Expression of cytokines in mouse hepatitis B virus X gene-transfected model / 华中科技大学学报（医学）（英德文版）

The expression profile in the mouse hepatitis B virus X (HBx)-transfected model was investigated in order to lay a foundation for further study on the implication of cytokines expression in hepatitis B virus (HBV) infection. Hydrodynamic injection method via the tail vein was used to establish the animal HBx-transfected model. By using microassay, the differential expression of gene in each group was analyzed, which was further confirmed by using real-time PCR and semi-quantitative PCR. Most of chemokine genes such as Ccl2, Ccl5, Ccl9, MIG and IP-10 were up-regulated in the HBx-transfected mouse model versus the control mice, which was coincided with the microarray results. Western blotting and immunohistochemistry were applied to detect the expression of MIG and IP-10 in the liver tissues. Simultaneously, ELISA was adopted to measure the content of IFN-γ in the liver tissues. DNA microassay revealed that the expression of 611 genes changed in HBx-transfected mice as compared with that in pCMV-tag2B-transfected mice, and most of the screened chemokines were up-regulated (including MIG and IP-10). Additionally, IFN-γ protein levels were increased by 20.7% (P<0.05) in pCMV-tag2B-HBx-transfected mice as compared with the untreated mice. IFN-γ protein levels were reduced by 53.9% (P<0.05) in pCMV-tag2B-transfected mice as compared with the untreated mice, which was consistent with the up-regulation of MIG and IP-10. It was suggested HBx transfection could induce the expression of MIG and IP-10 in the liver tissues, which might play the roles in HBV-related liver immunity and cytokines-mediated antiviral effect.

Expression of cytokines in mouse hepatitis B virus X gene-transfected model / 华中科技大学学报（医学）（英德文版）

The expression profile in the mouse hepatitis B virus X (HBx)-transfected model was investigated in order to lay a foundation for further study on the implication of cytokines expression in hepatitis B virus (HBV) infection. Hydrodynamic injection method via the tail vein was used to establish the animal HBx-transfected model. By using microassay, the differential expression of gene in each group was analyzed, which was further confirmed by using real-time PCR and semi-quantitative PCR. Most of chemokine genes such as Ccl2, Ccl5, Ccl9, MIG and IP-10 were up-regulated in the HBx-transfected mouse model versus the control mice, which was coincided with the microarray results. Western blotting and immunohistochemistry were applied to detect the expression of MIG and IP-10 in the liver tissues. Simultaneously, ELISA was adopted to measure the content of IFN-γ in the liver tissues. DNA microassay revealed that the expression of 611 genes changed in HBx-transfected mice as compared with that in pCMV-tag2B-transfected mice, and most of the screened chemokines were up-regulated (including MIG and IP-10). Additionally, IFN-γ protein levels were increased by 20.7% (P<0.05) in pCMV-tag2B-HBx-transfected mice as compared with the untreated mice. IFN-γ protein levels were reduced by 53.9% (P<0.05) in pCMV-tag2B-transfected mice as compared with the untreated mice, which was consistent with the up-regulation of MIG and IP-10. It was suggested HBx transfection could induce the expression of MIG and IP-10 in the liver tissues, which might play the roles in HBV-related liver immunity and cytokines-mediated antiviral effect.

