ObjectiveTo search the appropriate experimental conditions for using green fluores-cent protein (GFP) as a reporter gene in tumor gene therapy. Methods The plasmids carrying mu-tated GFP gene were transfected into the eukaryotic cells to observe transient gene expression. Thesestudies were conducted to 1. directly determine GFP expression and express stability in the COS- 7cells, 2. compare the transfection efficiency of two plasmids pcDNA3 - EGFP, pSVKa - S65T with dif-ferent promoters in different tumor cell lines, 3. determine two genes expression in a single cell usingLacZ cotransfected with GFP by FACS. ResultsFluorescence could be detected in intact viable cellsunder different sets of conditions. The expression of GFP might last two weeks or more and the ex-pressed fluorescence was stable. The transfection rate of pcDNA3 - EGFP expressed was different inthree tumor cell lines examined. But pSVK3 - GFP expressed similarly in four tumor cell lines exam-ined. FACS showed the probability of two genes entering a single cell is above >85% at the ratio 1:4.ConclusionThe above data indicate that the GFP can be visualized continuously and directly for geneexpression in living cells. GFP may also be used for quicklly selecting cells carrying a target gene.

The whole length mouse interferon-gamma (mIFN-?) cDNA was obtained using RT-PCR and was introduced into mouse hepatoma cell line H22 by retroviral vector. We examined the tumorigenicity of mIFN-? gene modified H22 in experimental animals. The results indicated that the gene modified tumor cells had less tumorigenicity than the parental tumor cells and the tumor vaccine had therapeatic effects on tumor-bearing animals.

The whole length cDNA of human interferon-?(IFN-?) gene including signal peptide was constructed to retroviral vector pLXSN and then packaged with PA317. A Human Hepetoma Cell Line(HHCL) and a human gastric cancer cell line (MKN-45) were infected by using supernatant containing retroviral vector with IFN-?. the gene-modified tumor cell. were isolated by G418 selection. Some changes were found in cell cycle, expression of HLA class Ⅰ and classⅡ, and tumorigenecity. Elevated immunogenecity and abrogated rumerigenecity suggest a means for generating gene-modified tumor cell vaccine. for the treatment of cancer.

Retroviral vector was used to introduce the interleukin - 2 (IL -2) gene into H22 cells, a BALB/c mouse hepatoma cell line. The IL-2 gene and marker gene (NeoR) were assessed by using PCR and RT-PCR methods. FACS analysis demonstrated that the cells with low DNA content in IL-2 gene modified H22 cells were more than these in control H22 cells. Electron microscopic morphological structure showed that some cells in IL-2 gene modified H22 went into apoptotic cell death. The study demonstrated that apoptotic cell death of H22 - IL-2 cells was induced by the IL-2 gene modification. It may be one of mechanisms of decreased tumorigenicity of IL - 2 gene modified tumor cells.

We compared the ability of tumorigenicity of TNF-? gene-modified and unmodified H22 tumor cells, and therapeutic functions of the irradiated cells. Results indicated that H22-TNF-? cells were less tumorigenic as compared to H22 and H22-LXSN cells. In case of treatment groups, injected with irradiated tumor vaccine in 3 or 6 days after inoculation of H22 cells, the tumor growth was suppressed.

In order to study the antitumor effect of human tumor necrosis factor alpha (TNF-?)gene modified fibroblasts, we have transfered TNF-? cDNA to fibroblasts NIH3T3 by a bicistronic retroviral vector pGCEN/TNF-?, which expressed TNF-? from the long terminal repeat and the selectable Neo~R from the encephalomyocarditis virus (EMCV) internal ribo-some entry site(IRES) . The results indicated that TNF-? modified NTH3T3, NIH3T3/TNF-? ,were able to express TNF-? at a high level for a long term culture. Furthermore, these modified fibroblasts could inhibit tumor progression signifi-cantly(P

Objectives: Use antisense VEGF to reduce the immunosuppression induced by tumor and enhance the antithumor effect of IL-2. Methods: After the MMT45.Li murine liver cancer cells were modified with antisense VEGF alone, or antisense VEGF combined with IL-2 gene, the tumorigenesis of modified MM45T.Li was studied. The apoptosis of cancer cell induced by antisense VEGF and IL-2 gene in vivo was also studied. The tumors were cryosmeared 3 weeks after the mice bearing tumor were treated correspondingly with Ad-antiVEGF or Ad-Il-2 or Ad-antiVEGF/IL-2 or Ad-lacZ, the immunohistochemical analysis of CD4 + , CD8 + was performed.Results: The tumorigenesis of MM45T. Li was reduced by antisense VEGF, and the number of CD4 + , CD8 + cell was increased in tumor tissue, and the immunosupression was also reduced; At the same time antisense VEGF also enhanced the antithumor effect of IL-2 gene.Conclusions: Antisense VEGF not only can inhibit the neovascularization, but also can reduce the immunosupression induced by tumor, and improve lymphocyte infiltration toward tumor site;when it was combined with Il-2,anti VEGF can enhance the antitumor effect greatly.

