Analysis of TCR Vβsubfamily for the diagnosis of MHC deficiency-induced subclinical graft-versus-host disease / 中华微生物学和免疫学杂志

Objective To analyze the possibility of using TCR Vβsubfamily as the diagnostic in-dicators for major histocompatibility complex( MHC) deficiency-induced graft-versus-host disease( GVHD) . Methods The BALB/c mice were given 9.5 Gy (950 rad) of irradiation and transplanted with 106 of T-cell depleted (TCD) bone marrow cells from C57BL/6 and DBA/2 mice with MHC Ⅱ deficiency.Two control groups were set up accordingly by injection of TCD bone marrow cells from wild type ( WT) C57BL/6 and DBA/2 mice.Several parameters including the body weight, the GVHD clinical score and the survival time of the recipients were monitored.Flow cytometry analysis and mixed lymphocyte culture test were performed for the evaluation of autoimmune responses.Histological examination was used to analyze the severity of GVHD.Results The MHC deficiency-induced GVHD was successfully induced in the irradiated BALB/c mice receiving MHC mismatched allogeneic hematopoietic cell transplantation ( allo-HCT ) . The MHC matched DBA/2 mice with MHC deficiency could be used as the mice model of subclinical GVHD.Changes of the TCR Vβ6 were consistent with the results of histopathological examination.Conclusion Highly ex-pressed TCR Vβ6 could be used as indicators for the diagnosis of MHC deficiency-induced subclinical GVHD.

Expression, purification and identification for fibronectin C-terminal heparin-binding domain polypeptide in Pichia pastoris / 生物工程学报

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.

Effects of Triptolide on HL-60 Cell in vitro and in vivo / 中国药理学通报

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.

Cell-specific expression of the diphtheria toxin A-chain coding sequence induces cancer cell suicide / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To test whether the diphtheria toxin A (DT-A) chain coding sequence linked to murine immunoglobulin Kappa light chain (IgKappa) promoter and enhancer have selective cytocidal effects on IgKappa producing cells.</p><p><b>METHODS</b>The diphtheria toxin A gene or beta galactosidase (beta-gal) gene were linked to a murine IgKappa promoter and enhancer to construct pcDNA3IgKappaDTA or pcDNA3IgKappaLacZ plasmids. These plasmids were transfected into IgKappa producing or non-producing cells by the liposome coated DNA method. Expression of beta-gal activity and effects on cell growth of transfected cells were assessed.</p><p><b>RESULTS</b>The beta-gal gene, under the control of cytomegalovirus (CMV) promoter, can express in all cell lines. Expression of beta-gal under the control of the IgKappa promoter was detected only in the IgKappa producing cell line, CA46. Expression of beta-gal was greatly suppressed when cotransfected with pcDNA3IgKappaDTA in CA46 cells. Cell growth of CA46 cells transfected with pcDNA3IgKappaDTA plasmid was significantly inhibited compared with CA46 cells transfected with pcDNA3IgKappaLacZ.</p><p><b>CONCLUSION</b>Selective killing of IgKappa producing cells can be attained by introducing the diphtheria toxin A gene under the control of IgKappa promoter and enhancer.</p>

Anticancer activities of curcumin on human Burkitt's lymphoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the anticancer activities of curcumin on human Burkitt's lymphoma and their molecular mechanism.</p><p><b>METHODS</b>The 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).</p><p><b>RESULTS</b>1. 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.</p><p><b>CONCLUSION</b>Curcumin 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.</p>

Expression of cytokines and their receptors in leukemia cell lines and normal blood cells / 中国病理生理杂志

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.

Effects of TGF-?_1 and TNF-? antisense PS-ODNS on e x vivo expansion of hematopoietic stem/progenitor cells(HSPC) / 中国病理生理杂志

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.

Fibronectin supports endotoxemia mice survival / 中国病理生理杂志

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.

Deficiency of TGF-?_1 in leukemic cells and the effects of exogenous TGF-?_1 gene on HL-60 cells / 中国病理生理杂志

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