| Malignant melanoma is a malignant tumor that derived from malignant melanoma cells and neural crest melanocytes; it can occur in the skin, mucous membranes, and malignant melanoma occurred in the oral mucosa is the highest level of oral malignant tumor, accounting for 1% to 2% in systemic malignancy. And In China and Southeast Asia occurred in oral mucosa were more inversely proportional to the facial skin, accounting about 80% of total. In recent years, many surveys indicate that the morbidity of malignant melanoma constantly increase, and about 70% early metastasis to regional lymph nodes, also by the blood flow distant metastasis to lung, liver, bone, brain and other organs, the distant metastasis rate up to 40%. Though, the traditional method of treating cancer (including:surgery, radiotherapy, chemotherapy, and adjuvant therapy) has achieved right effect, but the malignant melanoma cells is relatively insensitive to chemotherapeutic drugs, occur certain resistance to the radiation, besides the B16 melanoma derived from the melanoma tumor cells with high oncogenicity and low immunogenicity, so finding a safe and effective method of preventing and treating malignant melanoma is quite important.With the immunology and molecular biology are in-depth understanded, targeted therapy of cancer gene immune has become a research focus of tumor therapy. From 90 years of the 20th century, this concept of DNA vaccine was produced, DNA vaccines have entered a number of areas and have made great progress, and on the prevention and treatment of cancer genes, the types of DNA vaccines are numerous, of which suicide gene therapy is considered that one of the most promising cancer gene therapy at the present time. Suicide gene therapy of tumor (suicide gene therapy), also known as gene directed-enzyme prodrug therapy (GDEPT), is a new development gene therapy for solid tumors in recent years, exploit its killing effect on tumor cells and bystander effect to control the tumor. Herpes simplex virus type I thymidine kinase/ganciclovir (HSV-tk/GCV) system is a suicide gene therapy is the earliest studied, most mature, and most widely used. HSV-TK gene can specifically triphosphate non-toxic phosphate prodrug GCV into the high toxicity GCV, thereby inhibiting the activity of DNA polymerase, inducting anti-tumor effect.However, because the polymorphism, high cause tumor and low immunogenicity of tumor cells remains be an important reason which was difficult to overcome. How to break immune tolerance of tumor and immune escape mechanism of tumor cells, and activate the body's immune system to recognize and kill tumor cells, these which are the design of tumor vaccines need to address the fundamental problem. The heat shock protein (HSP) can combine with all non-self and self abnormal protein degradation products of cells, and form with the HSP peptide complexes (HSP-PCs) with all the characteristic fingerprint of the cells, so HSP-PCs are more much price, which is conducive to suppress the immune escape machinery, so as to enhance the immune function. Mycobacterium tuberculosis heat shock protein70 (mtHsp70) as a molecular chaperone process immune activational effect, mtHsp70 was not only as a carrier of antigen to antigen delivery to major histocompatibility complex (MHC) I and II molecules, but also can activate antigen presenting cells (dendritic cells DC), breaking the immune tolerance of tumor specific antigen. In addition, HSP70 can promote the transformation of Th cells to Th1 cells, adjust the immune status of cancer patients, and help control occur of the cancer.Another hindrance of cancer gene therapy is that it can not specifically transmit on anti-tumor gene to tumor tissues, which requires the establishment of an effective carrier system of gene that can divert the purposed gene to targeted cells. Viral carrier which is recently considerably much studies (eg adenovirus, retrovirus, adeno-associated virus, etc.), subsistent main drawback is that the most solid tumors exist the phenomenon of hypoxia will significantly weaken function of the virus carrier and expression of the purposed gene. The attenuated Salmonella are intestinal facultative anaerobes, the hypoxia phenomenon of solid tumor just provide a suitable place for growth and reproduction of the attenuated Salmonella; as the same time, The attenuated Salmonella is a kind of intracellular bacteria of invasion, primarily through M cells into the intestinal epithelium, that