| BackgroundMalignant melanoma (MM) is one of the most common malignant tumorsworldwide. It originates from the neural crest melanocytes, and is malignanttransformed from melanocytes. It is high aggressive and metastatic. Because of itscontinued increasing rates of morbidity and mortality, malignant melanomaincreasingly gained more attention. Conventional therapy is ineffective, and this isposing a serious threat to human health. Its incidence has been doubled in the past20years. According to incomplete statistics, the incidence of malignant melanomaaccounts for3%of all human malignancies, and human cutaneous malignantmelanoma accounts for7%to20%of the skin cancer, and the incidence growsannually.Human cutaneous malignant melanoma is a common type of plastic surgerysuperficial tumor, which originated from a malignant tumor of the epidermis ofnormal melanocytes or normal nevus cells, and its degree of malignancy is high, theprogress extremely rapid, and the prognosis is poor. Human cutaneous malignantmelanoma incidence and mortality rate has been increasing by2%to3%annually inthe past30years, and it has been attached great importance to by people.Currently, the treatments of human malignant melanoma include surgery,radiation therapy, chemotherapy, biological therapy, and the tumor vaccine treatment.The surgery is still the most common means for the treatment of human malignancies,and it has a good therapeutic effect on many early localized human malignantmelanoma.When it transferees diffused or infiltrate to vital organs in malignant melanoma,it is in the event of poor treatment outcome or even can not be treated by surgery. Forsuch patients, the main methods of treatments are radiation therapy and chemotherapy,but the treatment effect are poor because of its insensitivity to conventional radiation therapy and chemotherapy, and radiotherapy and chemotherapy will damage normaltissues and will lead to larger side effects. And the tumor resistance has restricted theapplication of chemotherapy greatly. For biological treatment, interleukin, interferon,granulocyte macrophage colony stimulating factor have been studied a lot,restrictions on the studied, but there are no uniform usage manuals can be followed.All in all, there are still no satisfactory treatments for human malignant melanoma.Recent years, with the rapid development of molecular biology, immunology,and biochemistry, biological behaviors of malignancies have been recognized further.It is known that cancer is a genetic correlated disease; and there are similarityhomology but at the same time heterogeneity between malignant tumor cells andnormal human tissue cells. Malignant cells evade immune clearance by immuneescape mechanisms, but they can produce immune response process different to thenormal tissue cells. By use of the characteristics of the tumor cells, tumor vaccine cankill and clear the malignant cells, but have little effect on normal tissue cells. Part ofthe tumor vaccines have been used in a Phase III clinical study, and it have been madean exciting progress.Monocyte chemoattractant protein1(MCP-1), also known as monocytechemoattractant factor protein CC chemokine ligand2(CCL2), belonging to thechemokine a member of the CC family, is the firstly found and the most studiedmember of CC chemokine family. It can be secreted by a variety of cells, such asleukocytes, monocytes, osteoblasts, fibroblasts, smooth muscle cells, endothelial cells,stellate cells, and some tumor cells. MCP-1can regulate the chemoattractant functionof monocyte memory T cells, NK cells and neutrophils and make these cells migrateand invade to the inflammation site. Studies have shown that through combining toCC chemokine receptor (CCR), MCP-1plays a variety of physiological effects, suchas induction the activation, differentiation and development of lymphocytes and NKcells, and particpation in the process of regeneration of blood vessels, which doesgood to the growth or inhibition of the tumor. In addition it also can increasemacrophage infiltration and macrophage-mediated angiogenesis.In recent years, the researchers have focused on not only the simple applicationof autologous or allogeneic tumor cell vaccine for treatment, but also the geneticmodification of the tumor cells, and combination with the immune adjuvant toincrease the immunogenicity of the tumor cells. Therefore, this project is intended touse the gene of MCP-1, to construct the gene-modified tumor vaccine by transfectingMCP-1to wild-type malignant melanoma cell lines, and to study the treatment ofmalignant melanoma from both immunotherapy and gene therapy, in order to found anew way for immunotherapy of human malignant melanoma.ObjectConstruct the eukaryotic expression vector of MCP-1and transfect murinemelanoma cell line B16to construct mouse malignant melanoma vaccine, and the lymphocyte chemotaxis of the malignant melanoma vaccine to be studied in vitro.Nude mouse has been chosen as the experimental animal model. Part of the immunefunction of the nude mouse has been rebuilded by transplanting thymic