Study For Human Choroidal Melanocyte Regulating Extracellular Matrix Remodeling

Purpose: The remodeling of uveal-sclera extracellular matrix is very important for many ocular diseases, such as prostaglandin F_2α decreasing intraocular pressure and myopia development. This process had been proposed to be regulated by scleral signal transduction mechanism. By exploring matrix metalloproteinases (MMPs) induction mechanism, we could be able to better understand the eyeball growth regulation, which would be helpful on understanding many eye diseases development. In this study, we investigated choroidal melanocyte expressing matrix metalloproteinases, induced by the relative singal transduction factors. This would help us to better understand the prostaglandin F_2α decreasing intraocular pressure, and to better undstand the retinal signals regulating the eyeball growth procedure during the myopia development.Materials and methods1. Human choroidal melanocytes were isolated by trypsin-collagenase digestion to the fresh human eye ball. Melanocytes were isolated and purified within F12 medium supplemented with fetal bovine serum, basic fibroblast growth factor, isobutyl methylxanthine and cholera toxin. Transmission electron microscope and immunochemistry were used to distinguish the characteristics of human choroidal melanocytes.2. Cultured choroidal melanocytes were randomly divided into three groups: control, PGF_2α treatment, and TGF-β₂ treatment. The PGF_2α concentration series were 0.1μg/ml, 1μg/ml, 10μg/ml, 100μg/ml;and TGF-β₂ concentration series were 0.1ng/ml, 1ng/ml, 10ng/ml, 100ng/ml. The morphology of three group cells was observed by inverse phase microscope, the number of melanocytes was counted by cytometry, and the activity was detected by MTT assay.3. Cultured choroidal melanocytes were randomly divided into three groups: control, PGF2a treatment, and TGF-P2 treatment. The extreme concentration of PGF201 was lOug/ml, and the one of TGF-p^was lng/ml. The treated cells were labeled with anti-matrix metalloproteinease-1, 2, 3, 9, tissue inhibitors of metalloproteineases-1, 2 IgG, the morphology was observed on the laser confocal microscope. Cell extracts were subjected on the western blot assay to compare the target protein expressing level.Results1. Separated choroidal melanocytes showed as brown spheroplasts, and anchored in 24h. Anchored cells spread in bipolar, tripolar or dentritic morphology. Yellow-brown pigment granules contained in the cytoplasm, and melamin content remained stable after several passages. Ultrastructurally, sparing microvillus existed on the surface of cells, and melanosomes in the cytoplasm displayed along membrane or in clump. 99% of culture-purified cells were positively labeled with anti-S-100 IgG, but negatively stained by anti-cytokeratin IgG, indicated that signal in this subsequencal detection was yielded by the melanocyte.2. No morphological change was found by inverse phase microscope after addition of PGF2a, so did the number and activity of melanocytes (P>0.05). After TGF-P2 treatment, the growth of melanocytes was partly affected, the number and activity showed decreasing, the difference had statistic significance (P<0.05 or P<0.01).3. Western blot and immunocytochemistry detection showed that non-treated choriodal melanocytes could express the low level MMP-2 only. With the PGF2tt induction, anti-MMP-1, 2, 3, 9 antibodies yield positive signals, among which the MMP-1 upregulating was the strongest. TGF-02 could induce the choriodal melanoctyes to express the MMP-2, 3, 9, but no TIMP-1, 2 signals could be determined.Conclusions1. PGF2a had no effect on the growth of human choroidal melanocytes in vitro, andTGF-02 inhibited the growth of human choroidal melanocytes in vitro.2. Some type of MMPs could be expressed by cultured choroidal melanocytes;while TIMPs could not be expressed. PGF2U could upregulate the expression of MMP-1, 2, 3, 9 in choroidal melanocytes in vitro. Addition of TGF-02 to the culture medium caused an increase of production of MMP-2, 3, 9 by cultured choroidal melanocytes.3. Choroidal melanocytes could produce certain subtypes of MMPs, which was regulated by the signal factors. This character would be altered within different diseases. Choriodal melanocytes would take part in the uveal-sclera extracellular matrix remodeling regulating pathway, which would become the important partner of the cellular therapeutic approaches.