Humans ; Spleen ; injuries ; physiology ; surgery ; Splenectomy

Aged ; Anastomosis, Surgical ; Duodenum ; blood supply ; surgery ; Female ; Gastrointestinal Hemorrhage ; etiology ; surgery ; Humans ; Hypertension, Portal ; complications ; Jejunum ; surgery ; Rupture, Spontaneous ; Varicose Veins ; complications

Alzheimer Disease ; metabolism ; pathology ; Amyloid beta-Peptides ; metabolism ; Animals ; Brain ; metabolism ; pathology ; Cerebral Amyloid Angiopathy ; metabolism ; pathology ; Fluorescent Dyes ; chemistry ; Humans ; Mice ; Peptide Fragments ; metabolism ; Staining and Labeling ; Thiazoles ; chemistry

OBJECTIVETo construct and purify heme oxygenase-1, GFP gene mediated by recombinant adeno-associated-virus and identify expression rate of GFP in transplanted liver in rats.

METHODSHeme oxygenase-1 gene of rat was cloned and subcloned to rAAV vector, the gene sequence was confirmed correct by restriction enzyme and DNA sequencing. The rAAV-HO-1 was then cotransfected into 293 cell line with accessory plasmid virus helper and AAV-cap-rep through CaCl2 coprecipitation. Virus particles were purified by heparin column chromatography and titre were detected by Real-time PCR. An orthotopic liver transplantation model by Wistar to Wistar was set up using Kamada's two cuff technique. Purified rAAV-GFP was injected into portal vein and incubated for 2 hours at the donor liver cold preserved stage, and then performed OLT. Recipients were killed and visceral organs were sampled at 1 and 3 months after operation respectively, frozen section (3-5 microm) were prepared and gene expression rate in different tissues was examined under fluorescence microscope.

RESULTSThe inserted segment of HO-1 was identified through restriction enzyme cutting followed with electrophoresis, the result of DNA sequencing was in accordance with which found in Genbank. The GFP expression rate was over 80% in allograft at 1 and 3 month after transfection whereas there was no GFP expression in heart, lung, spleen, kidney and small bowel.

CONCLUSIONSHigh titre rAAV carried HO-1 and GFP were constructed successfully. Steady and effective expression of GFP mediated by rAAV was demonstrated in liver allograft in rats.

Animals ; Dependovirus ; genetics ; Female ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; metabolism ; Heme Oxygenase-1 ; genetics ; metabolism ; Liver ; metabolism ; Liver Transplantation ; Male ; Plasmids ; genetics ; Rats ; Rats, Wistar ; Recombination, Genetic ; Transfection

OBJECTIVETo explore the effect of von Hippel-Lindau(VHL) gene on growth of EL-4 solid tumors in vivo.

METHODSC57BL/6 mice model of solid tumors was established by subcutaneous injection of EL-4 lymphoma cells. Mice were randomly divided into two groups as treatment group (n=6) and control group (n=6) when tumor diameter increased to 0.1 cm and 0.4 cm respectively. Plasmid pcDNA3-VHL was injected into solid tumor in treatment group, empty pcDNA3 vector in control group. The growth of tumor was observed. Immunohistochemistry and Western blot analysis were used to examine the transgenic expression of VHL, hypoxia inducible factor-1alpha (HIF)-1alpha, bcl-2 and VEGF. Microvessel density (MVD) and apoptosis index (AI) of tumors were also detected.

RESULTSVHL gene transfer eradicated tumors with small size (0.1 cm diameter), but it only retarded the growth of large tumors (0.4 cm diameter). VHL was overexpressed, the expression levels of VEGF, HIF-1alpha and bcl-2 were reduced in treatment group compared with those in the control group. The level of MVD was significantly lower in treatment group (P< 0.05), but AI was higher in treatment group compared with those in the control group (P< 0.01).

CONCLUSIONVHL gene therapy can inhibit the growth of EL-4 solid tumor in vivo.

