| Part Ⅰ:Cloning and expression of Hyper IL-15.Object:Construction of Hyper IL-15 (IL-15/sIL-15Ra complex)prokaryotic recombinant plasmid PET43.1-Hyper IL-15 and obtain its protein; construction of Hyper IL-15 eukaryotic recombinant plasmid pCDNA3.1-Hyper IL-15, and verify its biological function; Construction of recombinant minicircle plasmid Minicircle-Hyper IL-15 which contains a Igκ signal pepetide and could maintain a longer time in the body, then verify its biological function.Methods:1.Construction and expression of PET43.1-Hyper IL-15. Total RNA was extracted from mouse muscle by Trizol- chloroform method, then transcripted into cDNA. IL-15 and its soluble receptor (sIL-15Ra) gene were amplified by PCR respectively. IL-15 and sIL-15Ra were connected with a flexiable (26 Aa) linker by overlapping PCR. The IL-15/sIL-15Ra complex was inserted into pET43.1 vector and transformed BL-21 bacteria. After spontaneous expression, the target protein in supernantant was verified by SDS-PAGE electrophoresis and Western Blot.2. Construction and expression of pCDNA3.1-Hyper IL-15. Total RNA was extracted from mouse muscle by Trizol-chloroform method, then transcripted into cDNA. IL-15 and its soluble receptor (sIL-15Ra) gene were amplified by PCR respectively. IL-15 and sIL-15Ra were connected by overlapping PCR. The IL-15/sIL-15Ra complex was inserted into pCDNA3.1 vector. The recombinant plasmids were transfected into 3T3 cells to obtain the supernatant which contains Hyper IL-15 protein. The CTLL-2 cells that live depend on IL-2 or IL-15 were cultured with the supernatant. Observe the proliferation of CTLL-2 to testify the function of pCDNA3.1-Hyper IL-15. pCDNA3.1-Hyper IL-15 was injected into mouse by tail vein hydradynamic gene transfer,and test the IL-15 level in the serum.3. Construction and expression of Minicircle-Hyper IL-15. Design specific primers for Hyper IL-15 in which the original signal peptide of IL-15 was instead of IgGκ to increase protein secretion and a kozark sequence was also added in front of the ATG promoter to enhance gene expression. The target gene was amplied by PCR using pCDNA3.1-Hyper IL-15 as the template. Then insert it into the minicircle parental plasmid pMC.CMV-MCS-SV40polyA and transform ZYCY10P3S2T E. coli. Minicircle-Hyper IL-15 was obtained by Arabia sugar inducing, and purification. The Minicircle-Hyper IL-15 was injected into mouse by tail vein hydradynamic gene transfer to detect the expression of IL-15 in mouse serum which could verifiy the biological function of the recombinant plasmids.Results:1. By sequencing, we confirmed that we successfully constructed the prokaryotic expression plasmid PET43.1-Hyper IL-15.Western blot also showed we obtained the Hyper IL-15 protein.2. Successfully constructed the pCDNA3.1-Hyper IL-15 eukaryotic expression plasmid. CTLL-2 showed a better proliferation when cultured with the supernantant from 3T3 transfectd by pCDNA3.1-Hyper IL-15comparing to that transfectd by pCDNA3.1-Blank. The IL-15 expression level in the serum was much higher in pCDNA3.1-Hyper IL-15 hydradynamic gene transfer group mice compared with the control group.3. Successfully constructed the Minicircle-Hyper IL-15 parental plasmid. And the parental plasmid could produce Minicircle-Hyper IL-15 under the induction of Arabia sugar. The IL-15 expression level in the serum was much higher in Minicircle-Hyper IL-15 hydradynamic gene transfer group mice compared with the control group and could maintain transfectd by pCDNA3.1-Hyper IL-15 for at least 2 weeks.Conclusion:we obtained the Hyper IL-15 protein and functional recombinant plasmid pCDNA3.1-Hyper IL-15 and Minicircle-Hyper IL-15.Part II:The function of hyper IL-15 on mice immune systemin resting state.Object:To explore the effect of hyper IL-15 on immune system under physiological onditions in mice.Methods:1.pCDNA3.1-Blank,pCDNA3.1-IL-15and pCDNA3.1-Hyper IL-15 were injected into Balb/c mice plasmid by tail vein hydrodynamic injection,80ug/2ml/mice. Mice serum was collected at day 4 and day 14 respectively. Detect the levels of IL-15, IFN-γ and TNF-α in the mice serum by CBA kit. The lymphocytes from spleen and liver were also collected on day 4 and day 14 respectively and essayed by flow cytometry to detect the lymphocytes subsets and their status.2. Minicircle-Blank, Minicircle IL-15 and Minicircle-Hyper IL-15 were injected into mice by tail vein hydrodynamic injection,30ug/2ml/mice. Mice serum was collected 2 weeks later. Detect the levels of IL-15, IFN-γ and TNF-α in the mice serum by CBA kit. The lymphocytes from spleen and liver