[Effects of cisplatin on telomerase activity of human choroidal melanoma cells].

OBJECTIVE: To study the effects of CDDP (cis-dichlorodiamine platinum) on the telomerase of human choroidal melanoma cells and to investigate the toxic effects of CDDP on these cells. To study the relationship between these two effects and to explore the possibility of using CDDP in the chemotherapy of choroidal melanoma. METHODS: It was an experimental research. CDDP was added to the culture medium of primary cultured human choroidal melanoma cells at different concentrations (0.01, 0.1, 1, 10 and 100 mg/L, 72 h) and times (12, 24, 48, 72 and 96 h, 10 mg/L) and the results were compared with that of the controls. Toxic effects of CDDP were evaluated by MTT test and the level of telomerase was measured by PCR-ELISA assay. The correlation between these two effects was analyzed. RESULTS: The telomerase activity was inhibited by CDDP time dependently and dose dependently. Cell viability was decreased when the concentration of CDDP attained 0.1 mg/L and acted for 24 hours. The cell toxicity of CDDP was correlated negatively (r = -0.900, P = 0.037) with the inhibition of telomerase. The cell death was lagged behind the decrease of telomerase. CONCLUSIONS: CDDP is an effective telomerase inhibitor which can decrease the telomerase activity of cultured human choroidal melanoma cells significantly. This effect is dose and time dependent. CDDP can also cause the death of cultured melanoma cells.

[A preliminary study in establishment of mice model of experimental uveal melanoma].

OBJECTIVE: To explore the methods to establish a nude mice model of experimental uveal melanoma by the implantation the primary cultured cells into anterior chamber or subcutaneous injection. METHODS: 35 nude mice, which applied with the SPF grade standard by national healthy department, were divided into 3 groups: anterior chamber group (15), subcutaneous A group (10) and subcutaneous B group (10), respectively. (2 - 3) x 10(5) melanoma cells were inoculated into the anterior chamber of each nude mouse, while 2 x 10(6) cells were inoculated into every subcutaneous of the hind legs in A group. Besides this, the fresh mass of the tumor come from enucleated eye were transplanted into every subcutaneous of the hind legs in B group. Then the condition of transplanted tumor were observed under the slit lamp and naked eye, all nude mice were followed for 3 months the rates of tumor induction were compared. RESULTS: The results of the rates of tumor induction in the 3 groups were 7 (46.6%) of 15, 2 (20%) of 10 and 0 of 10, respectively. The discrepancy were of significance (chi(2) = 7.080, P = 0.029). CONCLUSIONS: Uveal melanoma can be induced successfully by inoculation the primary cultured human uveal melanoma cells into anterior chamber or subcutaneous in nude mice. The results suggest that the ration of the tumor growth is higher in subcutaneous mass transplant than any other groups.

[Effect of doxycycline on inflammation-related cytokines and apoptosis in human conjunctival epithelial cells].

OBJECTIVE: To evaluate the effects of doxycycline on the regulation of intercellular adhesion molecule-1 (ICAM-1, CD54), interleukin-1beta (IL-1beta), human leukocyte antigen DR (HLA-DR) and apoptosis in human conjunctival epithelial cells. METHODS: Human primary conjunctival epithelial cells were isolated and cultured from donors and identified by immunohistochemistry. Cultured epithelial cells were treated with either 0 U/ml IFN-gamma, 300 U/ml IFN-gamma, 300 U/ml IFN-gamma with 10 microg/ml doxycycline, 300 U/ml IFN-gamma with 20 microg/ml doxycycline, 300 U/ml IFN-gamma with 40 microg/ml doxycycline or 300 U/ml IFN-gamma with 100 microg/ml dexamethasone for 24 hours. The amount of CD54, HLA-DR and IL-1beta was measured by flow cytometry and western blot analysis. Apoptosis was evaluated by flow cytometry after the cultured epithelial cells were treated for 72 hours. RESULTS: Cultured conjunctival epithelial cells can express CD54 and IL-1beta. IFN-gamma increased the amount of CD54 and IL-1beta (P < 0.01). Doxycycline and dexamethasone inhibited the IFN-gamma induced increase of express of CD54 and IL-1beta of cultured conjunctival epithelial cells, and the inhibiting effect was dependent on the concentration of doxycycline (P < 0.01). Very little HLA-DR and apoptosis were detected before and after treatment with IFN-gamma. CONCLUSION: Doxycycline can suppress the expression of inflammatory cytokine such as CD54 and IL-1beta, which suggests that doxycycline may be a potent drug for the treatment of ocular surface inflammatory disease.

[Experimental study on the effects of interferon-gamma gene on Tenon's capsule fibroblasts in vitro].

OBJECTIVE: To investigate the effects of interferon-gamma (IFN-gamma) gene on Tenon's capsule fibroblasts in vitro. METHODS: Using lipofectAMINE, IFN-gamma gene was transferred to human Tenon's capsule fibroblasts with plasmid pcDNA3IFN-gamma in vitro. Cells treated with plasmid pcDNA3 without IFN-gamma gene were used as the controls. The transfected fibroblasts were screened by G418. The expression of IFN-gamma was determined by RT-PCR, immunohistochemistry and flow cytometry assay. The effects of IFN-gamma on the proliferation of Tenon's capsule fibroblasts were evaluated by flow cytometry and MTT. RESULTS: The Tenon's capsule fibroblasts transferred the IFN-gamma gene could express the IFN-gamma RNA and protein transiently. The proliferation of the fibroblasts transferred the IFN-gamma gene was inhibited. CONCLUSION: The proliferation of the Tenon's capsule fibroblasts in vitro can be inhibited by transferred IFN-gamma gene.

