| [Objective]To research the effect of different concentrations of iodine ion and iodine molecule on cultured fibroblast cells by various kinds of methods,including:cell culture,immunocytochemistry technique,flow cytometry and other interrelated biochemical techniques.This work can observe the effect of iodine on the proliferation cultured fibroblast cells and enrich the study on trace element.[Material and Methods]1.Cell strain:The fibroblast cells(Human Skin Fibroblast,HSF)were bought from Cell Culture Facility,Peking Union Medical College.The following expressions proceed when the cells passage steadily.2.Reagent and grouping:Potassium Iodide which is analytical reagent was dissolved to 1000mg/L by purified water and was diluted by DMEM for using. Iodine which is analytical reagent was dissolved by purified water.The concentration was determined by titrimetric method.It was prepared when it was demanded.There were one control group and many groups among 50～15000μg/L of iodine ion;There were one control group and many groups among 50～4000μg/L of iodine molecule.3.Cell counting and observation of morphology:The cells were seeded in 24-well culture plates and cultured in the DMEM which contains different concentration of iodine.Cell counting and observation of morphology were done after 4-hour,12-hour,24-hour and 48-hour. 4.MTT experimentation:The cells were seeded in 96-well culture plates and cultured in the DMEM which contains different concentration of iodine for 4-hour,12-hour,24-hour and 48-hour.20μl MT-F solution(5mg/ml)was added in each well,and continued to culture at 37℃for 4 hours.The supernatant was discarded subsequently,and 150μl DMSO was added to each well.The plates were vibrated to dissolve the crystals in the cells.The absorbance was determined by the microplate reader at 490nm.5.Flow Cytometry:The cells were cultured in the DMEM which contains different concentration of iodine for 48-hour.The single cell suspension was prepared and collected into centrifuge tube.The suspension was centrifuged to discard the supernatant.Cells were fixed by 70%alcohol.Supernatant was discarded after centrifuging.Cells were washed twice with PBS and stained with synthetic staining solution.Flow cytometer was used to analyze the cells.Data analysis was performed by ModFit software.6.Immunocytochemistry:Coverslips were sterilized and put in 6-well plates.Cells were seeded on the coverslips and cultured in the DMEM which contains different concentration iodine ion/molecule for 48-hour.The cells were washed with PBS buffer and fixed with acetone.The expression of Ki-67 was detected by PV method.7.Detection of SOD and MDA:Maleic Dialdehyde(MDA)and Superoxide dismutase(SOD)in supernate was detected by kits.[Results]1.Cell counting and MTT experimentation:When the iodine concentration was low (such as,iodine ion concentration was lower than 3000μg/L in 48-hour,and iodine molecule concentration was lower than 400μg/L in 48-hour),cell counting and proliferation activity increased with the increase of concentration. When the concentration of iodine was high(such as,iodine ion concentration was higher than 3000μg/L in 48-hour and iodine molecule concentration was higher than 400μg/L in 48-hour),cell counting and proliferation activity decreased with the increase of concentration,and change of cell morphology can be observed.2.Flow Cytometry:Proliferation index of 100 to 4000μg/L iodine ion or 50 to 700μg/L iodine molecule in experimental group were significantly higher than in the control group,while apoptosis rate were significantly lower than in the control group.There was no significant difference between proliferation index of 5000μg/L iodine ion or 800μg/L iodine molecule experimental group and proliferation index of the control group,while apoptosis rate were significantly higher in the experimental group.When iodine ion concentration is higher than 6000μg/L or iodine molecular concentration is much higher than 900μg/L,the proliferation index of experimental group cells was lower than the control group cells',while apoptosis rate was significantly high.3.Immunocytochemistry:Expression of Ki-67 in the experimental groups of 100 to 3000μg/L iodine ion or 50 to 500μg/L iodine molecular was much higher than in the control group;and expression of Ki-67 in the experimental group that iodine ion concentrations were higher than 5000μg/L or iodine molecular concentrations were higher than 700μg/L were lower than in the control group.4.Detection of SOD and MDA:Concentration of SOD in the experimental groups of 50 to 600μg/L iodine ion or 50 to 300μg/L iodine molecular increased with the concentration iodine increase,but it decreased with the concentration iodine increase when the concentration of iodine ion were higher than 800μg/L or iodine molecular were higher than 600μg/L.There was no significant difference of concentration of MDA between in the experimental group and in the control group when the iodine ion concentrations were lower than 800μg/L or the iodine molecular concentrations were lower than 500μg/L,while it was much higher when the iodine ion concentrations were higher than 1000μg/L or iodine molecular concentrations were higher than 700μg/L.[Conclusions]1.Iodine has the effects of improving and decreasing proliferation activities of fibroblast.The effects are related to time and concentration.In other words, there exists the relation of time-dose-effect.2.The effect of different concentrations of iodine ion or iodine molecule on proliferation activity of cultured fibroblast cells may be related to oxidation-antioxygen system,cell cycle and apoptosis.3.The effect of iodine molecular on fibroblast proliferation activity was much more obvious than the role of iodine ion,and it may be related to the oxidative of iodine molecular.
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