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The background and purpose of malignant melanoma is a derived from melanocytes, the nevus cells highly malignant, occur in the skin, accounting for a third of all skin malignancies, malignant melanoma, a high degree of malignancy, prone to blood metastasis. Malignant melanoma is usually used treatments include surgical removal, biological therapy, radiation therapy and chemotherapy, the effect is not ideal. Poor efficacy of traditional chemotherapy for malignant melanoma, the dacarbazine (dacarbazine, DTIC) remains the most effective drugs, its efficiency by nearly 20%, and cyclophosphamide (cyclophosphamide, CTX) effective rate of only 16% . Therefore, the search for good efficacy, toxic side effects of new treatments and drugs are the hot issues of the tumor scholars. Arsenic trioxide is the main ingredient of traditional Chinese medicine arsenic, its good role not only in the treatment of leukemia, but also have a significant role in the treatment of liver cancer, colon cancer, bladder cancer and other solid tumors. In recent years, arsenic as the research and application of anti-malignant melanoma drug has also been in-depth development, proven to inhibit the proliferation of arsenic trioxide on malignant melanoma A375 cells and B16 cells. α-interferon (interferon-α, IFN-α) is a strong anti-malignant melanoma activity of biological factors, their biological effect on malignant melanoma, including the surface tissue of the anti-cell proliferation and upregulation of MHC (Major histocompatibility complex, MHC) immune function. The combination of chemotherapy and immunotherapy in the treatment of malignant tumors, often compared with the effect of monotherapy. Arsenicals in combination with interferon for the treatment of malignant melanoma has not been reported. To this end, the purpose of this study is to establish a malignant melanoma B16 cells C57BL / 6 mice were tumor-bearing animal models, in vivo observation of arsenic trioxide in combination with interferon inhibition of malignant melanoma, and explore arsenic trioxide interferon resistant malignant melanoma possible mechanisms to provide experimental basis for clinical application. Method 1. Preparation of malignant melanoma B16 cell suspension. Henan Institute of Medical Sciences after stable passaged subcutaneously inoculated with malignant melanoma B16 cells 14 days of C57BL / 6 mice with tumor subcutaneously under sterile conditions from stripping tumor take was excluded fiber package film and necrotic tissue, take the good growth of the tumor tissue, and minced in a sterile petri dish, add appropriate amount of saline in the opposing tissue grinder, gently ground, a 200-mesh nylon mesh filter, to prepare a tumor cell suspension. The plate count of 0.4% trypan blue staining, cell count, cell viability greater than 95%, sterile saline adjusted the cell concentration of 5 × 10 6 cells / mL. 2. C57BL / 6 mice with malignant melanoma B16 cells inoculated, grouping and processing. (1) 50 mice with the tumor cell suspension 0.2mL (1 × 10 ~ 6 unit) containing tumor cells inoculated in the right axillary subcutaneous vaccination were randomly divided into five groups of 10 each. Grouped as follows: ① the Arsenic Trioxide group: intraperitoneal injection of arsenic trioxide 0.1mg/20g/d administered for 10 consecutive days; ② interferon group: 50000 U/20g/d subcutaneous injection of interferon, administered for 10 consecutive days; (3) the combination group above: arsenic trioxide and interferon dose, administered for 10 consecutive days; the ④ positive control group (CTX group): injection of CTX 100mg/kg/d only the first day of the administration; ⑤ negative control group: injection capacity sterile saline injection of 10 consecutive days. Daily observation of the growth of the mice in each group, every other day, weighing, recording the changes in body weight of mice in each group during administration of. Day after stopping mice were sacrificed, peeling tumor tissue weighed and retention of tumor tissue. Tumor inhibition rate = (negative control group average tumor weight - treatment group, the average tumor weight) / negative control group, the average tumor weight × 100% formula inhibitory rate was calculated. The above reservation tumor HE staining to observe the morphological changes of the pathology of the tumor tissue of mice in each group. (2) Another 50 mice inoculated with (1) the same method and grouping, the daily observation of the growth of mice in each group, and record the time of the natural death of the mice, the average survival time of each group of mice is calculated period. 3. Statistical analysis. The application of statistical software SPSS11.5 statistical analysis, statistical methods using t test and χ ~ 2 test. Α = 0.05 as the level of inspection. Results 1. Arsenic trioxide interferon on mice with malignant melanoma tumor weight inhibition of negative group 0.6532 ± 0.1562g, arsenic trioxide group tumor weight of .4055 ± 0.1287g, the inhibition rate was 37.92%; interferon group, tumor weight 0.5001 ± 0.0538g, the inhibition rate was 23.34%; tumor weight of the combined group is 0.3756 ± 0.1953g, the inhibition rate was 42.49%; the CTX group of tumor weight is .3435 ± 0.0843g its inhibition rate was 47.41 %. Arsenic trioxide group compared with the inhibition rate of interferon group, the difference was statistically significant (P <0.05). Combined treatment group compared with the inhibition rate of interferon group, the difference was statistically significant (P <0.05); combination group than the inhibitory rate of arsenic trioxide group and the positive control group, but no statistically significant difference (P> 0.05 ). 2. Arsenic trioxide group, compared to the combined treatment group and CTX group and negative control group tumor late, slow tumor growth. Arsenic trioxide group, the average survival period of the interferon group, the combined treatment group, CTX group and negative group were 32.1 ± 4.3 days, 26.9 ± 3.5 days, 37.2 ± 5.1 days, 29.0 ± 3.9 days, 23.1 ± 3.6 days. The survival time of the combination group compared with arsenic trioxide group, the interferon group and CTX group, the difference was statistically significant (P <0.05), the combination group prolonged survival of tumor-bearing mice are the most obvious. 3. Mouse malignant melanoma B16 cells in solid tumors is spherical nodular, tender, fish-shaped cut surface. The negative control group, tumor volume was significantly greater than the arsenic trioxide group, the combined group and the positive control group, the tumor showed mostly invasive growth, and some even invasion to sternum, clavicle, etc., and the surrounding tissue boundaries unclear, it is difficult to peel. Arsenic trioxide group and combined treatment group of mice the tumor is smaller, regular shape, easy to peel. Compared with other groups of mice, arsenic trioxide group and combined group the number of tumor blood vessels in mice was significantly less. The negative control group, tumor cells showed diffuse-like distribution, the cells were round, clear outline, and a large, prominent nucleoli, stromal vascular. Compared with the negative control group, arsenic trioxide group and combined treatment group tumor tissue necrosis, tumor cells karyopyknosis changes in cell morphology; interferon group no significant changes in cell morphology. Conclusion 1. The antitumor effect of arsenic trioxide on mouse melanoma B16 cells, but the tumor inhibition rate compared with CTX did not differ significantly. 2. The interferon inhibitory effect is small, but some prolong survival time. 3. Arsenic trioxide interferon on mouse malignant melanoma B16 cells in vivo antitumor effect, and inhibition rate of CTX group was not significantly different, but it can significantly extend the average survival time of tumor-bearing mice, and well tolerated .
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