were also collected at day 4 and day 14 respectively and essayed by flow cytometry to detect the lymphocytes subsets and their status.3. Minicircle-Blank, Minicircle IL-15 and Minicircle-Hyper IL-15 were injected into Balb/c mice plasmid by tail vein hydrodynamic injection,30ug/2ml/mice. The proliferation and apoptosis of various cell subsets were tested a weak later.Results:1. pCDNA3.1-Hyper IL-15 significantly increased the number of CD8+T cells, NK cells and NKT cells. It promoted CD8+T cell early activation, and enhanced NK and NKT cell maturation and activation. It also enhanced the ability of hepatic T cell to secret IFN-γ and TNF-α. IL-15 IFN-γ TNF-α levels in serum were elevated. pCDNA3.1-Hyper IL-15 seems superior to pCDNA3.1-IL-15, and it can maintain IL-15 production for 2 weeks.2. Minicircle-Hyper IL-15 showed a strong immune activation function 2 weeks after hydradynamic transfer. It significantly increased the number of CD8+T cells, NK cells and NKT cells, promoted CD8+T cell early activation, and enhanced NK and NKT cell maturation and activation,and enhanced the ability of hepatic T cell to secret IFN-γ and TNF-α. The effect of Minicircle-Hyper IL-15 was superior comparing to Minicircle IL-15.3. Minicircle-Hyper IL-15 increased the number of CD8+T, NK and NKT cells mainly by promoting their proliferation, reducing their apoptosis and maintaining their survival. Minicircle-Hyper IL-15 could slightly promote the proliferation of CD4+T cells but also increased its apoptosis, therefore the number of CD4+T cells remain the same.Conclusion:1. Hyper IL-15 can significantly activate the mice immune system in resing state. It can promote the proliferation of CD8+T, NK and NKT cells and effectively reduce their apoptosis and necrosis to maintain their survival, which dramatically increased the number of CD8+T, NK and NKT cells. Despit of the cell number increase, Hyper IL-15 can also enhance the function of CD8+T to produce more cytokines such as IFN-γ and TNF-α and promote NK and NKT maturation and activation.2. Minicircle-Hyper IL-15 plasmid showed a better performance than the pCDNA3.1-Hyper IL-15. In our study, Minicircle-Hyper IL-15 plasmid displayed stronger and longer effects with a lower dose than pCDNA3.1-Hyper IL-15 plasmid.Part Ⅲ:The function and mechanism study of Hyper IL-15 in the treatment of hepatocellular carcinoma.Object:To explore the therapeutic effect and its mechanism of Hyper IL-15 in primary hepatocellular carcinoma.Methods:First, establish mouse hepatocellular carcinoma model:1. liver in situ injection of tumor cell Hepal-6-luc.2. Tail vein hydrodynamic injection of tumor cells Hepal-6-luc. Then, Minicircle-Blank, Minicircle IL-15 and Minicircle-Hyper IL-15 plasmids were hydrodynamic injected into mice.Tumor volume and number were observed. The proportion and absolute number of the lymphoctes were detected by flow cytometry. The activation of the CD8+T, NK, and NKT were also accessed. Minicircle-Blank, Minicircle IL-15 and Minicircle-Hyper IL-15 plasmids were hydrodynamic injected into mice with CD8 neutralizing antibody clearance of mouse CD8+T cells and NK inhibitory antibody inhibit NK cells and tumor volume were observed.Results:In the in situ injection model, the volums of liver tumors from Minicircle-Hyper IL-15 treatment group were significantly smaller than that of the control group. In the tail vein hydrodynamic injection model, the numbers of liver tumors from Minicircle-Hyper IL-15 treatment group were significantly less than that of the control group. Flow cytometry showed that there were much more CD8+T cells, NK cells and NKT cells in the Hyper IL-15 treatment group. These CD8+T cells, NK cells and NKT cells were more activated than the control group. The number of effector CD8+T cells, memory CD8+T cells, activated NK and NKT, mature NK and NKT were significantly promoted. After depeletion of CD8+T cells with CD8 neutralizing antibody, the tumors of the Hyper IL-15 treatment group were retored. However, the tumor size did not significantly change when NK cell function was inhibited. It suggest that the CD8+T cells play a crucial role in the Hyper IL-15 mediated anti-tumor function in hepatocellular carcinoma.Conclusion:Hyper IL-15 is effective in the treatment of hepatocellular carcinoma. CD8+T cells play a critical role in the Hyper IL-15 mediated anti-tumor process. Hyper IL-15 promotes the proliferation, activation and cytokine production of the CD8+T cells which plays an important role in anti-tumor immune responses. |
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