Animals ; Female ; Genetic Therapy ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Mice ; Mice, Inbred C57BL ; Neoplasms, Experimental ; blood supply ; therapy ; Neovascularization, Pathologic ; therapy ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; Transfection ; Vascular Endothelial Growth Factor A ; metabolism ; Von Hippel-Lindau Tumor Suppressor Protein ; therapeutic use

OBJECTIVETo investigate the synergistic effects of antisense HIF-1alpha gene therapy combined with B7-1-mediated immunotherapy on cancer treatment.

METHODSAntisense HIF-1alpha and B7-1 expression vector were constructed. Lymphoma cells EL-4 were injected subcutaneously into C57BL/6 mice and transplanted lymphomas were established. The mice received either antisense HIF-1alpha, B7-1, or a combinational agent, complexed with DOTAP cationic liposomes. The tumor growth in the mice was monitored. Expression of HIF-1alpha, B7-1 and VEGF were detected by immunohistochemistry and Western blotting. The tumor blood vessels were immunostained with CD31- antibodies and the tumor vascular density was assessed by light microscopy.

RESULTSGene transfer of plasmid expressing the encoded antisense HIF-1alpha inhibited VEGF expression and reduced vascular density in the tumors, eradicated tumors in diameter smaller than 0.1 cm and only retarded the growth of larger tumors. Whereas combination of antisense HIF-1alpha gene therapy and B7-1 immunotherapy eradicated all tumors in diameter of 0.4 cm.

CONCLUSIONAntisense HIF-1alpha blocks tumor hypoxia pathway by downregulating VEGF expression, reduction of vascular density and enhances B7-1-mediated immunotherapy. Strategies that target HIF-1 may have therapeutic potential in cancer treatment and are worthy of further studying.

Animals ; B7-1 Antigen ; genetics ; therapeutic use ; Gene Transfer Techniques ; Genetic Therapy ; methods ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; therapeutic use ; Lymphoma ; immunology ; therapy ; Male ; Mice ; Mice, Inbred C57BL ; Neovascularization, Pathologic ; therapy ; Oligonucleotides, Antisense ; genetics ; therapeutic use

OBJECTIVETo study the morphology and functional character of blood-spleen barrier (BSB) and establish the concept of BSB.

METHODSThirty healthy Wistar rats were studied. Ten rats were injected with 1.5 ml mixed fluid of India ink and physiological saline through the tail vein. Histological changes of the spleen in all animals were observed with light and electron microscopy, including HE, Foot, Masson staining and immunohistochemistry of CD68 and CD34.

RESULTSMost of the carbon particles were within the splenic sinuses in marginal zone but not in the white pulp after 6 h. There was a characteristic distribution of the macrophagocytes, vessel endothelial cell, reticular tissue and collagen fiber in the BSB.

CONCLUSIONSBSB, surrounding the white pulp, is composed of macrophagocytes, marginal-sinus-endothelial cells and their basement membrane, the reticular tissue (reticular cells and reticular fibers) and collagen fibers. The role of BSB is to keep the microenvironment of white pulp stable. It becomes mature while the formation of germinal center of the white pulp. The permeability of BSB changes during its development.

Animals ; Basement Membrane ; ultrastructure ; Endothelial Cells ; ultrastructure ; Female ; Macrophages ; ultrastructure ; Male ; Rats ; Rats, Wistar ; Reticulocytes ; ultrastructure ; Spleen ; blood supply ; ultrastructure

To approach the effect of CCAAT/enhancer binding proteins (C/EBPs) on un-terminal differentiation of HL-60 cells after treatment with Arsenic Trioxide ( As_2O_3) . Methods The changes of cell morphology were observed by Wright staining, the alteration in the cell proliferation was determined by WST1 experiment and the NBT reduction assay was used to detect the differentiation condition of cells, determination and analysis cell cycle. The expressions of C/EBP? and C/EBP? mRNA in HL-60 cells exposed to ATRA and As_2O_3 were assayed by semi-quantitative RT-PCR. Results It was found that ATRA could up- regulate the mRNA expression of C/EBP? obviously, but down-regulate the mRNA expression of C/EBP?. As_2O_3 could up-regulate the mRNA expression of C/EBP? lightly, down-regulate the expression of C/EBP?. Conclusion Both of ATRA and As_2O_3 can down-regulate the mRNA expression of C/EBP?,but there is no significant difference between these two groups,ATRA and As2O3 can up- regulate the mRNA expression of C/EBP?, with significant differences (P

