ABSTRACT
Objective To investigate the effects of down-regulation of polo-like kinase-1 (PLK1) gene by RNA interference (RNAi) on the invasion of a human malignant melanoma cell line A375 and their possible mechanisms.Methods Cultured A375 cells were classified into several groups:blank control group receiving no treatment,liposome group transfected with lipofectamine only,and three siRNA groups transfected with three concentrations of a small interference RNA (siRNA) targeting PLK1 respectively.After additional culture,real time quantitative PCR and Western blot analysis were performed to quantify the expressions of PLK1 mRNA and protein in A375 cells respectively,Transwell invasion assay to evaluate the invasive capacity of A375 cells,agarose gel electrophoresis and terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labelling (TUNEL) to detect anoikis in A375 cells.The colony-forming capacity was also evaluated for A375 cells.Statistical analysis was carried out by one-factor analysis of variance.Results There was a significant decrease in PLK1 mRNA and protein expressions as well as in colony-forming units in the siRNA groups compared with the blank control group (all P ＜ 0.05).The invasive capacity of A375 cells was significantly inhibited in the siRNA groups with the number of migrating cells in Transwell assay being 39 ± 5,19 ± 5 and 9 ± 3 in A375 cells transfected with 3.125,6.250 and 12.500 nmol/L siRNAs respectively,compared to 56 ± 5 in the blank control group (all P ＜ 0.05).A characteristic DNA ladder was observed on agarose gel electrophoresis in the siRNA (6.250 nmol/L) group.Compared with the blank control group and liposome group,the three siRNA groups showed increased apoptotic index (3.86％ ± 0.35％ (3.125 nmol/L siRNA),7.35％ ± 0.36％ (6.250 nmol/L siRNA) and 17.56％ ± 0.38％ (12.500 nmol/L siRNA) vs.1.15％ ± 0.25％ (blank control group) and 1.18％ ± 0.22％ (liposome group),all P ＜ 0.05).Conclusions PLK1 siRNA can inhibit the invasion of malignant melanoma cells,likely by inducing anoikis in these cells.
ABSTRACT
Objective To study the effects of bFGF on the proliferation of cultured human melanocytes, and to seek a quick method for in vitro culture of human melanocytes. Methods Melanocytes were isolated from human foreskin, and divided into two parts to be cultured with or without the presence of bFGF (0.3 μg/L). Second-passage melanocytes were identified with immunochemical stain. The growth of melanocytes was observed every 3 days for 12 days. Third-passage melanocytes were treated with various concentrations (0.3 - 2.1 μg/L) of bFGF for 72 hours followed by the detection of proliferation of and trosinase activity in melanocytes. Results Human melanocytes were obtained from primary culture in medium containing certain concentrations of bFGF, which were identified with immunohistochemical stain. The morphology of cultured melanocytes varied with growth stage of cells. The bFGF-treated melanocytes appeared to grow more rapidly than untreated melanocytes. Further more, a significant increase was observed in the proliferation rate of melanocytes treated with bFGF of 0.3 and 0.6 μg/L (P＜0.05 or 0.01 ) and tyrosinase activity in melanocytes treated with bFGF of 1.5 and 1.8 μg/L (P ＜ 0.05 or 0.01 ) in comparison with the untreated melanocytes.Conclusions The addition of certain concentrations of bFGF to defined medium can benefit the primary culture of melanocytes and make it possible to get large quantities of purified melanocytes with high viability in short periods. Certain concentrations of bFGF can up-regulate the proliferation of and tyrosinase activity in melanocytes.
ABSTRACT
Objective To study the in vitro culture condition for melanoblasts from human foreskin tissue. Methods The skin tissue taken from foreskin of children was treated with 0.5% dispase Ⅱ to separate epidermis from dermis, then with trypsin to obtain single cell suspension, which was cultured in modified medium for melanoblasts, i.e., MCDB254 medium supplied with several cell growth factors. Finally, melanoblasts were obtained based on the difference of adhesion speed. The morphology and proliferation of cultured melanoblasts were observed under a light microscope. DOPA staining, immunostaining with anti- S-100 and -tyrosinase related protein 2 (TRP2) antibodies, and transmission electron microscopy were per- formed to identify the cultured melanoblasts. Results The cultured human melanocytes displayed a match-like shape, scattered arrangement, syrmnetric double poles, slim cell body, highly refractive nuclei; meanwhile, the melanoblasts exhibited plentiful cytoplasm, large volume, bipolar or irregular shape and clonal growth. Additionally, the melanocytes were positive for TRP2, S-100 and Dopa staining, while the melanoblasts were positive only for TRP2. Electron microscopy revealed the presence of mature melanin granules (stage Ⅲ-Ⅳ ) in melanocytes but immature melanin granules (stage Ⅰ ) in melanoblasts. Conclu- sion Stable pure culture of melanoblasts has been realized with the reformed medium, which may lay a foundation for the investigation into the mechanism of epidermal pigmentation.