| Introduction:It was considered that innate immunity to be low-level form response of immune system to external stimuli, but now studies have shown that innate immunity not only confront directly and quickly to external stimuli, but also play a direct role in the activation and guidance of acquired immune response. Among the humoral mediators mobilized by the innate immune responses, antimicrobial peptides play important roles in host immune defense by acting on diverse subpopulations of leukocyes, including killing of invading microorganisms directly, neutralization of harmful microbial products, inhibition of microbial entry into target cells, and enhancement of antigen-specific activation of B and T lymphocytes. At present, a number of studies have shown that the antimicrobial functions of antimicrobial peptides are highly sensitive to salt ion concentration under physiological conditions, and are directly inhibited by serum components; However, immunoregulatory function of antimicrobial peptides is not subject to the physiological concentration of salt ions, serum components or other factors. As a result of this, it was maintained that the immunoregulatory activity is very likely the main function of antimicrobial peptides under physiological conditions. Defensins are important components among antimicrobial peptides, which exist widely in higher organisms. Defensins not only kill pathogenic microorganisms with a broad-spectrum, but also has important immunoregulatory functions. One of the representative of defensins is the murineβdefensin 2(mBD2). The mBD2 can chemoattract immature dendritic cells through chemokine receptor CCR6, and act as a endogenous ligands of TLR4 to promote the maturation of immature dendritic cells, and produce a series of pro-inflammatory factors, chemokines, et al. The mBD2 also increase expression of costimulatory molecules. After immunization with plasmid encoding for a fusion protein of non-immunogenic lymphoma antigen and mBD2, an obvious anti-tumor activity can be induced in mice. It is indicated that mBD2 as an integral part of the innate immune response, can also assist non-immunogenic antigen in inducing a specific and effective acquired immune response in mice. It can be seen as a natural adjuvants. The mBD2 can be seen as bridge to connect innate immune response and acquired immune response, suggesting a new strategy for tumor immunotherapy. To make the use of activity of enhancing the antigen-specific immune response, We can work hard to develop a kind of mBD2-based natural immunoadjuvants. Many immunoenhancing agents have been investigated; however, most of them (with the exception of alum) can not be used in humans due to serious side effects. While the adjuvants potency of alum is limited and favors Th2 immune responses. Owning to the potent immunostimulatory activity of mBD2 as well as endogenous nature, it may be proved to be potent adjuvants without serious side effects.Malignant melanoma is a malignant tumor derived from melanocytes.It has a high degree of malignancy, an onset hidence and a high rate of misdiagnosis. The prognosis is poor. Malignant melanoma is one of the tumors with a fastest-growing incidence in the world. However, malignant melanoma is tolerate to general and comprehensive treatment measures, such as radiotherapy and chemotherapy. Treatment of melanoma in early stage mainly based on surgery, and prognosis of melanoma in late stage is poor. General speaking, there is no effective treatment, mainly in principle of individualized comprehensive treatment. Because malignant melanoma is a highly immunogenic tumor, specific immunotherapy is always one of the important measures in treatment of malignant melanoma. However, despite the immunotherapy is able to induce apparent protective effect of melanoma in a number of mouse models, the results of clinical trials showed that the response rate of human immunotherapy for malignant melanoma is far less than the experimental animals, only 10-30%. At present, the general agreement that the clinical effectiveness of the vaccine is not only determined by the type and the preparation methods of vaccines, but also the immunoadjuvants selected to enhance the effectiveness of the vaccines.We will make the use of adjuvants activity of the mBD2 in this research work, and apply it to the immunotherapy of malignant melanoma. First, we prepare mBD2 gene-modified melanoma cell vaccine, and then we used it in vivo to observe the anti-tumor effect and explore the anti-tumor immune protective mechanisms.Methods:1. Mouseβ-defensin-2 (mBD2) mature peptide gene fragment was amplified using RT-PCR technology from total RNA isolatd from C57BL/6 