OBJECTIVETo establish a syngeneic mouse model of liver tumor stably expressing hepatitis B virus (HBV) antigens.

METHODSMelanoma cell line B16 cells were transfected with pLXSN-2HBV. Cells (named B16/HBV) stably and persistently expressing HBV surface (HBsAg) and core (HBcAg) antigens were identified. The cells were injected into the hepatic subcapsular space of fifteen C57BL/6J mice. The mice were divided into 3 groups, receiving 100, 1000 or 5000 cells in a total volume of 5 µl per mouse, respectively, five mice in each group. Two weeks after the tumor cell inoculation, serum samples from the mice were collected weekly and the serum concentration of HBsAg and anti-HBs was quantified by ELISA. The tumor growth in the mouse liver was monitored by a high-resolution ultrasound system. Expression of HBsAg and HBcAg in the tumor tissues was determined by immunohistochemistry.

RESULTSLiver tumors were formed in all the mice receiving 1000 and 5000 B16/HBV cells per mouse, and in 80% of the mice receiving 100 B16/HBV cells. HBsAg and anti-HBs were detectable in their sera from 2 weeks after tumor cell inoculation. The mice receiving 100 cells per mouse began to die 4 weeks, those receiving 1000 cells per mouse began to die 3 - 4 weeks and those receiving 5000 cells began to die 2 - 3 weeks after the cell inoculation. All the tumor cells expressed HBsAg and HBcAg.

CONCLUSIONSThe B16/HBV cells stably and persistently express HBV antigens both in vitro and in vivo. A mouse model of transplanted liver tumor stably expressing HBV antigens has been successfully established by inoculation of those cells into the hepatic subcapsular space.

Animals ; Cell Line, Tumor ; Disease Models, Animal ; Female ; Hepatitis B Core Antigens ; metabolism ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; genetics ; metabolism ; Liver Neoplasms, Experimental ; immunology ; virology ; Melanoma, Experimental ; metabolism ; pathology ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Transfection

BACKGROUNDHepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide. In order to investigate the molecular biologic mechanism of HCC's development, we studied the expressions of SE-1, CD105 and CD31 in tumor endothelial cells (TECs) of HCC and in the serum of rats.

METHODSWe analyzed the expressions of SE-1, CD31 and CD105 in rat HCC tumor tissues using immunohistochemistry (IHC). Twenty HCC bearing rats and eighteen normal rats were examined for the expressions of SE-1, CD31 and CD105 antigens in serum by enzyme-linked immunosorbent assay (ELISA).

RESULTSSE-1, CD31 and CD105 antigens were detected both in HCC tissue and in normal liver tissue with higher expressions of CD31 and CD105 in HCC while the SE-1 antigen expression was higher in normal liver. Similarly, serum CD31 and CD105 in rats with HCC were significantly increased compared with normal rats (t = 2.8628, P = 0.0086; t = 4.4922, P < 0.0001, respectively). In contrast, SE-1 antigen in HCC rat serum was significantly decreased compared with normal rats (t = 3.4983, P = 0.0011).

CONCLUSIONSE-1, CD31 and CD105 are closely related with liver tumor angiogenesis, which is similar to their performances in terms of their expressions in the serum.

Animals ; Antigens, CD ; blood ; Carcinoma, Hepatocellular ; blood supply ; chemistry ; Endothelial Cells ; chemistry ; immunology ; Enzyme-Linked Immunosorbent Assay ; Immunohistochemistry ; Liver Neoplasms, Experimental ; blood supply ; chemistry ; Male ; Neovascularization, Pathologic ; blood ; Platelet Endothelial Cell Adhesion Molecule-1 ; blood ; Rats ; Rats, Inbred BUF

OBJECTIVETo investigate the antitumor immune response induced by dendritic cells vaccine coding AFPcDNA fragment with signal peptide (AFP(1)) and without signal peptide (AFP(2)), and to determine the inhibiting effect of the vaccine on the growth of hepatocarcinoma xenograft in Balb/c mice.

METHODSpcDNA3.1/AFP(1) and pcDNA3.1/AFP(2) were transfected into dendritic cells (DCs) by calcium phosphate nanoparticles and became DCs vaccine. Mouse spleen lymphocytes were stimulated by AFP(1)/DC and AFP(2)/DC. A Balb/c mouse model bearing mouse HCC xenograft was established on the day 14 after transplantation. Forty mice were divided equally into AFP(2)/DC group, AFP(1)/DC group and plasmid control group. The treated mice received DCs vaccine and the same amount of control plasmid.

