IL-3 has effects on a wide variety of cell types, including immature cells of the immune cells, and mature immune cells such as granulocytes. In the purpose of studying the immunoregulatory function of IL-3 and its potential role in cancer therapy, we established a IL-3-secreting tumor model using gene transfection to deliver locally IL-3 to tumor site. First, we constructed IL-3 expression vector BMGNeo-mIL-3, then transfected it into B16 murine melanoma cells. By G418 resistant screening and limiting dilution, we obtained a transfectant that expressed high levels of IL-3 (806U / ml) . The IL-3 expression of the transfectant was confirmed by Northern blot analyses. Although the wild-type B16 cells and the B16-Neo cells transfected with BMGNeo did not express IL-3, the IL-3 expression in B16 cells had no obvious effect on the in vitro proliferation of the transfectant. These results showed we had successfully established a IL-3-secreting tumor cell clone which would enable us to further study its in vivo tumorigenicity and immune function.

In the present study, we prepared cationic liposomes encapsulated IL-2 DNA or IL-6 DNA with reverse-phase evaporation to investigate their efficiency in mediating gene transfer. After transfection with IL-2 DNA liposome or IL-6 DNA liposome, the supernatants of NIH3T3 cell contained high levels of IL-2 or IL-6. The peritoneal cells from the mice injected i.p. of IL-2 DNA or IL-6 DNA liposome 10 days later could also be detected to secrete IL-2 or IL-6. The B16F10 melanoma cells from tumor-bearing mice which were injected intratumorally of IL-2 DNA or IL-6 DNA liposome exhibited resistant to G418 and secreted high levels of IL-2 or IL-6. The results show that the liposome can mediate cytokine gene transfer efficiently in vitro and in vivo.

The Ad-IL-2 and/or Ad-IL-3 were i. p. injected into experimental leukemic mice after high dose chemotherapy. When Ad - LacZ was i. p. injected into leukemic mice, there were leukemic cells in bone marrow, vessal, liver and spleen. After i. p. injection of Ad-IL-2 and/or Ad-IL-3, the leukemia - bearing mice showed significant inhibition of tumor growth. As i.p. injection of Ad-IL-2, the splenic NK, CTL activity increased; as i. p. injection of Ad - IL - 3, the number and cytotoxicity of peritoneal macrophages were significantly higher than that of the control groups. After i. p. injection of Ad - IL - 2 and Ad - IL - 3 at the same time, the anti - leukemia activity was the best. Though there were less leukemic cells in the bone marrow, there were no leukemic cells in the liver and spleen. Our results suggested that i. p. injection of Ad - IL - 2 and Ad - IL - 3 could significantly enhance the anti - leukemia activity of chemotherapy.

The human Interleukin-2 cDNA containing full-length of encoding region was cloned by RT-PCR from PBMNC and confirmed by DNA sequencing. The hIL-2 recombinant retroviral expressing vector (pLXSN-hIL2) was constructed by inserting hIL-2 cDNA into the BamHI cloning site of pLXSN retroviral vector. After packaged with CRIP packaging cell line, the hIL-2 retrovirions were produced at the titer of 7.6?105CFU/ml. Then a fibroblast cell clone(NIH3T3-hIL2) secreting 118.2U/ml hIL-2 was obtained by infecting the NIH3T3 fibroblast cells with hIL-2 retrovirions. The integration of hIL-2 provirus into the genome of NIH3T3-ML2 cells was confirmed by PCR analysis for NeoR gene. Our data showed that the hIL-2 retroviral vector was successfully constructed, which enables us to further the investigation of the hIL-2 gene therapy in clinical trial.

To evaluate whether in vitro and in vivo transfer of E. Coli cytosine deaminase gene will confer sensitivity of a solid tumor to prodrug 5-fluorocytosine(5FC), we used an adenovirus vector(AdexCMV. CD) carrying the cytosine deaminase gene driven by the CMV promoter, infected SMMC-7721 or HepG2 cells hepatocellular carcinoma cells in vitro, and found AdexCMV. CD vector could effectively suppressed the growth of SMMC-7721 and HepG2 cells. When the two cells were infected with AdexAFP. CD vector in which the CD gene was driven by the AFP gene 5'-flanking region, only HepG2 cells were conferred sensitivity to 5FC. (Infection with AdexCMV.CD, when as few as 20% of cells transfected the CD gene, SMMC-7721 cells were associated with a bystander effect when combined with 5FC in cell mixing studies.) Consistent with these in vitro observations, AdexCMV. CD was directly injected into established subcutaneous SMMC-7721 tumors in nude mice receiving 5FC,there was a 60% reduction in tumor size at day 8, 70% reduction at day 24. Our results suggested that adenovirus-mediated tumor-specific gene transfer of CD gene and concomitant administration of 5 FC may have potential as a strategy for local control of tumor growth.

