| Tumor is one of the major diseases that threaten human health. In recent years, with the deepening research on tumorous immunity, aetiology and molecular mechanisms, significant progress has been made in the research on treatent of tumour gene therapy. Tumour gene therapy is gradually becoming an expectation treatment means. Tumour gene therapy is used to induce interested gene into target cells,making them obtain specific function and then performing the functions of killing tumour cells or inhibiting tumor cell growth. As one class of small molecular polypeptide, Antimicrobial peptides have a fairly activity against bacteria and tumours. Because of their small gene fragments, easy to transfection and the peculiar mechanisms of their anti-tumor and the very low or no toxicity to normal cells,they are arouse interests in the study. In this study, based on the constructed recombinant of hCAP-18/LL-37, the unique known antimicrobial peptide of Cathelicidins family that exits in human body, we infected Lewis cells by way of transient transfection,using molecular biological techniques,aiming at studying the role of hCAP-18/LL-37 in the activity of anti-tumor and expression of immunosupressors.Objective:In this study,based on the constructed recombinant of hCAP-18/LL-37,we transfer it into Lewis cells to explore the mechanism of its anti-tumor activity and expression of immunosupressors.Methods:(1) The recombinant plasmid pcDNA4/Myc-His-LL-37 was transformed into JM109 bacterium and identified by restriction endonuclease analysis.(2)DNA of hCAP-18/LL-37 transient transfection mediated by Polyfect was employed.(3) proliferating ability of Lewis cells after 48h of transfection was measured by MTT colorimetric assay. (4) Cell apoptosis of transfected Lewis cells was detected by Annexin V-FITC/ pI double stainging and flow cytometry. (5) Nuclear apoptotic morphological changes were observed under the the fluorescence microscope. (6) The expression of Bax,Bcl-2 mRNA in Lewis after transfection was analyzed by RT-PCR. (7) The expressions of Bax and Bcl-2 in transfected Lewis cells were determined by cellular and immunohistochemical technique. (8) Effect of tumor cell supernatant on the splenocyte proliferation was measured by MTT colorimetric assay. (9) The expression of VEGF,TGF-βand IL-10mRNA in Lewis after 4transfection was analyzed by RT-PCR.Results:1.The recombinant plasmid pcDNA4/Myc-His-LL-37 identified by restriction endonuclease analysis. Purpose fragments are consistent with theoretic values.2.PCR result display the recombinant plasmid was transformed into Lewis cells.3. After 48 hand 72h of transfection, the proliferating viability of Lewis cells transfected by the recombinant plasmid was significantly inhibited, compared with the control(p﹤0.05,n=6). While the proliferating viability of the Lewis transfected by the free vector is non - specific insult, compared to control.4.The apoptosis rate were 4.4%, 5.2%, 7.6% in respectivly by FCM measurement, apoptosis in tumor cell was arise.5.The expression of Bax and Bcl-2 mRNA in Lewis after transfection was analyzed by RT-PCR.The expression of Bax mRNA in transfected Lewis with recombinant was increased, there was no significant difference in the expression of Bcl-2 mRNA, compared with the control.6.After 48h of transfection, the protein expression of Bax in transfected Lewis cells was increased (p﹤0.05,n=10), while there was negative expressions of it in control group and the free vector-transfected group, which were no significant difference in the expression of Bcl-2. The expression of Bcl-2 in recombinant-transfected MCF-7 was positive.7. The supernatant was diluted 1:2 times after 48h of transfection.The results showed that the recombinant group cell culture supernatant can not inhibit rat splenocyte proliferation, while the control group and the free vector cell culture supernatant can inhibit rat splenocyte proliferation (p﹤0.05), compared with the splenocyte cell group control.8.The expression of VEGF and TGF-βmRNA in transfected Lewis with recombinant was decreased, while there was no significant difference in the expression of IL-10 mRNA, compared with the control.Conclusions: 1.The recombinant plasmid pcDNA4/Myc-His-LL-37 was successfully transformed into Lewis cells by liposome transfection method.2. hCAP-18/LL-37 can induce apoptosis of Lewis by the up-regulation of Bax and the down-regulation of Bcl-2 and inhibit proliferating ability of Lewis cells.3.hCAP-18/LL-37 can control the expression of VEGF and TGF-βmRNA in Lewis and regulate immune response which induced by tumour cells.
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