RESULTSAFP(2)/DC stimulated T lymphocytel proliferation in vitro and improved CTL activity. The effects were better than AFP(1)/DC. The tumor-bearing mice injected intralesionally with AFP(1)/DC and AFP(2)/DC at a dose of 0.5 ml per mouse showed inhibition of tumor growth and prolongation of survival time. The tumor inhibition rate of the AFP(2)/DC group was 79.2% and the AFP(1)/DC group was 39.7% at 2 weeks after treatment. The tumor volume of AFP(2)/DC group was (726.7 +/- 298.2) mm(3), significantly smaller than the (1486.2 +/- 457.2) mm(3) of the AFP(1)/DC group and (2137.2 +/- 547.2) mm(3) of the plasmid control group (P < 0.05). The mean survival time of mice in the AFP(2)/DC group [(58.5 +/- 4.2) d] and AFP(1)/DC group [(45.2 +/- 4.8) d] were significantly longer than that of plasmid control group [(30.6 +/- 6.2) d, P < 0.05]. Bax-positive cell percentage was increased in the xenografts of AFP(2)/DC-treatment group compare with that of plasmid control group.

CONCLUSIONAFP(2)/DC and AFP(1)/DC vaccines show evident inhibiting effect on the growth of H22 xenograft in Balb/c mice through inducing efficient and specific immune response against the hepatocarcinoma cells.

Animals ; Calcium Phosphates ; pharmacology ; Cancer Vaccines ; immunology ; Cell Line, Tumor ; Cell Proliferation ; DNA, Complementary ; genetics ; immunology ; Dendritic Cells ; immunology ; Immunization ; Liver Neoplasms, Experimental ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Nanoparticles ; Neoplasm Transplantation ; Peptide Fragments ; Spleen ; cytology ; T-Lymphocytes ; pathology ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; alpha-Fetoproteins ; genetics ; immunology

OBJECTIVETo investigate the inhibitory effect of dehydroepaimdrosterone (DHEA) on the growth of transplanted Morris hepatomas (7288CTC) in vivo in rats.

METHODS21 Buffalo rats were randomly devided into 4 groups, including one blank control group (n = 5), one group for tumor-bearing control (n = 6), and 2 experimental groups with DHEA (n = 6) or DHEA-s (n = 4). DHEA or DHEA-s was fed to the rats for 4 weeks immediately after Morris hepatomas (7288CTC) was implanted in both flanks. Phenotypes of the spleen lymphocytes were examined by flow cytometry, Akt and PTEN expression in tumor cells was detected by Western blot and immunohistochemistry.

RESULTSTumor weights of DHEA treated group were less than those of the control (P less than 0.05), the inhibitory rate was 43%. The results of Western blot and immunohistochemistry showed that in DHEA tumor group,the expression of phosphorilated Akt protein was decreased, the expression of PTEN was enhanced, the percentage of CD3 positive cells and the ratio of CD4/CD8 were increased (P less than 0.05).

CONCLUSIONDHEA can inhibit tumor growth, possibly via the inhibition of the Akt signaling pathway as well as modulating the immune function.

Animals ; Antineoplastic Agents ; pharmacology ; CD4-Positive T-Lymphocytes ; immunology ; CD8-Positive T-Lymphocytes ; immunology ; Dehydroepiandrosterone ; pharmacology ; Dehydroepiandrosterone Sulfate ; pharmacology ; Flow Cytometry ; Immunohistochemistry ; Liver Neoplasms, Experimental ; immunology ; metabolism ; pathology ; Neoplasm Transplantation ; PTEN Phosphohydrolase ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Random Allocation ; Rats ; Rats, Inbred BUF ; Signal Transduction ; drug effects

OBJECTIVETo investigate the antitumor effects of koumine in mice bearing H22 solid tumor and its effect on the immune system of the mice.

METHODSThe changes in spleen and tumor weights and blood cell count were observed after koumine treatment in BALB/c athymic mice bearing H22 solid tumor, using normal saline solution and 5-Fu as the controls.

RESULTSKoumine significantly inhibited the tumor growth in a dose-dependent manner. The spleen index and blood cell counts in koumine group showed no significant differences from those in the saline control group, but higher than those in 5-Fu group.