Arsenic trioxide downregulates the expression of annexin II in bone marrow cells from patients with acute myelogenous leukemia / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Most patients with acute myelogenous leukemia (AML) suffer from disordered hemostasis. We have previously shown that annexin II (Ann II), a high-affinity co-receptor for plasminogen/tissue plasminogen activator, plays a central role in primary hyperfibrinolysis in patients with acute promyelocytic leukemia (APL). The expression of Ann II in cells from patients with major subtypes of AML and the effect of arsenic trioxide (As2O3) on Ann II expression in AML cells were investigated to determine whether As2O3-mediated downregulation of Ann II could restore hemostatic stability.</p><p><b>METHODS</b>A total of 103 patients (48 females and 55 males; age, 19 - 58 years) were included. Plasma samples were collected before and after treatment as well as after complete remission. Ann II and plasminogen activation were measured in leukemic cells during treatment with 1 micromol/L As2O3.</p><p><b>RESULTS</b>Before As2O3 treatment, Ann II mRNA expression (real-time PCR) was the highest in M3 cells (P < 0.05), higher in M5 cells than that in M1, M2, M4, and M6 cells (P < 0.001), and positively correlated with Ann II protein expression (flow cytometry) (r = 0.752, P < 0.01). Exposure for up to 120 hours to As2O3 (1 micromol/L) had no significant effect on Ann II protein in M1 and M2 leukemic cells, but decreased Ann II protein expression twofold within 48 hours of exposure in M3 cells (P < 0.05) and twofold within 96 hours in M5 cells (P < 0.05). The rate of plasmin generation was higher in APL, M5, and M4 cells than in M1, M2, and M6 cells.</p><p><b>CONCLUSIONS</b>As2O3 may reduce hyperfibrinolysis in AML by downregulation of Ann II. Furthermore, As2O3 affects more than one form of AML (APL, M4 and M5), suggesting its potential role in their management.</p>

Effect of arsenic trioxide on the expression of apoptosis-related genes in NB4 cells / 中国实验血液学杂志

The aim of this study was to investigate the gene expression profiles of acute promyelocytic leukemia (APL) cell line NB4 treated with arsenic trioxide (As2O3) by using cDNA microarray. cDNA probes were prepared through reverse transcription from mRNA of NB4 cells treated with or without arsenic trioxide. The probes were labeled with Cy3 and Cy5 fluorescence dyes individually, hybridized with cDNA microarray representing 201 different human genes, and their fluorescent intensities were scanned. The genes were screened through the analysis of the difference in the gene expression profile. The results showed that after the treatment of arsenic trioxide (2 micromol/L), 6 genes were up-regulated, and 12 genes related to apoptosis and signal transduction were down-regulated. The p21, survivin, cdc2 and Wee1Hu genes may be related to the differentiation and/or apoptosis of NB4 cells induced by As2O3. It is concluded that p21, survivin, cdc2 and Wee1Hu may play an important role in the mechanism underling arsenic trioxide-mediated NB4 cell apoptosis.

Effects of arsenic trioxide or retinoic acid on mRNA and protein expression of tissue factor and thrombomodulin and procoagulant activity in NB4 cells / 中国实验血液学杂志

To investigate the effect of arsenic trioxide (As(2)O(3)) or all-trans retinoic acid (ATRA) on the mRNA and protein expression of tissue factor (TF) and thrombomodulin (TM) and procoagulant activity (PCA) in NB4 cells. The NB4 cells were cultured in vitro and treated with As(2)O(3) or ATRA, expression of TF and TM antigen, and PCA change of treated NB4 cells were detected with ELISA, TF and TM mRNA transcription on the NB4 cells was assayed with reversed transcription polymerase chain reaction (RT-PCR). The results showed that 1 micromol/L As(2)O(3) and 1 micromol/L ATRA both gradually downregulated the expression of TF antigen and mRNA on NB4 cells, a human promyelocytic leukemia cell line, in time-dependent manner, as compared with control. The levels of TF antigen expression in AS(2)O(3) group were 13.3 +/- 1.8, 8.6 +/- 1.9, 10.8 +/- 1.5, 2.0 +/- 0.6 and 2.6 +/- 0.9 ng/10(7) respectively; while the levels of TF antigen expression in ATRA group were 12.4 +/- 1.1, 11.3 +/- 1.8, 5.7 +/- 1.7, 2.8 +/- 0.8 and 2.0 +/- 0.6 ng/10(7) at 24, 48, 72, 96 and 120 hours respectively (P<0.05). The procoagulant activity (PCA) of NB4 cells was decreased, blood coagulation times were 123.5 +/- 10.5, 156.3 +/- 11.6, 179.3 +/- 15.3, 248.9 +/- 20.1, 312.0 +/- 29.8 seconds in As(2)O(3) groups, respectively; 76.4 +/- 5.6, 146.8 +/- 10.9, 198.2 +/- 15.6, 265.8 +/- 20.6 and 363.8 +/- 31.9 seconds in ATRA groups respectively at 24, 48, 72, 96 and 120 hours (P<0.05). ATRA upregulated TM antigen expression on NB4 cells. It is concluded that the As(2)O(3) and ATRA decrease mRNA transcription of TF, downregulate expression of TF and reduce procoagulant activity in NB4 cells. The TM transcription and expression upregulated by ATRA may alleviate dysfunction of coagulation in APL.