is swallowed by macrophages and dendritic cells into the lymph nodes, spleen and other organizations. These two characteristics provide possibility for the attenuated Salmonella as targeting carrier of oral tumor gene. And then it also has attacking to tumor cells, limited replication in tumor cells, and is easily cleared by antibiotics, with good biological safety.With more and more in-depth study of tumor gene therapy, single-gene therapy exposed the more and more deficiencies, the most important element is easy to cause drug resistance. To solve this problem, many scholars using combined gene therapy and have achieved fairly good results. But the majority are the form of the fusion gene, which exist many shortcomings, the uppermost defect is the instability of the fusion protein, it is prone to degradation, secondly fusion protein is not easy to by fold correctly, so it is difficult to ensure that all components remain maintain the original activity after the integration. Using attenuated Salmonella as a genetic vaccine vector, it can transfer the single gene into Salmonella, avoiding the mutual interference of the fusion gene when express the two types of genes; and it also make that the two types of gene product synergies, eliminating drugs tolerance of the tumor cells.To combine the above reasons, the subject use attenuated Salmonella typhimurium as carrier, immuno-adjuvant effect of Mycobacterium tuberculosis Hsp70 on the antigen and the killing effect of the suicide gene on tumor cellt and bystander effect, preparating DNA vaccines Oral of recombinant attenuated Salmonella:SL7207/mtHSP70/HSV-TK dual-gene co-expression, seting up the animal model of B16 mouse melanoma, exploit the prepared recombinant Salmonella Oral to treat bearing B16 mice, and observing the targeting to tumor, the inhibited effect on tumor and its interrelated mechanismThe study is divided into two chapters:the first chapter:the oral recombinant Salmonella carrying EGFP on tumor; the second Chapter:Inhibited effect of oral recombinant Salmonella carrying mtHSP70/HSV-TK to mouse melanoma.Chapter One:The targeted effect Oral recombinant Salmonella carrying EGFP on tumor1. Objective:To Study targeting of attenuated Salmonella carrying EGFP (SL7207/PIRES-EGFP) to tumor-tissue. 2. Methods:Using recombinant Salmonella with enhanced green fluorescent protein (EGFP) that has been prepared and identified by our discussion group to recovery, preparating the recombinant Salmonella (SL7207PCMV-/PIRES-EGFP) Oral that using EGFP as reporter gene.100μl of 109 CFU/mL recombinant salmonella typhimurium was given tomice bearing B16 melanoma mice by oral (gavage method). Through the frozen sections of tissue and tissue spare pulp count method of bacteria colony to observe bacterial distribution in tumor tissue and normal tissue at different time points.3. Result:Two days after the oral, there is a small number of colony growth in liver, spleen and tumor tissue's spare pulp. And four days after the oral, the number of colony growth of liver and spleen tissue's spare pulp increased more significantly than after two days; and tumor tissue were more significantly increased in colonies as liver and spleen tissue. However, to the seventh day, the number of colony growth of liver and spleen tissue's spare pulp significantly reduced; but bacterial colony of tumor tissue group in a large of numbers. At the same time, Frozen sections by fluorescence show the same results as tissue's spare pulp.Chapter Two:Inhibited effect of oral recombinant Salmonella carrying mtHSP70/HSV-TK to mouse melanoma. 1. Objective:To study the targeted effect of oral attenuated Salmonella typhimurium as carrier of Mycobacterium tuberculosis heat shock protein 70 (mtHSP70) and human herpes simplex virus thymidine kinase (HSV-TK) DNA vaccine of double-gene coexpression on mouse melanoma and the possible mechanism of the anti-tumor vaccines.2. Methods:Using identified recombinant Salmonella that our discussion group has had prepared earlier stage:SL7207/PCMV-mtHSP70-IRES-TK,SL7207/PCMV-mtHSP70-IRES-EGFP,SL7207/PCMV-TK-IRES-EGFP,SL7207/PIRES-EGFP, to recovery, and were prepared with attenuated Salmonella typhimurium SL7207 as a carrier for oral DNA vaccine. First of all, 0.1mL mouse melanoma B16 cells(cell concentration 107/mL) were subcutaneously inoculated into the back on the right side of C57BL/6J mice, so as to established animal model of mouse melanoma. When tumor volume reached about 