CONCLUSIONKoumine can significantly inhibit the growth of H22 solid tumor without obvious inhibitory effect on the immune system in mice.

Animals ; Antineoplastic Agents, Phytogenic ; therapeutic use ; Female ; Gelsemium ; chemistry ; Indole Alkaloids ; isolation & purification ; therapeutic use ; Liver Neoplasms, Experimental ; drug therapy ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Phytotherapy

OBJECTIVETo observe the mucosal immune mechanism of anti-tumor action of Ganoderma lucidum polysaccharides (GLP).

METHODSThe concentration of H22 cells in suspension were adjusted to 1 x 10(9)/ L, and 0.2 mL of the cell suspension was injected subcutaneously in the right oxter of Kunming mice. Then the H22 bearing mice were randomly divided into 4 groups: the GLP group, the Cytoxan (CTX) group, the CTX + GLP group and the untreated model group, 8 mice in each group. Besides, a blank control group was set up. Starting from the 2nd day of modeling, GLP, at the dose of 1.02 g/kg was given to GLP group and GLP + CTX group by gastrogavage once a day for 12 successive days; CTX at the dose of 100 mg/kg was administered via peritoneal injection to the CTX group and the GLP + CTX group on the 1st day and the 6th day of the experimental course; but to the model group and the blank group, only equal volume of distilled water was given. All mice were sacrificed on the 14th day, the ileum at 1 cm upper to cecum was taken, through 4% paraform fixation and paraffin section, it was used for immunohistochemical detecting expressions of immunoglobulin A (IgA), tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2) and interleukin-10 (IL-10) in ileum. Besides, the lymphocyte subsets in the intraepithelial lymphocyte (IEL), lamina propria lymphocytes (LPL), and Peyer's patch lymphocytes (PPL) were analyzed by immune fluorescence technique and flow cytometry.

RESULTSCompared with the blank control group, the phenotype of lymphocytes and the expression of cytokines in ileum in the model group changed significantly; and the phenotype was variant in different regions. Compared with the model group, both indexes were adjusted in the GLP, CTX and GLP + CTX group to different degrees.

CONCLUSIONThe adjustment of GLP on intestinal mucosal immune is probably another path for its anti-tumor action.

Animals ; Antineoplastic Agents, Phytogenic ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Immunoglobulin A ; metabolism ; Interleukin-2 ; metabolism ; Intestinal Mucosa ; immunology ; Liver Neoplasms, Experimental ; immunology ; Male ; Mice ; Mice, Inbred Strains ; Polysaccharides ; isolation & purification ; pharmacology ; Random Allocation ; Reishi ; chemistry ; Tumor Necrosis Factor-alpha ; metabolism

BACKGROUNDIt is generally accepted that spleen plays a complex role in the tumor immunity, which would change in the different periods of cancer. In this study, we investigated the changes in the function of splenic macrophage (Mphi) in different stages of liver cancer induced by diethylnitrosamine (DEN) in rats. The aim was to support the characteristics of "two-way" and "phase" of spleen in tumor immunity.

METHODSThe model of pulmonary metastasis of liver cancer was established in forty male SD rats by DEN. In the 8th, 13th and 16th week, 10 rats were randomly chosen and sacrificed, and divided into cirrhosis, liver cancer and pulmonary metastasis groups depending on the pathological result, respectively. The other 10 rats were taken as control group. The Mphi was isolated by anchoring cultivation. The changes in ultrastructure, phagocytosis, cytokine secretion, antigen processing and presenting, and viability of splenic Mphi were detected by transmission electron microscopy, Vybrant(TM) Phagocytosis Assay, DQ(TM) Ovalbumin, and rat TNF-alpha ELISpot kits.

