| The survival rate of patients with cancer has improved, with the advent of effective anticancer treatments such as chemotherapy and radiotherapy. However, the majority of patients with metastatic disease will not be cured by these measures and will eventually die of their disease. New and more effective treating methods are required urgently. Differing from other measures, theraputic tumor vaccine could induce specific antitumour response by stimulating immune system. DNA vaccine is an attractive tool for vaccination studies because of its simple and unique given methods and substantial immune efficacy. It can induce not only specificantibodies production, but also specific cytotoxic T lymphocytes in animal experiments. This is the key point for prevention and treatment of cancer.Polymorphic epithelial mucin, encoded by the MUC1 gene, is a type I transmembrane protein, which presents at the apical surface of glandular epithelial cells. It is over-expressed and aberrantly glycosylated in many carcinomas resulting in an antigenically distinct molecule and a potential target for active specific immunotherapy. pcDNA3.1(+)vector, which has both CMV promoter and SV40 polyadenylation signal, can efficiently enhance transfection of foreign gene and then expression of recombinant protein. To study the specific anticancer role of MUC1 DNA as a vaccine, we constructed a eukaryotic expression vector pcDNA3.1(+)-MUCl containing the coding region of human full length MUC1 gene , and discussed immunological properties of MUC1 mucin and the efficacy tumour rejection induced by MUC1 DNA vaccine with and without GM-CSF adjuvant. Another, to study the changes expressions of MUC 1 in different tumor tissues and its clinical implications, a detailed immunohistochemical analysis of MUC1 protein expression was performed with MUC1 monoclonal antibody.First, The MUC 1 gene was obtained by cutting out the MUC 1 cDNA using Hindlll and Xhol and it was ligated into a clone vector pGEM-3zf. After identification and sequenthing , the gene was subcloned into eukaryotic expression vector pcDNA3.1(+). Restriction analysis and DNA sequencing showed that the recombinant plasmid contained the coding region of human full length MUC1 gene.The eukaryotic expression vector containing the MUC1 gene was succesfully constructed.Then, the recombinant pcDNA3.1(+)-MUCl was transfected into COS-7 cell by electroporation. After 56h transfection, Immunoflourescence and FCM were used to detect MUC1 gene expression in COS-7 cells. Fluorescence could been seen obviously in the transfected COS-7 cells membrane, whereas not in untransfected cells . Transfection experiment verified that MUC1 gene could be expressed in COS-7 cells and MUC 1 is a transmembrane protein molecule.Female BALB/c mice were immunized intramuscularly with 100 microgram MUC1 cDNA 3 times at 3-weekly intervals,. On 1, 3 and 5 days after Intra-muscular immunization, GM-CSF lOOul (lug/100ul) was given s.c. 3 times. Three weeks after the last immunization tumor challenge experiments were performed by using MUC1 expressing tumor cell line EMT6 ,H22. Tumor growth inhibition and body protection was observed two weeks later. Both humoral and cell-mediated MUC1-specific immune responses were detected with Immunohistochemical staining and 51Cr assays of CTLs, respectively. T-lymphocyte phenotype was detected by FCM. Histological analysis of tumor tissue was carried out with HE staining.After 45d of challenge experiments, the volume of EMT6 tumor in MUClcDNA, MUClcDNA+GM-CSF, pcDNA3.1(+), and PcDNA3.1(+)+GM-CSF group was (145?3.8)mm3, (250?4.3)mm\ (538?3.6)mm\ (596?8.2)mm3, respectively; the volume of H22 tumor in MUClcDNA, MUClcDNA +GM-CSF, pcDNA3.1 (+), pcDNA3.1 (+)+GM-CSF group was(293?6.3)mm3^ (547?8.6)mm3> (1207?48)mm\ (1220?59)mm3 , respectively. Compared with pcDNA3.1(+) control group, EMT6 tumor as well as H22 tumor growth in mice of MUClcDNA group were suppressed obviously(p<0.01). Co-delivery of GM-CSF adjuvant enhanced the antitumour immunotherapeutic effects.
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