Cao Xuetao

Effects of anesthesia-induced modest hypothermia on cellular radiation sensitivity

To assess the mechanisms of modest hypothermia (MH) and its effects on cellular radiation response, a model of anesthesia-induced modest hypothermia (AIMH) in the adult mice and a model of pure MH in the newborn mice were established. The survival rate of lethally irradiated mice was increased to 72% through AIMH before irradiation. Both apoptosis and necrosis of human fetal bone marrow CD34+ hematopoietic stem cells cultured under MH were significantly decreased as detected by MTT and flow cytometry, with three-color labeled by PE-CD34+/ FITC-AnnexinV /7AAD. The survival and proliferation of mouse bone marrow MNC treated with MH after irradiation were also increased. The MH exerted similar protective effects on the leukemia cell lines A20, HL60, K562 to the normal bone marrow cells, but it enhanced the radiation sensitivity of leukemia cell line FBL3 and mouse melanoma B16F10. No effects have been found on the radiation sensitivity of those cells treated with MH before irradiation. The results also showed that MH mediated the effects on radiation sensitivity, in addition to increasing the oxygen tension. These results show different effects of MH on different cells: (i) AIMH exerts a protective effect on the normal hematopoietic stem cells, some leukemia cell lines A20, HL60, K562, and some neoplasma 3LL, LOVO. And MH exhibits a synthetic effect with anesthetic. (ii) MH enhances the radiation sensitivity of another leukemia and neoplasma cell lines FBL3, B16F10 and CT26. Therefore, AIMH has a potential to enhance the effects of radiation-therapy and decrease side effects on some tumors.

CONSTRUCTION OF THE DICISTRONIC ADENOVIRUS VECTOR EXPRESSING BIOACTIVE HUMAN INTERLEUKIN-12*

The full-length cDNA encoding the subunits p40 and p35 of human interleukin-12(hIL-12) were cloned separately by RT-PCR, linked together by internal ribosomal entry site (IRES) of encephalomyocarditis virus which initiates cap-independent translation to form a dicistronic gene fragment. The dicistronic fragment was placed between the cytomegalovirus (CMV) promoter and SV40 polyA signal to form a dicistronic expression cassette. Subsequently, the dicistronic expression cassette was inserted into El region of Ad5 genome in cosmid vector pAxlcw of El-substitution type. By homologous recombination with EcoT22I-digested Ad5 DNA-TPC in 293 cells, the replication-deficient recombinant adenoviruses of ML-12 were generated efficiently. After infected with hIL-12 recombinant adenoviruses in vitro, 293 cells, human hepatocellular carcinoma cells HepG2, and primary human skin fibroblasts expressed and secreted hIL-12 at comparable levels (30⊃60ng/106cells/24hr), which could stimulate the proliferation and IKN-γ production of human lymphoblasts. These suggest that the dicistronic adenovirus vector of hIL-12 could effectively mediate the expression of bioactive hIL-12 and might be used in cancer gene therapy.

The biological characteristics of adenovirus-mediated il-18 gene-modified murine colorectal adenocarcinoma cellin vivo andin vitro

