ABSTRACT
Objective Studies are rarely reported on the effect of short peptides of the pigment epithelium derived factor (PEDF) on the proliferation of human cutaneous squamous (SCL-1) cells. The purpose of this study is to investigate segmented cloning and expression of the PEDF protein and observe its effect on the proliferation of human SCL-1 cells. Methods The target genes of PEDF1, PEDF2 and PEDF3 were amplified by PCR and the recovered fragments subjected to double digestion of NheⅠ and Hind Ⅲ and inserted into the pET28a(+) plasmid. The product was transformed into human E coli BL21 and induced to express, followed by isolation and purification of the fusion protein. CCK-8 assay was used to detect the proliferation of the SCL-1 cells with PEDF1, PEDF2 and PEDF3 at 100, 400, 800 and 1000 nmol/L at 24, 48 and 72 hours. Results The prokaryotic expression vectors of PEDF1, PEDF2 and PEDF3 were successfully constructed, and their fusion proteins prepared, with the molecular weight of 18 000, 17 000 and 13 000, respectively. The proliferation of the SCL-1 cells was significantly decreased in the 800 and 1000 nmol/L PEDF3 groups compared with that in the 0 nmol/L PEDF3 group at 24 hours (0.16 ± 0.03 and 0.78 ± 0.07 vs 1.00 ± 0.00, P < 0.05), inhibited in a concentration-dependent manner in the 400, 800 and 1000 nmol/L PEDF3 groups at 48 hours (P < 0.05), markedly lower in the 800 and 1000 nmol/L PEDF3 groups at 72 hours (0.53 ± 0.05 and 0.51 ± 0.05) than in the in the 400 and 0 nmol/L PEDF3 groups (0.60 ± 0.05 and 1.00 ± 0.00) (P < 0.05). Conclusion The PEDF fusion proteins were successfully segmentally cloned and expressed and PEDF3 inhibited the proliferation of SCL-1 cells, which has paved the ground for further screening of active functional short peptides of PEDF.
ABSTRACT
<p><b>OBJECTIVE</b>To further confirm release of adenosine triphosphate (ATP) from cultured marginal cells in vitro of stria vascularis in neonatal rat, and to explore the mechanism of ATP release from marginal cells.</p><p><b>METHODS</b>Isolation and in vitro culture of marginal cells of neonatal rats' cochlea. ATP released by marginal cells in extracellular fluid were detected using bioluminescence assay when add regain separately as follow: bafilomycin A(1), didecyl adipate (DDA), extracellular K(+), thapsigargin, extracellular Ca(2+), U73122, and aristolochic acid.</p><p><b>RESULTS</b>The concentrations of ATP in the extracellular fluid significantly and gradually decreased along with increasing concentrations of bafilomycin A(1). The concentrations of ATP in the extracellular fluid were in linear increased with DDA was added to marginal cell suspensions. ATP concentrations increased as the concentration of extracellular K(+) was increased, and reached the peak with a K(+) concentration of 9.15 mmol/L. At higher K(+) concentrations, ATP concentrations decreased. With the addition of increasing concentrations of thapsigargin to test marginal cells, ATP concentrations were significantly decreased. When extracellular Ca(2+) was completely chelated, marginal cells continued to release ATP. Moreover, as extracellular Ca(2+)increased, the release of ATP decreased. However, the amount of ATP releas remained to a baseline when extracellular concentration of Ca(2+) reached 1.25 mmol/L or above. When concentrations of U73122 remained within the range of 0.25 to 1.25 µmol/L, as U73122 increased, the release of ATP decreased. When concentrations of aristolochic acid ranging from 12.5 to 100.0 µmol/L were added to the marginal cells suspension, the release of ATP was significantly decreased. However, with concentrations of aristolochic acid less than 100.0 µmol/L, the release of ATP tended to be not significantly different from the amount of ATP released by control group.</p><p><b>CONCLUSIONS</b>ATP could be release from marginal cells cultured in vitro of vascular stria in neonatal rats. ATP release from marginal cells has relevant with calcium pump, K(+) channel state and signaling pathway related enzymes.</p>