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The Immunotherapeutic Potential And Anti-tumor Effects Of Recombinant Mycobacteria

Posted on:2003-05-08Degree:DoctorType:Dissertation
Country:ChinaCandidate:J Z YaoFull Text:PDF
GTID:1104360095461216Subject:Surgery
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Intravesical instillation of BCG is one of the most effective treatment and recurrence prevention approach of bladder cancer. It is considered that the clinical results is significantly improved if combined with intravesical perfusion of rIL-2. However, as the BCG and IL-2 dose are large and administration is frequent, so this therapy is associated with some complications such as disseminated mycobacteriosis disease. M.semgmatic, isolated from soil, is fast-growing, non-pathogenic cmpared with BCG . With the devolpment of molecular biology and DNA recombination technique in recently year, recombinant mycobacterial allows us to explore the possiblity that the inherent immunostimulatory properities of mycobacterial could be modified or potentiated through the secretion of cytokines and further more to develop a new method for treating and preventing the recurrence of transitional bladder cancer.In the present study, first, the human IL-2 mycobacterial shuttle-expressing plasmid was constructed, and recombinant forms of M.semgmatic which express and secrete biologically active IL-2 was developed. Then, Babl/c mice were immunized with this strains by intraperitoneal injection, cell-mediated immune responses in spleenocyte and responses of mice to infection with it was observed. Meanwhile, antiproliferative effect against transitional cell carcinoma lines and cytotoxicity of these strain were investigated respectively in vitro, and the morphological or surface phenotype changes on transitional cell carcinoma lines were observed. Moreover, its anti-tumor effects on subcutaneous tumor model in T739 micewas studied and the effect on immunocompetent cells of tumor tissuse were observed. The main results and conclusions are as follows:1. The Shuttle plasmid pR-IL-2 was constructed. It includes hsp70 promoter and an antigen signal peptide gene that targets IL-2 for secretive expression, which was amplified using the PCR method. The pR-IL-2 was identified by restriction map, DNA sequencing. The results showed that ligation of promoter ,an -antigen signal peptide gene and IL-2 is correct. Shuttle plasmid pR-IL-2 is successfully constructed.2. The pR-a-IL-2 was introduced into the M.semgmatic by electroporation and the recombinant forms of M.semgmatic was identified by restriction map, DNA sequencing and IL-2 bioassay. The secretion of IL-2 from recombinant of M.semgmatic was detected by Western blot and MTT colorimetry and the IL-2 activity in supernatant reached 118.5U/ml.The stability of the recombinant M.semgmatic in vitro was studied, the result showed that the recombinant M.semgmatic has more stable when it growed with Kan resistence in vitro,it can contiune propagate for 20 generation without Kan resistence and did not lost plamid.3. Our observation revealed of M.semgmatic vector- M.semgmatic and recombinant M.semgmatic all have antiproliferative directly on bladder tumor cell growth in vitro, the recombinant M.semgmatic is most effective in all. The tumoricidal activity of SMAK effector cells of the recombinant M.semgmatic and LAK cells are high. However, no significant difference between SMAK effector cells of the recombinant M.semgmatic and LAK cells was observed. After the recombinant of M.semgmatic on bladder tumor cell, we find tumor cell become proliferative slowly, detach, quantity decreasing and death under microscope, and various degree degeneration on most of tumor cell, disorganization on organell, aggregation on chromatin, pycnosis on nucleolus, and some Cytoplasm Lysis, nucleus fragmentation, cell coat disruption, organell escape were observed on tumor cell under transmission electronmicroscopy. In vitro experiments on a panel of bladder tumor cell lines (EJ, BTT739) using the flow cytometry analysis indicate that all forms M.semgmatic could induce or up-regulate the expression of HLA-DR and ICAM-1, the recombinant of M.semgmatic are most effective in all. These results suggest that the recombinant of M.semgmatic have more immunomodulatory properties, ant...
Keywords/Search Tags:bladder cancer, Interleukin-2, Mycobacterium, recombinant mycobacterial, BCG, immunogenetherapy, shuttle plasmid
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