The human CD80 full-length encoding cDNA was cloned by RT-PCR, and inserted into El-substituted aden-ovirus vector pAxlcw. Subsequently, the hCD80 recombinant adenovirus vector was cotransfected into 293 cells together with EcoT22I-digested Ad5-TPC, and the replication-deficient hCD80 recombinant adenovirus was generated efficiently by homologous recombination, with the tilers of 4?10~9pfu/ml. Infected with prepared hCD80 recombinant adenovirus in vitro, Hela cells expressed high levels of hCD80 48 hours after infection. These suggest that COS/TPC is an efficient method to prepare recombinant adenoviruses and the prepared hCD80 adenovirus could be used in cancer gene therapy.

Objective: To investigate the effects of DC derived from IL-12-induced erythroleukemia cells on the induction of CTL and protective antituinor immunity. Methods: The cytotoxicity of CTL was assessed by 4h 51 Cr-release assay. HE staining and eleclromicroscopy were used to observe the histological changes after immunized with erythroleukemia-derived DCs. Results: After incubation with naive T cells, the IL-12-induced FBL-3 cells could apparently induce the generation of CTL which exihibit the specific cytotoxicity against wild-type FBL-3 cells in vitro. We further assayed the cytotoxicity of CTL in vivo after immunized with erythroleukemia-derived DCs. CTL cytotoxicity of mice immunized with IL-12-in-duced FBL-3 cells was higher than that of mice immunized with IL-I2-uninduced FBL-3 cells. C57BL/6 mice immunized with IL-12-induced FBL-3 cells could resistant the subsequent rechallenge with the wild-type FBL-3 cells. The tumor growth was efficiently inhibited. Histological observation showed that more inflammatory cells infiltrated into the tumor tissue and necrosis of leukemia cells existed. By transmission electron microscopy, apoptositic phenomena were observed in the tumor of group immunized with IL-12-induced FBL-3 cells. However, these changes were not observed in the group immunized with IL-12-uninduced FBL-3 cells. Conclusion: These data indicate that erythroleukemia cells-derived DCs could induce specific CTL efficiently in vitro and in vivo, and may be used as new vaccine to activate antituinor immune responses.

To observe the effect of IL-2 gene modification on the character of biology and function in dendritic cells( DC) and to investigate the immune mechanism of specific anti-tumor of IL-2 gene modification in DC. Methods: DCs were prepared from mouse bone marrow and genetically modified by IL-2 adenovirus. Then observe the changes of DC morphology by scanning electro-microscopy, analyzed molecules on DC by FACS, examined the expression of IFN-? mRNA in DC by RT-PCR.The stimulatory capacity of DC to T cells detected by MLR their capacity of antigen present were measured by 3H-TdR mix into assay. Results: After IL-2 gene modification, the morphology of DC was changed, its pseudopod was more and longer. The expression of Ia, B7-1, B7-2 and CD40 molecules was more on DC surface. The IFN-7 mRNA was expressed in the DC-EL-2 and DC-rhIL-2.DC-IL-2 could stimulate allogeneic T cells more potently and IL-2 gene-modified DC could induce more potent antigen-specific autogeneic CTL. Cooclusioo: IL-2 genetic modification can promote DC growth and up regulate their expression of membrane immune molecules that are relevant for antigen presentation of DC,and enhanced the biologic activity of the DC.

Low self-certainty of patient satisfaction evaluation in patient satisfaction survey is found in the survey to be the main cause for the failure that satisfaction scores cannot precisely indicate the correct satisfaction level of patients for medical services received; it is stated in the study that psychological bias in the survey can be reduced by means of reasonable choice of survey scenario, clear notice of survey's objective, and independent completion of survey questionnaire by the patients; it also stated the theoretic references and research clues in studying patient satisfaction uncertainty, as a mathematic model is built for such study, for the purposes of better advancement of the theoretic study and practice of patient satisfaction.

Objective To study the effect of benzo(a) pyrene on DNA of human embryonic fibroblast under inhibition of DNA repair,and to explore the mechanism of DNA repair involved in the DNA damage induced by xenobiotic chemical carcinogens. Methods DNA damage of human embryonic lung fibroblast (HELF) induced by benzo(a) pyrene was observed when DNA repair was inhibited by treating HELF with arabinosylcytosin(ara-C) to inhibit the activity of polymerase ?/? in the cells. With S9 mixture added as metabolic activation system in vitro,HELF was treated for 2 hours with ara-C at the doses of 0 and 100 ?mol/L combined with-C at the doses of 0,10,20,50 ?mol/L by the 2?4 factor-factorial analysis.Comet assay was used to assess the DNA damage. Results Compared with the control group,the comet rate and Oliver tail moment of groups treated by B(a)P increased significantly (P