Objective To investigate the expression of exogenous γ-interferon gene in human hepatocellular carcinoma cells following retroviral transduction and the effect on the expression of surface HLA class Ⅰ molecules.Methods Retroviral vector pLXSN was used to introduce human γ-interferon (IFN-γ) gene into four different human hepatocellular carcinoma cell lines (HCC). The G418-resistant colonies were isolated and cloned. The integration and expression of IFN-γ gene were determined by PCR and RT-PCR analysis. A bioassay method was used to test the amount of IFN-γ secreted by gene modified HCC cells. The expression of HLA class Ⅰ molecules in HCC cells were analyzed by flow cytometry using indirect fluorescence staining.Results Four different HCC cell lines were successfully transduced with human IFN-γ gene using retroviral vector. The integration and expression of IFN-γ gene were shown only in the transduced cells. All four genetically modified HCC cells can secrete varied amount of IFN-γ and demonstrate a significant up-regulation of surface HLA class Ⅰ antigens. One specific HLA class Ⅰ antigen, HLA-A2, has almost the same degree of increase as that of the total HLA class Ⅰ molecules after transduction with IFN-γ gene.Conclusions Gene modification with IFN-γ gene can significantly enhance the expression of HLA class Ⅰ molecules in HCC cells and may increase its immunogenicity. These gene modified tumor vaccines can be helpful in tumor biotherapy.

OBJECTIVETo investigate the expression of aquaporin-1 (AQP1) in the human peritoneum and to evaluate the effect of peritoneal dialysis (PD) on its expression.

METHODSPeritoneal biopsies were obtained from normal subjects (n = 10), uremic nondialysis patients (n = 12) at catheter insertion and PD patients (n = 10) at the time of catheter removal, reinsertion or renal transplantation. Western blot, immuno-histochemical staining and reverse transcript-polymerase chain reaction (RT-PCR) techniques were used to investigate AQP1 expression.

RESULTSAll peritoneal samples expressed AQP1 at both mRNA and protein levels. Western blot revealed a major band at 28 kD as well as more diffuse bands between 35 and 50 kD. The 28 kD band represents the nonglycosylated form of the protein while the 35 - 50 kD bands correspond to glycosylated AQP1. Immunohistochemical staining found the positive deposits were distributed in the mesothelial cells, endothelial cells of capillaries, venules and small veins, whereas no signal was detected in the arterioles. Semi-quantitative analysis showed that AQP1 expression was remarkably stable in all samples, whatever their origin (P > 0.05).

CONCLUSIONSOur findings suggested that AQP1 is the molecular counterpart of an ultra small pore during PD. Secondly, the peritoneal mesothelial cell might also be involved in peritoneal transcellular water transport. As regards whether or not the structural or distributional alterations of AQP1 in the peritoneum may be more obviously expressed during PD, further study is needed.

Adult ; Aquaporin 1 ; Aquaporins ; analysis ; Blood Group Antigens ; Female ; Humans ; Male ; Middle Aged ; Peritoneal Dialysis ; Peritoneum ; chemistry ; physiology ; Uremia ; physiopathology

Objective To study the roles of TNF-α secretion of PBMC in patients with chronic hepatitis C and Nonalcoholic fatty liver. Methods The level of TNF-α secretion of PBMC in patients with chronic hepatitis C and Nonalcoholic fatty liver was detected by ELISA after culturing for 72 hours in vitro, as well as patients with chronic hepatitis C and the normal control. Results (1) Compared with the normal control, the level of TNF-α in group with chronic hepatitis C and in group with chronic hepatitis C and Nonalcoholic fatty liver notably increased. (2) Compared with the group with chronic hepatitis C, the level of TNF-α in group with chronic hepatitis C and Nonalcoholic fatty liver also notably increased. Conclusion It suggested TNF-α takes important roles in the infection course of chronic hepatitis C with Nonalcoholic fatty liver.