Objective: Interleukin 18 (IL-18) is a strong activator of NK cells and promotes the generation of IL-2, IFN-γ and GM-CSF. In the present study, we constructed adenovirus encoding IL-18 gene (AdIL-18), and observed the biological characteristics of IL-18 gene-modified murine colorectal adenocarcinoma cell (CT26)in vivo andin vitro. Methods: Gene modification was mediated by adenovirus. The proliferation of the cells was determined by MTT and IL-18 was assayed by ELISA. The cytotoxicity of NK and CTL was detected by four-hour51Cr release assay. Results: IL-18 gene modification had no effect on the proliferation and morphology of CT-26 cellsin vitro, but the growth of IL-18-modified CT26 cells was obviously inhibitedin vivo. In addition, although IL-18-modified CT26 cells could form tumor nodulesin vivo as well as LacZ-modified CT26 cells or wild-type CT26 cells, the mean survival time of the mice inoculated with IL-18-modified CT26 cells was significantly prolonged as compared with that of control groups. Thus, the anti-tumor immune responses were induced in the group of mice inoculated with IL-18-modified CT26 cells, which might be related to the activation of NK cells and CTL. However, all the three groups ultimately died of tumor.Objective: Interleukin 18 (IL-18) is a strong activator of NK cells and promotes the generation of IL-2, IFN-γ and GM-CSF. In the present study, we constructed adenovirus encoding IL-18 gene (AdIL-18), and observed the biological characteristics of IL-18 gene-modified murine colorectal adenocarcinoma cell (CT26)in vivo andin vitro. Methods: Gene modification was mediated by adenovirus. The proliferation of the cells was determined by MTT and IL-18 was assayed by ELISA. The cytotoxicity of NK and CTL was detected by four-hour51Cr release assay. Results: IL-18 gene modification had no effect on the proliferation and morphology of CT-26 cellsin vitro, but the growth of IL-18-modified CT26 cells was obviously inhibitedin vivo. In addition, although IL-18-modified CT26 cells could form tumor nodulesin vivo as well as LacZ-modified CT26 cells or wild-type CT26 cells, the mean survival time of the mice inoculated with IL-18-modified CT26 cells was significantly prolonged as compared with that of control groups. Thus, the anti-tumor immune responses were induced in the group of mice inoculated with IL-18-modified CT26 cells, which might be related to the activation of NK cells and CTL. However, all the three groups ultimately died of tumor.Conclusion: IL-18-modified CT26 cells could induce the anti-tumor immune responses incompletely, which required other factors for effective anti-tumor responses.

COMBINED IL-2/IL-3 GENE THERAPY FOR G422 MOUSE GLIOBLASTOMA BY INTRATUMORAL INJECTION OF RECOMBINANT ADENOVIRUSES 1

Recombinant adenoviruses encoding murine IL-2 gene or IL-3 gene were directly injected into established subcutaneous tumor model of G422 glioblastoma cells. After treatment, the tumor size and survival of the glioblastoma-bearing mice were observed. The splenic NK and CTL cytotoxicities were detected by standard 4-hour51Cr release assay. We also examined the histopathological changes of tumor by hematoxylin and eosin staining. The results showed that intratumoral injection of adenoviruses encoding murine IL-2 gene or IL-3 gene significantly inhibited the growth of G422 glioblastoma and prolonged the survival period of glioblastoma-bearing mice. The CTL cytotoxicity of the gene therapy groups was significantly higher than that of the control groups, but NK activity remained unchanged, indicating that specific immunity contributes to the in vivo antitumor effect of the direct gene therapy. There were much more tumor necrosis and inflammatory cell infiltration in the tumor of the gene therapy groups. Combined IL-2/IL-3 gene therapy could induce higher level of CTL and enhance the therapeutic potential further. The results suggest that intratumoral injection of recombinant adenoviruses encoding certain kind of cytokines may be a useful approach in the treatment of a malignancy of the central nervous system.

The effector functions of macrophages transfected with interferon-gamma gene mediated by recombinant adenovirus

Macrophages(M(φs) are not only a kind of immune effector cells but also a kind of antigen-presenting cells(APC). In order to improve their antitumor effect, we transfected interferon-gamma(IFN-γ) gene into Mφs by recombinant adenovirus because IFN-γ is a kind of potent macrophage-activating factor(MAF). High level of IFN-γ could be detected in the supernatants of Mφs after IFN-γ gene transfection and IFN-γ secretion peaked at 20 hour. The cytotoxicity of IFN-γ gene-transfected Mφs increased significantly. The secretion of TNF, IL-1, nitric oxide(NO) also increased to some extent. The results demonstrated that recombinant adenovirus-mediated IFN-γ gene transfection could improve the effector functions of Mφs efficiently.

Construction and identification of retro viral vector expressing human interleukin-17 gene

Human interleukin 17(IL-17) is a newly found cytokine secreted by activated T lymphocytes. Many of its characteristics remain unknown. To provide a way for understanding its role in immunology and hematology further, we constructed a recombinant retroviral vector PL17SN containing the human IL-17 gene about 1.3kb with the coding region. The constructed retroviral vector packaged in CRIP cells could infect human primary fibroblasts, and could stimulate the primary fibroblasts secreting human GM-CSF and IL-6. The retroviral vector containing the human IL-17 gene we constructed may present an efficient way to understand the biological functions of human IL-17 and to investigate applications of IL-17 in cancer gene therapy.