Molecular Pathologic Studies In Uveal Melanoma And Primary Culture Of Uveal Melanoma Cells

Objectives: To investigate the mutation of BRAF gene in uveal melanoma. Study the expression of MMP-2, MMP-9 and ^p-ERK1/ERK2 in uveal melanoma and the relationship between their expression intension and prognosis. To culture human uveal melanoma cells and study their biologic characteristics.Methods: The mutation of BRAF gene (exon 15) in 52 specimens of uveal melanoma were screened with PCR-SSCP technology and the PCR products of BRAF gene were gel purified and sequenced on ABI 3000 to confirm the presence of mutation. The protein expression of MMP-2, MMP-9 and ^p- ERK1/ERK2 in uveal melanoma were checked with SP method of immunohistochemistry in 52 specimens of uveal melanoma. According to different indicators of prognosis, all cases were classified into different groups and the difference of expression intension was analysed with Image-Pro Plus system. The human uveal melanoma cells derived from 4 patients with uveal melanoma were primarily cultured with different methods and confirmed by S-100, HMB45 immunohistochemistiy stain. The growth conditions of uveal melanoma cells were observed throughinvert microscope and the growth curve was made out by MTT method. Results: (1) No mutation of BRAF gene(exon 15) were checked out from all the 52 specimens of uveal melanoma. (2) The positive expression rate of MMP-2 > MMP-9 and P"ERK1/ERK2 in 52 specimens of uveal melanoma was separately: 88. 5%, 100% and 92. 3%. (3) The expression intension of MMP-2 > MMP-9 and P'ERK1/ERK2 was related to the largest tumor diameter(Ltd), tumor invasion to sclera ang cell type (PO.05) . The expressin intension was higher in specimens with larger Ltd and positive invasion of sclera than those with smaller Ltd and negative invasion of sclera (P<0.05) . Epithelioid type showed higher expressin intension than other types (P<0.05). (4) The human uveal melanoma cell was successively cultured and was confirmed by S-100, HMB45 immunohistochiemistry stain. (5) In vitro, the cultured human uveal melanoma cells showed three types: spindle type, polygon type and epithelioid type. Melanin was found in cytoplasm. The cell growth curve showed as "S" type. Conclusion: (1) Uveal melanoma lack mutation of BRAF gene and mutation of BRAF gene has nothing to do with the pathogenisis of uveal melanoma. (2) The positive expression rate of MMP-2, MMP-9 and P"ERK1/ERK2 in uveal melanoma is high. Their expression intension relates to the pathogenisis and prognosis of uveal melanoma and they can indicate the pathogenisis of uveal melanoma. (3) The human uveal melanoma cells are successively cultured and they are ideal experimental model for to us study the pathogenisis of uveal melanoma.