T cells of thesame type of mouse. Establish nude mouse malignant melanoma animal models tostudy whether the vaccine has the antineoplasmic activity in vivo, and to explore itsmechanism.Methods1. Acquisition of target gene MCP-1Extract total mouse liver RNA, reverse transcribed into cDNA, according to theGeneBank accession mouse MCP-1cDNA sequence (SEQ ID No. AF065929), theprimers of mouse MCP-1are designed by using the software Premier5. And theobjective gene are amplified by PCR. After1hour agarose gel electrophoresis, theamplified fragment can be observed in the UV lamp and the target gene can berecovered and purified.2. Construction of recombinant plasmid pEGFP-N1-MCP-1Prepare E. coli, and the target gene is transformed into a vector plasmidpEGFP-N1, and then recombinant plasmid pEGFP-N1-MCP-1were extracted byalkaline lysis method, and identified by the digestion of restriction endonuclease.3. Transfection of Recombinant plasmid pEGFP-N1-MCP-1to murine malignantmelanoma cell line B16The transfection experiment was conducted according to the instructions ofLipofectamineTM2000kit. The experiment was divided into three groups: theexperimental group, the empty vector control group and blank control group, andthere were three holes for each group. For experimental group4μL recombinantplasmid pEGFP-N1-MCP-1were used, for empty vector group the same amount ofempty plasmid pEGFP-N1were used, and for blank control group, an equal volumeof sterile PBS solution were used.4. Determination the effect of transfectionForty-eight hour after transfection, three groups of cells were observed byfluorescence microscopy and the rat of transfection can be determined by observationof the expression of the green fluorescent protein.5. Select stable cell line B16-MCP-1Mouse melanoma cell line B16was selected by G418to get the optimalselection concentration.48h after transfection, B16cells were cultured in culturesolution containing the final concentration of G418. Select resistant clones andculture such cells.6. Identification of recombinant cells and study cell proliferation After cultured for3weeks, the resistant clones in each group were taken and thetotal RNA was extracted for RT-PCR. The recombinant cells were identified bymeasuring the transcription of MCP-1. Protein of MCP-1was detected by ELISA.Cell proliferation was conducted by using MTT assay.7. Detection of the in vitro function of mouse chemokine tumor cell vaccineB16-MCP-1MCP-1was collected from the cultivation supernatant of the B16-MCP-1cellsby using a multi-step centrifugation. Dissect healthy nude mice to get spleen, andmononuclearcells were obtained by density gradient centrifugation. The in vitrochemotactic activity of the tumor cell vaccine was measured by using Transwellchamber invasion assay. The numbers of cells migrating to the back of themicroporous membrane were counted in inverted microscope. Five randomhigh-power fields were counted in each group. Using the following formula tocalculate chemotactic index=the number of cells migrating to the back of themicroporous membrane in the experimental group/the number of cells migrating tothe back of the microporous membrane in the control group.8. Isolation of T cells from BABL/c mouseMice were sacrificed by cervical dislocation under sterile conditions, andprepare the cell suspension from the thymus. Lymphocytes were separated by usingdensity gradient centrifugation and then cultured in complete medium at37℃,5%CO2culture for2hours. Nonadherent cells were aspirated and filtered by nylon woolcolumn to get T lymphocytes9. Rebuilding part of the immune function in the nude mouse and preventivevaccinationGrouping of nude mice: Forty-five nude mice were divided into three groupsrandomly,15each group: The first group was saline blank control group (Nacl group),the second group was the empty vector pEGFP-N1transfection cells control group(wild-type B16vaccine group), the third group is the tumor vaccine B16-MCP-1treatment group (chemokine-type B16-MCP-1vaccine group).Rebuilding part of the immune function in nude mice: The isolated mousethymus T cells were injected intraperitoneally into the nude mice with3×107permouse (0.5ml).Immunization nude mice by the method of the left inguinal subcutaneousinjection, for the Nacl group injection with0.2mL NaCl, for the second groupinjection with0.2mL empty vector pEGFP-N1transfected cells (containing2×104cells), and for the third group injection with0.2mL chemokine-type tumor vaccineB16-MCP-1(containing2×104cells). Immunization performed once a week, for atotal of three times.10. Observation of nude mice bearing melanoma animal modelOne week after the3rd immunization, nude mice bearing melanoma model wereestablished by a single point injection with0.2mL of wild-type mouse melanoma cellsB16(containing2×104cells) in the right Shoulder and back of the nude mice. 11. The effects of the CTL activity on mouse spleen lymphocytes by vaccination withtumor cell vaccine B16-MCP-1One week after the3rd immunization, five nude mice of each group were killedby cervical dislocation to