Establishment Of Highly Metastatic Models Of Melanoma In Mice In Vivo Screening And Cell Lines

Objective: To establish highly metastatic murine and hominal melanoma models by means of selection in immunodeficiency mice vivo and tumor resection, observing the correlated bionomics and investigate their biological characteristics so as to provide a useful animal model for the study of tumor precaution, diagnosis and the treatment of cancer.Methods: 1. Establishment of a highly metastatic model of murine melanoma in mice in vivo screening and cell line: Histologically melanoma B16 intact tissues were subcutaneously implanted into BNX mice with immunodeficiency. Firstly three generatin screening were developed according to the method of subcutaneous implantation→tumor resection→lung metastasis→subcutaneous inoculation.Then screening went on in last three generation.to estabalish a high metastastic spontaneous tumor model,observing growth ,lung metastasis and pathohistology of tumor.Used primary culture to establish a high metastastic cell line,observing morphology,growth velocity, Chromosome analysis ,flow cytometry, basement membrane invasion assay .Then the tumor growth status and lung metastastic were observed after tansplanting the cell line subcutaneouly.2. Establishment of a highly metastatic model of murine melanoma in mice in vivo screening and development of lung metastasis:One of six has lung metastasis , we tansplanted visible metastatic focus into SCID mice.then according to the method of subcutaneous implantation→lung metastasis→subcutaneous inoculation,to establish a highly metastatic model of human melanoma in SCID mice in vivo selection and correspond cell line,which is by haema and lymphatic metastasis ,observing cell line and studying their biological characteristic. Meanwhile we dynamicly detected the micrometastasis by the technology of molecular biology after inoculating tumor into BNX mice by Alu-PCR. Results: 1. Establishment of a highly metastatic model of murine melanoma in mice in vivo screening and cell line: We successfully established a highly metastasis melanoma B16 model by the exairesis thrice of neoplasm and selection in immunodeficiency mice vivo.Tumor can growth subcutaneouly at 100%.At 35 days ,lung metastasis canbe sdetected at 91.7(11/12),metastasisi degree achieved at +++ .Meanwhile a highly metastastic cell line named B16-sci were established,which showed doubling generation time at 46.07h, significant lower apoptosis and better invasion than B16 cell.Its chromosome analysis indicated that its chromosome has the characteristic of murine malignant tumor.In vivo assay indicateded that tumor can growth subcutaneouly at 100% after injection of B16-sci cell subcutaneously,and lung metastasis can be detected at 100%(6/6).At the same time, the potential lung metastasis were elevated after injecting B16-sci into C57BL/6J subcutaneously. Then after 3 generations in vivo without exairesis, the carcinoma metastasis rate accounted to 91.7 percent(11/12), metastasis extent reached +++ after 35 days. It indicated that B16-sci has similar characteristic with B16 by the assay about histology, cell growth curve,flow cytometry , Chromosome analysis and basement membrane invasion. 2. Establishment of a highly metastatic model of murine melanoma in mice in vivo screening and development of lung metastasis: Successfully established a mice model with high metastasis in lung and lymph node, decurtating incubative stage from 7 or 14 days to 5days, survival life from 60 or 75 to about 45 days. The neoplasm of the SCID mice for selection all well developed survival after subcutaneouly inoculation in 243 SCID mice .And the rate of lung metastasis were sublimated in the screening generation one by one. Meanwhile a highly metastastic cell line named SCI-375 were established, showed doubling generation time at 21.60h, better invasion thanA375 cell.Its chromosome analysis indicated that its chromosome has the characteristic of hominal malignant tumor. In vivo assay indicateded that obviously metastasis can be found in different immunodeficiency mice, such as BNX mice at 100% (13/13), nude mice at 90.91%(10/11). Alu-PCR showed that none lung metastasis can be detected at 2 weeks.Lung metastsis were can detect at (37.5%)3/8,(75%)6/8,(100%)10/10 by Alu-PCR,at (0%)0/8,(37.5%)3/8,(50%)5/10 by pathology.In thus,micrmetastasis of SCI-375 in lung of BNX mice after 3 weeks, which has more sensitively than pathology. Conclusions: 1. we successfully established a highly metastasis melanoma B16 model and homologous cell line, which was high, steady. 2. We observed the high metastasis mouse model of human melanoma and homologous cell line, which have two metastatic path of blood and lymph fluid. And it was applied in nude mouse. By amplification of specific gene, we reviewed the dynamic state of SCI-375 micrometastasis in BNX mice. 3. The result provided a useful tool for the study of metastatic mechanism and treatment of carcinoma.