| DsNKG2D-IL-15 fusion protein composed by two of NKG2D extracellular domains and a IL-15 domain. Previous research has confirmed that NKG2D highly expresses on the surface of most tumor cells which can recognize MICA and other ligands, and meanwhile IL-15 has the function of stimulating NK and CD8+T cells, the fusion protein has dramatic anti-tumor effects. The nanometer material of Chitosan is the emerging advantage drug carrier which plays a more and more important role in the transfer process of gene vaccine and a variety of antitumor drugs. With the advantage of high biocompatibility, low toxicity and biodegradability, chitosan is the good choice for us to use as the material to prepare nanoparticles. In our study, the recombinant plasmid of pcDNA3.1-dsNKG2D-IL-15 and the fusion protein of dsNKG2D-IL-15 both had been already prepared. Besides HTCC(2-Hydroxy-N,N,N-trimethyl-l-propanaminium chloride) chitosan is the material we use to load the agencies. And then we aimed to evaluate the anti-tumor effection of the nanoparticles through experiments in vitro and in vivo.Our study can be divided into two parts.Part I. Preparation of the nanoparticle loading with recombinant plasmid pcDNA3.1-dsNKG2D-IL-15 and its antitumor activity.Objective:To make the antineoplastic nanoparticle with the chemically modified HTCC chitosan and the recombinant gene (pcDNA3.1-dsNKG2D-IL-15) prepared in our early work. To test part of the physical and chemical properties of the nanoparticle and observe whether the recombinant gene can be transferred into the cells and express NKG2D, IL-15. To find out its influence on lymphocyte especially NK and CD8+T cells and the influence on B16BL6-MICA melanoma in the C57BL6 mice.Methods:Firstly, mix HTCC with recombinant plasmid in the mass ratio of 1:2,2:1,1:1. After shaking and loading, centrifuge the mixture and get the supernatant. Detecting the concentration of DNA in the supernatant with spectrophotometry and calculating the encapsulation efficiency by the following formula (total DNA-free DNA)/total DNA* 100%. Take the prepared nanoparticles and test its diameter and electric potential after dilution. Then test its toxicity to RAW264.7 and B16BL6 cells with MTS/PMS kits. Compared with lipidosome and monomeric protein, transfer the nanoparticles into the CT-26 cells, test the express of NKG2D by FCM after 72 hours. In addition, analyse the concentration of IL-15 in the supernatant of the transferred cells. Use the transferred cells co-incubation with the spleen cells from mice, analyse the frequency and activity of NK and CD8+T cells morrow. Finally, the in vivo experiment was performed by subcutaneous injection of B16BL6-MICA transplantation tumor into C57BL/6 mice. Inject each 4 group with PBS, HTCC, nanoparticle intramuscularly and intraperitoneally respectively. Record the subsistence of tumor-bearing mice and the growth of tumor everyday for three weeks. Eventually, execute the mice and analyse the change in frequency and activation of NK and CD8+T cells in the spleen of tumor-bearing mice. Detect the express of CD69, NKG2D, CD44 by FCM.Results:The HTCC material can load with the pcDNA3.1-dsNKG2D-IL-15 recombinant gene and the loading efficiency can reach 92.8±0.37%. It has certain cytotoxicity to tumor cells in a high concentration. Besides, the nanoparticle can transfer the recombinant gene to CT-26 and RAW 264.7 cells. The expressed NKG2D and IL-15 can be detected by FCM and ELISA respectively. The frequency of the NK and CD8+T cells in the spleen of tumor-bearing mice also increased. And so does the NKG2D, CD44 enhanced. The life of tumor-bearing mice treated by the nanoparticle is extended, and the growth of transplanted tumor can be inhibited.Conclusions:The nanoparticle loading with pcDNA3.1-dsNKG2D-IL-15 recombinant gene can be endocytosed by tumor cells. In vivo nanoparticle can stimulate NK and CD8+T cells function andPart Ⅱ. Preparation of the nanoparticle loading with dsNKG2D-IL-15 fusion protein and its antitumor activity.Objective:To prepare the nanoparticle loading with dsNKG2D-IL-15 fusion protein and test part of its physicochemical property and toxicity. Observe its anti-tumor effect in vivo and the influence on tumor-bearing mice. To observe the influence about frequency and phenotype of lymphocyte such as NK, CD8+T cells.Methods:Mix HTCC solution and dsNKG2D-IL-15 fusion protein to prepare nanopartcile. Test the concentration of free protein in the supernatant of mixture with spectrophotometry and calculate the encapsulation efficiency by the following formula:(total protein-free protein)/total protein* 100%. Besides, the diameter of the nanoparticle is measured by the dynamic light scattering and the electric potential variation on the surface of nanometer materials can be observed by zeta potential meter. Test the toxicity of our nanoparticle with MTS/PMS kit. Then, the dialysis release experiment is used to test whether the nanoparticle could release the fusion proein, the result is checked by protein gel electrophoresis. After that, inject B16BL6-MICA transplantation tumor into C57BL/6 mice subcutaneously. Inject each 4 group with PBS, HTCC chitosan, nanoparticle and fusion protein respectively. Record the subsistence of tumor-bearing mice and the growth of tumor everyday for three weeks. Eventually, execute the mice and analyse the change in frequency and activation of NK and CD8+T cells in the spleen of tumor-bearing mice. Detect the express of CD69, NKG2D, CD44 by FCM.Results:The HTCC chitosan can load the recombinant protein in the solution. When the mass ratio is 1:1, it reaches its utmost load efficiency:95.7 ± 0.36%. The nanogel carrying dsNKG2D-IL-15 is poisonous to some tumor cells such as B16BL6 while less poisonous to mice macrophage RAW 264.7. The nanometer material of Chitosan releases recombinant preoteins effectively in 24 hours in vitro release test. In vivo test, the group treated by nanogel is in contrast to the group with PBS and chitosan which can inhibit the growth of B16BL6- MICA melanoma in the C56BL/6 mice and prolong the life of tumor-bearing mice to a certain extent. Our team finds the increase in the frequency of the NK and CD8+T cells in the spleen of tumor-bearing mice, and phenotypes such as NKG2D, CD44 enhanced.Conclusions:The nanoparticle loading with dsNKG2D-IL-15 fusion protein has the ability to release protein in vitro. Moreover in vivo with the treatment of nanoparticle the transplantation tumor can be inhibition. That mainly because with the expression of fusion gene, the NK and CD8+T cells are activated effectively. |
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