dissect the mouse spleen, and the spleen cells wereseparated by the method of density gradient centrifugation used as effector cells,mouse melanoma cells B16used as target cells, the two groups of cells were added to96-well cell culture plate in accordance with the rate of effector cells: target cells was25:1, and then cultured in37°C,5%CO2incubator. Lymphocyte killing rate wascalculated in according to the following formula.Lymphocyte killing rate (%)=(1-(OD of the effector cell+OD of the target cells–OD of experimental group)/OD of the target cells)×100%Results1. Mouse MCP-1gene RT-PCRTotal RNA was extracted from mouse liver tissue, after reverse transcription andPCR amplification, a480bp amplified product can be observed after1%agarose gelelectrophoresis, which was exactly the same with the expected size.2. Identification of the recombinant plasmid pEGFP-N1-MCP-1The recombinant plasmid pEGFP-N1-MCP-1was digested by EcoR Ⅰ andXbaI and the results were observed by1%agarose gel electrophoresis. It was showedthat there are two bright bands with the size of4.7kb at480bp, which were the samewith the plasmid vector and the target gene. In the empty plasmid group there onlywas a bright band with the size of4.7kb.3. Identification of the recombinant cellAfter transfection by the method of liposome and selection by G418, theresistant clones of the mouse melanoma cell line B16were collected to detect theexpression of the target gene. After1%agarose gel electrophoresis, there was a clearstrip with480bp in the experimental group, which was the same with the positivecontrol group. There was no target gene in the empty plasmid transfected group. Theresults of ELISA showed that: there was expression of MCP-1in the experimentalgroup, while there were no positive results in the control group ones.4. Detection of the in vitro function of mouse chemokine tumor cell vaccineB16-MCP-1.The numbers of cell invasion in the third group were significantly higher thanthat in the first and second group. This shows that the chemokine type vaccineB16-MCP-1can attract lymphocytes in vitro.5. Observation of each group of mouse model after mmunizationFour days after the establishment of animal models, formed in part of the nude mice,30days later, all the nude mice in the first and second group of mice grewvisible tumors, and the tumor forming rate was100%in30days. But for the thirdgroup the tumor forming rate was20%in three days and60%in50days. There were4nude mice in the third group haven’t formed palpable tumors in during the time ofobservation. At the30rd day, there were2mice in the first group emerged tumorulceration, and there were5nude mice tumor ulceration during the time ofobservation. There was1nude mouse emerged light tumor ulceration in the thirdgroup, and in the experimental process, there is no tumor ulceration in the rest of thenude mice.In the first and the second group the nude mice showed the state of cachexiasuch as significant weight loss, weakening of activity, and the survival state of thethird group was significantly better than the first two groups.6. Observation of tumor growthIn2weeks of the establishment of nude mice bearing melanoma animalmodels, there were no significant difference (P>0.05) in the sizes of the tumorvolume in each group, but two weeks later to all the observation time, the tumorvolumes in the third group were significantly smaller than those of the other groups(P <0.05), From19d to all the observation time, there was significantly difference (P<0.05) among each group.7. Observation of the mice survival time1nude mouse died in the first group four days after the establishment of animalmodels, and which occurred at the10thday in the second group and35thin the thirdgroup. At the55thday, all the nude mice in the first group and the second group died.At the80thday, there were still4nude mice survival in the third group.Survival time in the third group was significantly longer (P <0.01) comparedwith the first group and the second group, and the survival time of the first group andthe second group are basically the same (P>0.05).8. The effects of the CTL activity on nude mouse spleen lymphocytes by vaccinationwith tumor cell vaccine B16-MCP-1The in the third group was significantly stronger than the other group (P <0.01),while there were no significant specificity cytotoxic function in the first and secondgroup, and there was little significant difference between the two groups(P>0.05).This indicates that transfection of MCP-1gene increased the CTL activity of mouseand strengthened the properties of anti-tumor.Conclusions1. Successfully constructed eukaryotic expression vector pEGFP-N1-MCP-1.2. Successfully constructed mouse chemokine tumor vaccine B16-MCP-1, and thevaccine can abstract mouse mononuclear cells in vitro. 3. The chemoattractant vaccine has a significant immune effect on nude mice bearingmelanoma animal models, and can alleviate the symptoms of the tumor-bearing nudemice and prolong the survival time of the tumor-bearing nude mice. |
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