mouse kidney tissue; Mouse IgK signal peptide sequence was added at the 5’end of mBD2 mature peptide gene using overlap PCR method; After T-A cloning, constructed secreted eukaryotic expression vector of mBD2 containing the IgK signal peptide sequence: pcDNA3.1(+)-IgK-mBD2, and confirmed its correctness through DNA sequencing.2.pcDNA3.1(+) empty vector and eukaryotic expression vector pcDNA3.1 (+)-IgK-mBD2 containing the target gene was transfected into B16 melanoma cells using Lipofectamine 2000, and access to the two stable expressional cell lines after G418 screening. The two stable expressional cell lines were named B16-p, and B16-mBD2. RT-PCR was used to identify the neo gene expression and confirmed the sucessful transfection of the two plasmids. The mBD2 gene expression was identified using RT-PCR from RNA level; The protein level of mBD2 expression was identified using Western-Blot.3. MTT method was used to determine the cell growth curves of the three cell lines: B16, B16-p and B16-mBD2 during 120 hours; Flow cytometry by PI staining was used to detect cell cylcle distribution of the three cells mentioned above; Flow cytometry by FITC-conjuncated anti-mouse CD80, CD86, MHC I and MHC II and other control antibody staining was used to detect the immune related molecules, such as CD80, CD86, MHCⅠand MHCⅡexpressional level in above-mentioned three cells.4. To prepare cell vaccines with the three cells:B16, B16-p, B16-mBD2 by radiation method,respectively.To do immunotherapeautic and immunoprotective experiments in C57BL/6 mice.5.Procedures of immunoprotective experiments:Female C57BL/6 mice of 6-8 weeks old were randomly divided into four groups, and 10 to 12 mice in each group, respectively. The four groups were named saline control group, B16 control group, B16-p control group and B16-mBD2 experimental group, respectively. All animals in each group, in accordance with their respective groups, were given subcutaneously in the left armpit 0.1 mL saline and 0.1 mL radiated B16, B16-p and B16-mBD2 cell suspension with a concentration of 107/mL respectively. Seven days after immunization, all mice in each group were given 5×104 wild-type B16 cells to induce tumor. To observe the survival status daily of mice in each group. To draw tumor growth curves in vivo with Excel. To do survival analysis with SPSS 13.0. To detect histotogical changes of the tumor and important organs, such as: liver, kidney, spleen in all groups with HE staining. To detect infiltration of lymphocytes in tumor with HE staining.6.Procedures of immunotherapeautic experiments:Female C57BL/6 mice of 6-8 weeks old were randomly divided into four groups, and 10 to 12 mice in each group, respectively. The four groups were named saline control group, B16 control group, B16-p control group and B16-mBD2 experimental group, respectively. All animals in each group were injected subcutaneously in the left armpit for 105 wild-type B16 cells in logarithmic growth phase to induce tumor. All the mice in each group according to their respective groups, were given subcutaneously 0.1mL saline and 0.1 mL radiated B16, B16-p and B16-mBD2 cell suspensions with a concentration of 107/mL to begain the first therapy from the same day. The immunotherapy was given twice a week, consecutive two weeks. To observe the survival status daily of mice in each group. To draw tumor growth curves in vivo with Excel. To do survival analysis with SPSS 13.0. To detect histological changes of the tumor and important organs, such as:liver, kidney, spleen in all groups with HE staining. To detect infiltration of lymphocytes in tumor with HE staining.7. Concentration of IFN-y, IL-12 and IL-4 in spleen lymphocytes culture supernatants of mice immunized with B16、B16-p and B16-mBD2 cell vaccines was measured with ELISA. NK cells and CTL cytotoxicity in each mice immunized with B16、B16-p and B16-mBD2 cell vaccines were assayed with Non-Radioactive cytotoxicity assay.8. Using SPSS 13.0 for statistical analysis. Cell growth curve, tumor growth curves in vivo by repeated measures ANOVA; Analysis for cell cycle, apoptosis, cell surface expression of immune molecules, NK and CTL cell killing activity by factorial design-single variable analysis of variance. When homogeneity of variance, multiple comparisons using LSD method, variance missing, the multiple comparisons using Dunnett T3 method. P<0.05 indicated statistical significance.Results:1. Successfully constructed secreted eukaryotic expression vector containing Murine IgK signal peptide sequence, which was named pcDNA3.1 (+)-IgK-mBD2. The sequence of pcDNA3.1 (+)-IgK-mBD2 was confirmed to be correct with DNA sequencing.2. pcDNA3.1(+) and pcDNA3.1(+)-IgK-mBD2 