RESULTSUnder the electron microscope, the Mphi in the control group had some pseudopodium-like prominences, and mitochondria, ribosome, rough endoplasmic reticulum, lysosome can be found in the cytoplasm, and phagocytized RBC. In the liver cirrhosis and liver cancer group, Mphi had more prominences, meanwhile much more mitochondria, ribosome, rough endoplasmic reticulum, lysosome can be found in the cytoplasm, especially in the liver cancer group. In the pulmonary metastasis group, the Mphi was swelling, with few organelle. As compared to the control group, the function of splenic Mphi increased in cirrhosis and cancer groups, but decreased in metastasis group (phagocytosis rate: (84.7 +/- 1.9)%, (89.5 +/- 3.1)%, and (36.0 +/- 2.6)% vs (75.6 +/- 1.7)%, P < 0.05, P < 0.01; viability: (1.53 +/- 0.15)%, (1. +/- 0.14)%, and (1.12 +/- 0.29)% vs (1.48 +/- 0.17)%, P < 0.05, P < 0.01; TNF-alpha secretion: (741.0 +/- 52.9)%, (1126.2 +/- 174.5)%, and (313.8 +/- 50.8)% vs (626.6 +/- 24.6)%, P < 0.05, P < 0.01; positive cell rate of antigen processing and presenting: (24.03 +/- 1.87)%, (27.95 +/- 2.63)%, and (10.46 +/- 2.16)% vs (16.45 +/- 1.86)%, P < 0.01).

CONCLUSIONSIn the stage of cirrhosis and early cancer, the immune functions of splenic Mphi were reinforced. It may promote the non-specificity tumor immunity. On opposite, in the stage of pulmonary metastasis, the immune functions of splenic Mphi were impaired. It may lead to the decrease of tumor immunity.

Animals ; Cells, Cultured ; Diethylnitrosamine ; toxicity ; Disease Models, Animal ; Liver Cirrhosis ; immunology ; pathology ; Liver Neoplasms, Experimental ; chemically induced ; complications ; immunology ; ultrastructure ; Lung Neoplasms ; immunology ; secondary ; ultrastructure ; Macrophages ; pathology ; ultrastructure ; Male ; Microscopy, Electron, Transmission ; Rats ; Rats, Sprague-Dawley ; Spleen ; pathology ; ultrastructure

OBJECTIVETo investigate the effects of dendritic cells (DCs) infected with adenovirus vector encoding mTERT on induction of mTERT antigen specific immunity against H22 hepatoma in vivo.

METHODSForty Bal B/c mice were subcutaneously immunized with Ad-mTERT infected DC. Cytotoxicity of mTERT specific CTL was determined by 51Cr release assay. IL-2 and IFN-gamma were tested by ELISA. IFN-gamma ELISPOT assays were performed for measuring antigen specific IFN-gamma production by T cells. Tumor size and survival of the immunized mice were recorded and evaluated whether preexisting hepatoma metastases could be supressed after immunization with mTERT-expressing DCs.

RESULTSThe lytic activity of CTL, IL-2 (871.25 pg/ml), IFN-gamma (169.15 ng/ml) and IFN-gamma secreting cells (378/10(6) spleen cells) elicited by the Ad-mTERT infected DCs were much stronger and higher than that by Ad-GFP group (131.6 pg/ml, 15.4 ng/ml, 36/10(6) spleen cells, P<0.05), DC group (71.3 pg/ml, 10.5 ng/ml, 21/10(6) spleen cells, P<0.05), PBS group (65.8 pg/ml, 7.4 ng/ml, 18/10(6) spleen cells, P<0.05). In prophylaxis and treatment experiment the Ad-mTERT/DCs immunized mice lived significantly longer than other groups, demonstrating that primary DCs were genetically modified to express the mTERT antigen and could suppress the tumor growth.

CONCLUSIONAdenovirus vector mediated mTERT infected DCs can effectively induce mTERT antigen specific antitumor activity, and can induce protective and therapeutic antitumor immunity.

Adenoviridae ; genetics ; Animals ; Cell Line, Tumor ; Dendritic Cells ; immunology ; metabolism ; Female ; Genetic Vectors ; Immunization ; Interferon-gamma ; Interleukin-2 ; Liver Neoplasms, Experimental ; immunology ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Recombinant Proteins ; genetics ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; Telomerase ; immunology ; metabolism ; Tumor Burden

OBJECTIVETo study the immunological suppressing effect of recombinant adenovirus vector rAD-mTERT promotor-m4-1BBL (rAD-mTERT) on mouse hepatoma cell line Hepa1-6 cells in co-culture with T lymphocytes.

METHODSAdding recombinant adenovirus rAD, rAD-CMV-m4-1BBL (rAD-CMV) and rAD-mTERT to Hepa1-6 and L929 cells, respectively, to observe the effect of these adenoviruses on growth and apoptosis of these cells in co-culture with T lymphocytes.

