Effects Of Extracellular Flouride Concentrations On Founction Of The Cultured Vscular Endothelial Cells

Objective To research the fluoride effects on cultured human umbilical vein vascular endothelial cells for the first time by various kinds of methods: cell culture techNOlogy , immuNOcytochemistry techniques , Flow Cytometry and other interrelated biochemical techniques. To observe the relationships of fluoride and the cultured human umbilical vein vascular endothelial cells. It will have great supports on the study of fluoride with body and enrich the study on trace element. Material and Methods1,Cell cultivation: The vascular endothelial cell lines HUECV-304 were routinely grown in DMEM cultivation medium containing 10% fetal calf serum, penicilin and streptomycin in a humidified incubator at 37℃ and 5% CO2.2,Prepare a single cell suspension with density at 0.8-1.0×10~5 cells per mililiter. Plant cells in the tissue culture plate with 24 wells.3,MTT experimentation: To evaluate the proliferation of endothelial cell lines HUECV-304. The HUECV-304 were exposed to NaF at concentrations of 120, 240, 360, 480, 600, 720, 840 and 960μmol/L, and finish cultivation after 4 hour,8 hour, 12 hour,24 hour,48 hour exposure. Pipette out DMEM culture medium and discard carefully. Add 0.25% trysinase and ensure that the entire moNOlayer is covered with the trysinase solution. Add 150μl DMSO to each well until the cells begin to detach,then concuss it for ten minutes. Remove plate cover and measure the absorbance ineach well, including the blanks, at 570 nm in a microtiter plate reader.4, ImmuNOcytochemistry: To sterilize glass coverslips and put them in 24-well plates.Seed 100,000 cells per well and add NaF after cell attached, we finished cultivationafter 12 hours. Remove the media and wash with PBS buffer, add primary antibodyand secondary antibody. Pick up coverslips with forceps and drain away excess buffercarefully before add DAB. At last observe staining of the HUECV-304.5,Flow Cytometry: Samples were washed with phosphate-buffered saline (PBS) (pH7.2), fixed, suspended in PBS and then analyzed using flow cytometer. Data analysiswas performed with Cell Quest software. We use PI represent the activity of cellproliferation, PI = (S+G2/M) / (G0/G1+S+G2/M) .6,Test of cell function: Test MDA, Superoxide dismutase(SOD) and NO in medium.Results1 , Fluoride significantly increased endothelial cells proliferation activities in certainranges of concentration120～240μmol/L) and at certain time(four hours to forty-eighthours). Afterwards, the proliferation activity of endothelial cells was decreased alongwith the time of culture and the increase of doses of fluoride.2, After administration of fluoride, the expression of PCNA and Ki-67 was thehighest in group 120～240μmol/L of fluoride at 24h, whereas the lowest in the groupof 600～960μmol/L of fluoride.3, The cell function was influenced by fluoride administration to adapt to newconditions. The adaptation phase is characterized by the increased MDAconcentration and the decreased NO concentration, while SOD increased firstly andthen decreased.Conclusions1, The fluoride effect on the proliferation activity of endothelial cells is dose and timedependent.2, The "swing" of MDA, SOD and NO relates to endothelial cell growth and plays animportant role in cell proliferation. 3, How did the fluoride influence rdirectly or indirectly still needs lucubrate.