In vitro anti-tumor effect of CTL induced by HSP70-Id complex-modified dendritic cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To prepare the tumor antigen peptide complex (HSP70-1d) of HSP70 and idiotype (Id) from SmIg ScFv fragment in patients with Chronic B cell leukemia (B-CLL), and to study the anti-tumor effect of cytotoxic T lymphocyte (CTL) induced by HSP70-Id complex-modified dendritic cell (DC) in vitro and explore their immune mechanism.</p><p><b>METHODS</b>Purified HSP70 was combined into peptide complex (HSP70-Id) with the prepared Id-ScFv from B-CLL cells in vitro by using biochemical technique. The plastic-adherent monocytes from human peripheral blood were cultured and induced into DC with rhGM-CSF and rhIL-4 using cell culture and separation technique. The cultured DC were harvested and pulsed by HSP70-Id complex. DC morphology was observed under converted phase microscope and its phenotype was characterized by FCM on 8th day as well as their secreting cytokines were measured. Host lymphocytes were stimulated by DC loaded with HSP70-Id complex and co-cultured in the medium containing IL-2. The activation and proliferation of lymphocytes were examined by MTr test, which was also used to assay cytotoxicity of CTL elicited by modified DC to Daudi, K562 and HepG2 tumor cells, and FCM analyzed the changes of T lymphocyte subsets.</p><p><b>RESULTS</b>Mature DCs were obtained successfully, showing typical morphology and phenotypic properties, the expression ratio of cellular surface molecules, CD1a was 20% - 30%, CD83 was more than 72% , both CD86 and HLA-DR over-expressed obviously in the complex-loaded DC group secreting cytokines of Thl type, IL-12 and TNF-alpha. The culturing lymphocytes that were activated by modified DC could more effectively and specifically kill Daudi (71. 24%), but not K562 and HepG2 tumor cells. Results of FCM assay demonstrated that percentage of CD4+ and CD8+ T lymphocytes cocultured with complex-modified DC increased notably to 56.51% and 70.21%, respectively. CD4+ T/ CD8+ T proportion was changed from 1.49 to 0.81. The dose of peptide would be reduced to 1/50 if specific CTL induced by complex-modified DC instead of directly by peptide complex.</p><p><b>CONCLUSION</b>DCs modified by HSP70-Id complex exhibit powerful biological activities, and could induce CTL to specific cytotoxicity against carcinoma cells. It might be produced by cooperation of CD4+ T, CD8+ T lymphocytes and DC. The results also suggested that DC modified by HSP70-Id complex can present antigen and induce CTL with high efficacy and specificity.</p>

Study on the mimic epitopes screening program severe acute respiratory syndromes antigen with random phage peptide library / 中华流行病学杂志

<p><b>OBJECTIVE</b>To screen the severe acute respiratory syndromes (SARS) mimotopes with random phage peptide library and to investigate their immunogenicity.</p><p><b>METHODS</b>Using SARS sera as selective molecule, a 12 mer phage peptide library was biopanned and positive clones containing the mimic epitopes were selected. The immuno-characteriation of the epitopes were then investigated.</p><p><b>RESULTS</b>2 positive clones that having specific affinity to SARS sera were obtained. The DNA sequencing data showed no homology between the sequences of the deduced amino acid of the two mimic antigen peptides and the sequence of SARS.</p><p><b>CONCLUSION</b>SARS mimotopes were obtained by phage peptide library screening. This method might provide a new approach for SARS therapy and vaccine development.</p>