100mm3, the C57BL/6J mice were randomly divided into five groups and each group hanve ten mices. for the experiment, with 10 tumor bearing mice for each group. For experimental group and control group, 100μL of 109CFU/mL recombinant salmonella typhimurium was given by oral (gavage method). The blank control group oral 100μL PBS. qd×2day.48h after the first oral, GCV 50mg/Kg in 0.5mL PBS was intraperitoneally injected, twice a day for five consecutive day.Each treatment course lasted for seven days. In total three successive treatment course were given. During treatment, each group was measured with vernier caliper once every three day. Calculating inhibitory rate based on measured tumor volume. At the end of the last treatment, five B16 bearing mice were sacrificed randomly from each group, The remaining tumor-bearing mice survival time for observation. Using HE staining, immunofluorescence measuring tumor tissue and surrounding changes in immune cells; under aseptic state getting immunized mice spleen preparated spleen cell suspension, LDH release assay checking CTL and NK activity; detecting IFN-γlevels of spleen cell supernatants.3. Result:Five groups of B16 melanoma in mice by recombinant Salmonella treatment three cycles, the tumor volume in each group were analyzed by Statistics, results showed that differences between the five groups possess significance (F=599.949, P=0.000), treatment group with different recombinant exist interaction between time (F=331.643, P=0.000); significant differences in tumor volume when comparing HT group to every control groups (P=0.000); H group and T group exist significant difference in tumor volume (P=0.000); H group and T group tumor volume difference was not statistically significant between Sa group and PBS group(P=0.398); significant differences in tumor volume when comparing Sa group to PBS group(P=0.000); In relation to PBS group, all recombinant Salmonella groups in the end of the third course of treatment, the inhibitory rates were:HT tumor inhibition rate was 78.47%, H tumor inhibition rate was 63.02%, T tumor inhibition rate was 61.63%, Sa tumor inhibition rate was 55.61%; with Log-Rank method proceed survival analysis showed that the median survival time:HT group was 35.60 days, H group was 31.17 days, T group was 31.50 days, Sa group was 29.73 days, PBS group was 27.53 days; survival rates between five groups exist differences (x2=12.591, P=0.013), HT group survival was significantly longer than the other groups (P＜0.05). LDH release assay results showed that:in a different effective target, the difference of CTL killing activity between five groups was significant (x2=11.867, P=0.018), the order of HT group, H group, T group, Sa group, PBS group in turn are:13.07,10.33, 8.33,5.67,2.00, the difference of CTL killing activity between different effector to target ratios was not significant (x2=1.820, P=0.403); the difference of NK killing activity between five groups was not significant (x2=7.121, P=0.130), the differences between different effector to target ratio was significant (x2=6.316, P=0.043), effector to target ratio of 50:1,25:1,12.5:1, the order in turn are:11.60,7.90,4.50. Elisa assay was performed to detect IFN-r concentration in tumor tissue of bearing mice after treatment of recombinant vaccine, the resuls ware as follow:all groups does not meet homogeneity of variance, so, with welch way:the overall difference between groups was significant (F=54.441, P=0.000), further using Dunnett T3 compared to two groups:difference in HT group, H group, T group between PBS group was significant (P＜0.05), HT group, H group, T group, Sa group group There was no significant difference (P＞0.05). HE staining and immunofluorescence results showed that:the tumor tissue of tumor-bearing mice that obtain immune by recombinant treatment was clearly necrotic, in tumor tissue and it's circumambience which there are a lots of inflammatory cells, CD8+T lymphocyte infiltrated into.Conclusion:(1) Oral attenuated Salmonella typhimurium SL7207 is targeting to tumor tissue and can express the target gene in tumor cells, is an ideal tumor targeting gene therapy vectors.(2) Oral recombinant Salmonella (SL7207/mtHSP70/HSV-TK) can induce CD8+ T cells to produce specific cellular immunity, activated CTL and NK cell activity, promot spleen cells to perspire IFN-y, inhibit C57BL/6J mice B16 tumor growth and prolong B16 mouse survival.
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