The objective of this study was to investigate the effect of hexamethylene bisacetamide (HMBA) on differentiation of HL-60 cells and its possible molecular mechanism. HL-60 cells were co-cultured with different concentrations of HMBA (0.5, 1, 2 mmol/L) for 4 days, then the proliferation was assayed by MTT at different time points. Wright-Giemsa staining was used to observe the change in morphology. Cell differentiation antigen CD11b expression and the altered distribution of cell cycle in HL-60 induced by HMBA were analyzed by flow cytometry. The expressions of c-myc, mad1, p21, p27, hTERT and HDAC1 mRNA were detected by RT-PCR. The results indicated that the proliferation of HL-60 cells was inhibited by HMBA in a time-and-dose-dependent manner. Upon 2 mmol/L HMBA treatment, the HL-60 cells arrested at G(0)/G(1) phase and differentiated into granular line in morphology, with the up-regulation of CD11b expression. The expression of c-myc and hTERT mRNA obviously down-regulated, the expression of p21, p27 and mad1 mRNA up-regulated, while there was no change of the expression of hTERT mRNA. It is concluded that effect of HMBA on the differentiation of HL-60 cells may partly contribute to switch from c-myc to mad1 expression, to up-regulate expressions of p21 and p27 mRNA, and down-regulate hTERT mRNA expression, while there is no relation with the expression of HDAC1 mRNA.
Acetamides ; pharmacology ; Antineoplastic Agents ; pharmacology ; Cell Cycle ; drug effects ; Cell Differentiation ; drug effects ; genetics ; Cell Proliferation ; drug effects ; HL-60 Cells ; Humans

OBJECTIVESix1 and Six4 are expressed in several tumors, and associated with tumor progress and poor prognosis. The aim of this study was to investigate the expression of Six1 and Six4 in esophageal squamous cell carcinoma (ESCC), and to evaluate their correlation with the clinicopathological factors and prognosis.

METHODSTissue microarray technology and immunohistochemical method (EnVision) were used to detect the expression of Six1 and Six4 in the tumor tissues and corresponding adjacent normal epithelium of esophagus from 292 ESCC patients.

RESULTSAmong the 292 ESCC patients, the positive rates of Six1 and Six4 protein expression in tumor tissues were 72.9% (213/292) and 56.2% (164/292), respectively, significantly higher than the expression rate of 33.2% (97/292) and 32.5% (95/292) in adjacent normal epithelium of esophagus (P < 0.05). Chi square test showed that the expression of Six1 protein was related to tumor size, depth of tumor invasion and patient survival status; higher Six4 protein expression level was related to poor differentiation and increased depth of invasion. Single factor Log-rank analysis revealed that gender, TNM stage, Six1 protein expression level were related to the overall survival of ESCC patients (P < 0.05), while the five-year survival rate was significantly higher in the Six1-negative group than the Six1-positive group [51.9% (41/79) vs. 43.7% (93/213)]. Multi-factor Cox proportional risk model analysis showed that TNM stage and positive expression of Six1 were independent prognostic factors for ESCC patients (P < 0.05).

CONCLUSIONSSix1 and Six4 are highly expressed in ESCC. Their expression levels are closely related to the progress and prognosis of ESCC. Over-expression of Six1 is related to poor prognosis in ESCC patients. Thus, Six1 could be used as an important prognostic indicator for ESCC patients.

Adult ; Aged ; Carcinoma, Squamous Cell ; metabolism ; pathology ; surgery ; Esophageal Neoplasms ; metabolism ; pathology ; surgery ; Female ; Follow-Up Studies ; Homeodomain Proteins ; metabolism ; Humans ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Invasiveness ; Neoplasm Staging ; Prognosis ; Proportional Hazards Models ; Risk Factors ; Survival Rate ; Trans-Activators ; metabolism ; Tumor Burden