukemia. TGF-?_1 gene was transduced into HL-60 cells by lipofectin-mediated DNA transfection. In the presence of G418, the HL-60 clone expressing TGF-?_1 was selected. The effects of exogenous TGF-?_1 gene on the proliferation and apoptosis of HL-60 cells were studied by leukemic colony assay, tumorigenicity in athymic nude mice, DNA fragmentation and cell cycle analysis. The expression of intrinsic TGF-?_1, [STBX]bcl-2 oncogene, hTERT mRNA on the apoptosis of HL-60 cells induced by exogenous TGF-?_1 gene were detected by RT-PCR. RESULTS: The levels of TGF-?_1 were obviously lower in the supernatant of leukemia cell lines and primary cells from patients with acute leukemia, as compared with normal controls (P

Objective To study the temporo-spatial changing laws of calcitonin gene-related peptide(CGRP)in the rat model of sciatic nerve ligation.Methods Totally 48 Sprague-Dawley rats were randomly divided into sham-operation group(n=6)and experimental groups which survived for 1,3,5,7,14,21,28 d respectively after sciatic nerve ligation(n=6 at each time point).The distribution and quantity of CGRP in sciatic nerve,dorsal root ganglion(DRG)and spinal cord were detected by immunohistochemistry and image analysis methods.Results A large amount of CGRP piled up in the sciatic nerve at 1 d after the ligation,significantly higher in proximal segment than that in distal segment,gradually dropping till disappear basically at 14 d in proximal segment and at 7 d in distal segment.The expression of CGRP in DRG at 7 d began to drop after the ligation,at 21 d to the minimal value and at 28 d lower than normal.The CGRP-immunoreactivety in ipsilateral spinal dorsal horn at 14 d decreased after operation,but the immuno-positive areas of CGRP were of no changes.There are no changes in the CGRP-positive spinal ventral motoneurons of all groups.Conclusion The changes of CGRP presented a temporo-spatial pattern following peripheral nerve legation,maybe resulting from the deficiency of neurothrophins from target organs.

OBJECTIVETo study the anticancer activities of curcumin on human Burkitt's lymphoma and their molecular mechanism.

METHODSThe effect of curcumin on the growth of CA46 cells and apoptosis were studied through Trypan blue exclusion, MTT assay, cell cycle, DNA fragmentation analysis and detection of TdT-mediated dUTP nick end labeling (TUNEL). The effect of curcumin on the expression of c-myc, bcl-2, mutant-type p53 and Fas protein and mRNA was studied by flow cytometry (FCM) and reverse transcription-polymerase chain reaction (RT-PCR).

RESULTS1. Curcumin inhibited proliferation of CA46 cells in a time- and dose-dependent manner, 2. CA46 cells treated with curcumin showed G(0)/G(1) or G(2)/M phase increase and S phase decrease, 3. CA46 cells apoptosis induced by curcumin was confirmed by DNA fragmentation and TUNEL and 4. The expression of c-myc, bcl-2, mutant-type p53 protein and mRNA was decreased sharply in CA46 cells treated with curcumin, while Fas protein and mRNA was increased.

CONCLUSIONCurcumin is able to inhibit the proliferation of CA46 cells and induce the cell apoptosis by down-regulating the expression of c-myc, bcl-2, mutant-type p53 and up-regulating the expression of Fas.

Antineoplastic Agents ; pharmacology ; Apoptosis ; Burkitt Lymphoma ; Cell Cycle ; drug effects ; Cell Division ; drug effects ; Curcumin ; pharmacology ; Gene Expression Regulation ; drug effects ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-myc ; biosynthesis ; genetics ; Tumor Cells, Cultured ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics ; fas Receptor ; biosynthesis ; genetics

To express and identify fibronectin C-terminal heparin-binding domain (FNCH BD) polypeptides in Pichia pastoris expression system and study its function, the fragment of FNCHBD was amplified by PCR and inserted into pGEM-T vector. After sequenced, the fragment was inserted into pAo815SM vector, and then cloned into the expression vector pPIC9k. The recombinant plasmid was linerarized with restrict enzyme Sal I and transferred into the yeast host cell KM71 and GS115. The positive yeast clone was screened by G418 resistant, and the target protein was induced to express in the medium containing 0.5% methanol. The culture supernatant was collected and then was purified with membrane ultrafiltration and ion exchange chromatography. The purified product was analyzed with mass spectrogram, SDS-PAGE, Western blotting and heparin affinity chromatography. The results showed that the target protein was around 32 kDa and the purity of the product was above 95%. FNCHBD could be specifically recognized by fibronectin polyclonal antibody. These results suggest that FNCHBD could be expressed and purified successfully in Pichia pastoris, which provides a good strategy to further studies.
Fibronectins ; biosynthesis ; chemistry ; genetics ; Genetic Vectors ; Heparin ; metabolism ; Peptides ; genetics ; metabolism ; Pichia ; genetics ; metabolism ; Protein Binding ; Protein Structure, Tertiary ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics

A recently identified protein, FAN1 (FANCD2-associated nuclease 1, previously known as KIAA1018), is a novel nuclease associated with monoubiquitinated FANCD2 that is required for cellular resistance against DNA interstrand crosslinking (ICL) agents. The mechanisms of FAN1 regulation have not yet been explored. Here, we provide evidence that FAN1 is degraded during mitotic exit, suggesting that FAN1 may be a mitotic substrate of the anaphase-promoting cyclosome complex (APC/C). Indeed, Cdh1, but not Cdc20, was capable of regulating the protein level of FAN1 through the KEN box and the D-box. Moreover, the up- and down-regulation of FAN1 affected the progression to mitotic exit. Collectively, these data suggest that FAN1 may be a new mitotic substrate of APC/CCdh1 that plays a key role during mitotic exit.
Anaphase-Promoting Complex-Cyclosome ; Bone Neoplasms ; metabolism ; pathology ; Cadherins ; genetics ; metabolism ; Cdc20 Proteins ; Cell Cycle Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Exodeoxyribonucleases ; genetics ; metabolism ; HEK293 Cells ; Humans ; Mitosis ; Osteosarcoma ; metabolism ; pathology ; Ubiquitin-Protein Ligase Complexes ; genetics ; metabolism