were sucessfully transfected into B16 cells, and the stable cell lines named B16-p and B16-mBD2 were succesfully constructed.3. Compared with wild-type B16 cells and B16-p, proliferation of B16-mBD2 cell was decreased significantly (P<0.05, F=144.256). Compared to the wild-type B16 cells, the proliferation of B16-p has no significant change. These results were obtained through cell growth curve analysis.4. Flow cytometry analysis showed that 24 hours after inoculation, compared to wild-type B16 and B16-p cells, the proportion of S phase in B16-mBD2 cells was increased significantly (P<0.05, F=8.952). The expression of cell membrane surface molecules, such as CD80, CD86, MHCⅠand MHCⅡin the three cell lines B16, B16-p and B16-mBD2 was not significantly different.5. In the immunoprotective experiments, all the mice in the saline control group, B16 control group and B16-p control group died within 49 days, and there was no significant difference between each two control groups; The median survival in each control groups are 35,37,33 days reapectively. In vivo tumor growth rate of mice in B16-mBD2 immunization group decreased significantly (P<0.05, F=118.387, and survival time of these mice was significantly prolonged compare to other three control groups (P<0.05,χ2=18.857); The median survival is 55 days. Survival state of mice in B16-mBD2 immunization group were better than mice in other control groups during the same period of survival. To the experimental observation end of 150 days after tumor induction, there are still 37.5% of mice were tumor-free in B16-mBD2 immunization group.6. In immunotherapeautic experiments, all the mice in saline control group, B16 control group and the B16-p control group died in 44 days, and there is no significant difference between each two control groups; The median survival in each groups are 32,34,31 days respectively. In vivo tumor growth rate of mice in B16-mBD2 immunization group decreased significantly(P<0.05, F=289.615),and survival time of these mice was significantly prolonged compare to other three control groups (P<0.05,χ2=22.006); The median survival is 59 days. Survival state of mice in B16-mBD2 immunization group were better than mice in other control groups during the same period of survival. To the experimental observation end of 150 days after tumor induction, there are still 25% of mice were tumor-free in B16-mBD2 immunization group.7. During the immunopretective and immunotherapeautic experiments, a large number of infiltrating lymphocytes into the tumor tissue were detected in mice of B16-mBD2 immunization group; Germinal center of spleen of mice in B16-mBD2 immunization group was increased in number and area. All the mice in saline control group, B16 control group and B16-p control group were not found to be induced such initiative anti-tumor immune response.8. Compared with the B16 control group and B16-p control group, immunization with B16-mBD2 cell vaccine can promote augmented production of IFN-y and IL-12, the difference was statistically significant (P<0.05, F=506.814 or 83.637).The concertration of IL-4 was not different between the four groups. Compared with the B16 and B16-p control groups, immunization with B16-mBD2 cell vaccine can induce augumented CTL killing activity and NK cell killing activity in mice, the difference was statistically significant (P<0.05, F=44.376 or 119.750)9. The B16-mBD2 cell vaccine immunization didn’t induce any pathological changes in main organs, such as liver, kidney, spleen, lung, etc.Conclusions:mBD2 gene genetically modified B16 cell vaccines has an obvious anti-tumor effect on malignant melanoma B16. Immunization with B16-mBD2 cell vaccine can induce enhanced NK cells killing activity, CTL killing activity, and increased production of Thl-type cytokines, including IFN-y and IL-12. B16-mBD2 cell vaccines can also induce accumulation of lymphocytes into the tumor organization, and induced proliferation of spleen lymphonodules. The protective effect of mBD2 gene-modified B16 cell vaccine maybe not related to Th2 type cytokines, IL-4. Additionally, the B16-mBD2 cell vaccine immunization didn’t induce any side effects according to our results. In conclution, mBD2 gene-modified B16 cell vaccine can activate both the innate immune system (NK cell activity) and the acquired immune system (CTL cytotoxicity), and both the local tumor immunity (infiltration of lymphocytes into tumor) and the systemic immunity (enhanced production of IFN-γand IL-12) to strengthen immunoprotection against malignant melanoma. This research work developed a novel way in immunotherapy to malignant melanoma. |
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