RESULTSAdding adenovirus significantly suppressed the growth and slightly increased apoptosis of the two types of cells (P < 0.05). rAD-mTERT promotor-m4-1BBL showed only pro-apoptotic effect on Hepa1-6 cells. When co-cultured with T lymphocytes, rAD-CMV-m4-1BBL showed promoting effect on apoptosis of the cells. Compared with that of T cells pre-co-culture, CD4(+) and CD8(+) T cells were proliferated, and the ratio of CD4/CD8 was significantly reduced (from 1.27 to 1.08).

CONCLUSIONAdding the recombinant adenoviruses only suppresses the cell growth, but not promotes their apoptosis. In co-culture with T lymphocytes, recombinant adenovirus vector rAD-mTERT promotor-m4-1BBL can targetingly suppress the growth and induce apoptosis of Hepa1-6 cells. The apoptosis is induced through the immunological killing effect of T lymphocytes.

4-1BB Ligand ; genetics ; physiology ; Adenoviridae ; genetics ; Animals ; Apoptosis ; CD4-CD8 Ratio ; Cell Line ; Cell Line, Tumor ; Cell Proliferation ; Coculture Techniques ; Fibroblasts ; cytology ; Genetic Vectors ; Liver Neoplasms, Experimental ; immunology ; pathology ; Mice ; Mice, Inbred C57BL ; Promoter Regions, Genetic ; Recombinant Proteins ; genetics ; T-Lymphocytes ; immunology ; Telomerase ; genetics ; Transfection

OBJECTIVETo construct a dendritic cell vaccine transduced by murine alpha-fetoprotein (mAFP) gene, and evaluate its immunoprotective effect on C57BL/6J mice during the induction of hepatocellular carcinoma by diethylnitrosamines, carbon tetrachloride and ethanol.

METHODSDendritic cells (DCs) were induced and augmented by murine IL-4 and GM-CSF, and transfected by recombinant adenovirus engineered with mAFP gene. Major MHC class I and II, B7.1 (CD80), B7.2 (CD86), CD18a, and CD54 molecules on DC were analyzed by FACS. 80 C57BL/6J male mice were randomly divided into 4 groups (20 mice per group): Simple DC inoculated group, pAdBM5-mAFP-DC inoculated group, pAdBM5-mAFP plasmid inoculated group, and PBS control group. They were immunized once with 5 x 10(5) DCs (0.1 ml)/mouse administered s. c. in the left flank or 100 mg pAdBMS-mAFP plasmid/mouse administered i. m. in the left tibialis anterior muscle. Inoculation was conducted once a week for 4 weeks after 3 times consecutive immunization initially. At the same time of immunization, DEN/CCl4/ethanol were given to induce hepatocellular carcinoma. Tumor incidence was assessed after 20 weeks.

RESULTSA transgenic DC vaccine was successfully constructed and the mAFP transgenic DCs expressed high level molecules of major MHC class I and II , B7.1, B7.2, CD18a, and CD54. After the 20-week induction, the incidence of primary hepatocellular carcinoma (PLC) was 70.0% in simple DC inoculated group, 25.0% in pAdBMS-mAFP-DC inoculated group, 65.0% in pAdBM5-mAFP plasmid inoculated group, and 75.0% in PBS control group. There was a significant difference between group B and other groups (P < 0.05).

CONCLUSIONmAFP transgenic DC tumor vaccine inoculation may induce strong immunoprotection against liver carcinogenesis and tumor development and reduce PLC incidence induced by DEN/CCl4/ethanol.

Adenoviridae ; genetics ; Animals ; B7-1 Antigen ; metabolism ; Cancer Vaccines ; Carbon Tetrachloride ; Cells, Cultured ; Dendritic Cells ; cytology ; immunology ; metabolism ; Diethylnitrosamine ; Ethanol ; Genetic Vectors ; Histocompatibility Antigens Class I ; metabolism ; Histocompatibility Antigens Class II ; metabolism ; Intercellular Adhesion Molecule-1 ; metabolism ; Liver Neoplasms, Experimental ; chemically induced ; immunology ; prevention & control ; Male ; Mice ; Mice, Inbred C57BL ; Random Allocation ; Recombinant Proteins ; genetics ; metabolism ; Transfection ; alpha-Fetoproteins ; genetics ; metabolism