[Histopathological and ultrastructural characteristics of oil-associated complications in silicone oil-filled human eyes].

OBJECTIVE: To observe morphological changes of oil-associated complications in silicone oil-filled human eyes, and to further explore their pathogenesis. METHODS: The morphology analysis and immunohistochemistrical study were performed in 25 specimens including 8 eyeballs, 1 ocular content, 4 preretinal membranes, 4 corneal buttons and 8 lens from human eyes with silicone oil tamponade. Two of preretinal membranes acquired freshly were also evaluated by transmission electron microscopy (TEM). RESULTS: Endothelium cell loss (90%) and band keratopathy (83%) were the most typical changes in silicone oil-associated keratopathy;while epithelial cell fibrosis was the most frequent histopathological features in silicone oil-associated cataract. In 8 eyeballs and 1 ocular content, it was found that damages to normal retinal layers and formation of preretinal or subretinal membrane with extensive silicone bubbles were obvious in the cases of silicone oil-associated retinopathy, which included loss and degeneration of neuron cells. Moreover, in 3 eyeballs with silicone oil for more than 60 months, retinas were completely replaced by fibril membranes, and the oil vacuoles were also found in sclerocorneal scar, trabecula, iris, ciliary body, choroid, optic nerve and its tunica vaginalis. These finding demonstrated that the longer the silicone oil was retained in eyeballs, the more severe its complications were. Different sizes of silicone bubbles in 2 preretinal membranes were noted easier by TEM than light microscopy. There were some macrophages marker (CD68) positive staining cells in the tissues filled with silicone bubbles, such as preretinal or subretinal membrane and optic nerve. Partial of the membranes surrounding the oil bubbles was positive for GFAP staining, and other part was positive stained for Vimentin. CONCLUSIONS: Intraocular silicone oil can damage the normal tissue structures and function if it is retained in eyeballs too long. This results suggest that silicone oil should be removed timely after the retinal reattachment stabilized and can not be used as a kind of long term intraocular tamponade.

[Effect of bcl-2 antisense oligonucleotides on multidrug resistance of cultured uveal melanoma cells].

OBJECTIVE: To evaluate the chemotherapeutic sensitivity of uveal melanoma in vitro and reverse its drug resistance by the delivery of bcl-2 antisense oligodeoxynucleotide (AS-ODN). METHODS: The drug sensitivity tests of primary cultured uveal melanoma cells toward 5-flurouracil (5-FU), thiophosphamide (TSPA), cisplatin (DDP), adriamycin (ADM), vinblastine (VLB), dacarbazine (DTIC) were performed with 3, -4, 5 dimethyliazol-2,5 diphenyl tetrazolium bromide (MTT). AS-ODN bcl-2 were delivered with cationic lipid to down-regulate bcl-2 expression. Immunohistochemistry and Western-blot methods were used to detect bcl-2 expression. According to the principle of multi-drug mutual action, the influence of anti-apoptosis gene bcl-2 on tumor cell drug sensitivity was measured. RESULTS: Uveal melanoma was resistant to different chemotherapeutic drugs in different degrees. AS-ODN bcl-2 could down-regulate bcl-2 expression, was synergetic with all tested drugs and partially reversed the multi-drug resistance. CONCLUSIONS: Clinically, with ordinary dosages the above 6 drugs have little cytotoxicity to uveal melanoma cells, bcl-2 over-expression is associated with multi-drug resistance and AS-ODN bcl-2 can partially reverse the multi-drug resistance.

[The inhibition effect of interleukin-1 receptor antagonist on interleukin-1 alpha-induced intercellular adhesion molecule-1 expression on orbital fibroblasts and adhesion of peripheral blood mononuclear cells].

OBJECTIVE: The aim was to identify whether interleukin-1 receptor antagonist (IL-1ra) inhibits interleukin-1 alpha (IL-1 alpha)-induced intercellular adhesion molecule-1 (ICAM-1) expression on cultured orbital fibroblasts, and adhesion of peripheral blood mononuclear cells (PBMC) to orbital fibroblasts, and to investigate the clinical application potential of IL-1ra in the treatment of Graves' ophthalmopathy (GO). METHODS: Cultured orbital fibroblasts from patients with GO and controls were challenged with IL-1 alpha or/and IL-1ra. Immunocytochemical staining was used to examine the changes of ICAM-1 in response to IL-1ra treatment; fluorescent photomicroscope was used to measure the adhesion between the labeled PBMC and orbital fibroblasts. Neutralizing antibody against ICAM-1 was used to demonstrate the role of ICAM-1 in the IL-1 alpha-induced adhesion. RESULTS: IL-1ra inhibits IL-1 alpha-induced ICAM-1 expression in cultured orbital fibroblasts both from GO patients and controls; IL-1ra inhibits IL-1 alpha-induced adhesion of PBMC to orbital fibroblasts in a concentration and time dependent manner. Moreover, a monoclonal anti-human ICAM-1 antibody produced a concentration dependent inhibition of the IL-1 alpha-induced adhesion of PBMC to the fibroblasts. CONCLUSIONS: IL-1ra inhibits IL-1 alpha-induced ICAM-1 expression in cultured orbital fibroblasts and the adhesion of PBMC to fibroblasts. IL-1 alpha-induced ICAM-1 expression may play an important role in the adhesion process. IL-1ra may be useful in the prevention or treatment of GO.