Expression of heme oxygenase-1 and GFP gene mediated by recombinant adeno-associated-virus in transplanted liver in rats / 中华外科杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Heme 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSIONS</b>High 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.</p>

Antisense hypoxia inducible factor-1alpha and B7-1 combination gene therapy for mouse lymphoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the synergistic effects of antisense HIF-1alpha gene therapy combined with B7-1-mediated immunotherapy on cancer treatment.</p><p><b>METHODS</b>Antisense 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.</p><p><b>RESULTS</b>Gene 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.</p><p><b>CONCLUSION</b>Antisense 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.</p>

Gene transfer of von Hippel-Lindau inhibits the growth of transplanted solid tumors / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To explore the effect of von Hippel-Lindau(VHL) gene on growth of EL-4 solid tumors in vivo.</p><p><b>METHODS</b>C57BL/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.</p><p><b>RESULTS</b>VHL 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).</p><p><b>CONCLUSION</b>VHL gene therapy can inhibit the growth of EL-4 solid tumor in vivo.</p>

The study on the morphology character of blood-spleen barrier / 中华外科杂志

<p><b>OBJECTIVE</b>To study the morphology and functional character of blood-spleen barrier (BSB) and establish the concept of BSB.</p><p><b>METHODS</b>Thirty 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.</p><p><b>RESULTS</b>Most 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.</p><p><b>CONCLUSIONS</b>BSB, 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.</p>