The construction of transferrin receptor- mediated HSV-TK gene transfer system and its effect on human hepatocellular carcinoma cells in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To construct the localization system involving anti-TfR monoclonal antibody (McAb) and AFP promoters and assess its effect on human hepatoma cell lines.</p><p><b>METHODS</b>The conjugate of anti-TfR McAb and polylysine (PLL) was made by SPDP and purified by molecular screen chromatography. DNA blocking test determined that the ratio of one pEBAF/tk to six Ab-PLL was the most suitable to couple them. The pEBAF/tk recombinant plasmid bearing HSV-TK gene was coupled to Ab-PLL by noncovalent bond. The pEBAF/tk was transferred into human hepatoma cell line HepG2, SMMC7721 and pulmonary cancer cell line A549 by receptor-mediated gene delivery (Ab-PLL-DNA) and liposome procedure. The growth inhibitory rates of HepG2, SMMC7721 and A549 cells were measured by MTT assay.</p><p><b>RESULTS</b>The inhibitory rates of HepG2/tk in 100 mg/L and 1 mg/L of GCV were 60.5% and 24.3%, respectively. The inhibitory rate of GCV to SMMC7721 was 23.2% in 3 days. The pulmonary cancer cell A549, A549/tk (Ab) and A549 /tk (lipo) could not be inhibited by the addition of GCV.</p><p><b>CONCLUSION</b>The localization system employed in this paper has high specificity, effectiveness and safety for gene therapy. It would be a promising strategy for gene therapy.</p>

Survivin antisense RNA enhances taxol-induced apoptosis in leukemia cell line HL-60 / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the effects of survivin antisense RNA on taxol-induced apoptosis in leukemia cell line HL-60.</p><p><b>METHODS</b>A survivin antisense eukaryotic vector pcDNA3-SVVas was transferred into HL-60 cells by electroporation. The live fraction was determined by trypan blue dye exclusion assay. Cell counting and MTT assay were performed to evaluate the sensibility of the transfected cells to taxol. Apoptosis was detected by DNA gel electrophoresis and nuclear staining.</p><p><b>RESULTS</b>Two positive cell clones, HL-60 SVVas and HL-60 neo were obtained. Compared to HL-60 and HL-60 neo cells, HL-60 SVVas cells growth was significantly reduced (P < 0.05). By MTT assay, the IC(50) of taxol to HL-60 SVVas, HL-60 neo and HL-60 cells were (14.4 +/- 1.87) ng/ml, (31.9 +/- 6.38) ng/ml and (32.0 +/- 3.52) ng/ml, respectively, the difference was significant by statistic analysis (P < 0.01). Agarose gel electrophoresis of genomic DNA from HL-60 SVVas showed typical DNA ladder, but DNA from HL-60 neo and HL-60 did not. Nuclei become condense in HL-60 SVVas cells.</p><p><b>CONCLUSION</b>Survivin antisense RNA could enhance taxol-induced apoptosis in leukemia cell line HL-60. This may lay an experimental foundation for further research of gene therapy in leukemia.</p>

Construction of expression vector for recombinant annexin II and characteristics of its fibrinolysis / 中国实验血液学杂志

The study was designed to investigate annexin II resulting in molecular pathological mechanism of the primary fibrinolysis and establish annexin II vector model for further research on disturbance of coagulation. A target gene was amplified from human umbilical vein endothelial cells (HUVEC) by RT-PCR. Annexin II gene fragment was purified and ligated with molecular biological recombinant technology. The recombinant of plasmid annexin II was transfected into HL-60 cells and its distribution in the cell and structure characteristics of annexin II protein were evaluated by multi-photon excitation laser scanning microscope. By means of flow cytometry (FCM) and Werstern blot technique, the protein expression was qualitatively and quantitatively analyzed. Transfected cells were treated in vitro with annexin II antisense oligonucleotide (AS) targeting to the start site of annexin II cDNA. The results showed that the recombinant pZeoSV2(+)/ANN II was constructed successfully and expressed in HL-60 cells. The protein expression was distributed on the surface of cell by fluorescence assay. After transfection for 48 hours, the cells occurred higher level of expression. The level of the plasmin was significantly enhanced in the present of annexin II. The FCM and Western blot analysis showed the annexin II expression was similar both in transiently and stably transfected in HL-60 cells. Annexin II antisense oligonucletide and McAb significantly inhibited the activity of plasminogen. It was concluded that annexin II plays an important role in the fibrinotysis. Annexin II vector was defined as a expression tool for further studying fibrinolysis and coagulopathy in malignant disease.