Effect Of Cobalt(?) Ions On The Cytotoxicity And The Bax, Bcl-2 Expression In Human Renal Tubular Epithelial Cells In Vitro Test

Background:Beacuse of good meachanical performance, wear-resisting, simple manufacture and reasonable price, Cobalt alloys are used as common denture materials to restorate diseases such as defected teeth, partial edentulous jaws, edentulous jaws,jaw defects and so on. Related study showed casting alloys were prone to electrochemical corrosion and released metal ions, for the long-term contacting with the environment of physics, chemistry and biology. Metal ions released from electrochemical corrosion wound contact with the neighboring cells or tissues and be distributed throughout the body by the blood circulation, which made the patients be prone to gingival discoloration, gingivitis, periodontitis, allergy and other adverse reactions.The corrosion of Cobalt alloys mainly release Co2+. Whether Co2+ will cause damage to human body health systems or not, there are few reports at home and abroad. The vast majority of Co2+ in the human body are excreted by the kidney,and reabsorbed in the renal tubules after filtration by the glomerulus, so Co2+ is more likely to be accumulated in renal tubular epithelial cells. This study takes the human renal tubular epithelial cells as research object, and uses MTT method and RT-PCR method to evaluate the renal toxicity of Co2+, in order to provide experimental basis for the clinical choice of Cobalt alloys restotations.Objectives:1. To study the cytotoxicity of cobalt ions on human renal tubular epithelial cells.2. To investigate the effect of cobalt ions on the expression of Bax, Bcl-2 in human renal tubular epithelial cells.3. To evaluate the bicompatibility of cobalt ions, and explore the mechanism of cobalt ions' effect on the growth of human renal tubular epithelial cells.Methods:1. HK-2 cells were cultured in vitro, then the cells were incubated with five concentrations(0?M, 2.5?M, 5?M, 7.5?M, 10?M)of Co2+ medium for 12 h, 24 h and48h.2. The growth state of cells were observed under inverted phase contrast microscope at each time,and the change of cellular morphology was recorded.3. The absorbance values of each concentration culture group at each time were detected by MTT method, then the relative growth rates of cells were calculated.4. The m RNA expression of Bax, Bcl-2 of each concentration culture group at each time were detected by RT-PCR method, and the effect of Co2+on the apoptosis of renal tubular epithelial cells was explored.5. The experimental datas were analysised by using SPSS 17.0 statistic software for one-way ANOVA.Results:1. The morphology of cells were observed by inverted phase contrast microscope: The normal morphology of renal tubular epithelial cells were oval or short fusiform, arranged closely as paving stone and adhered well. With the increase of concentration and duration of Co2+, cells' gap increased, and the number reduced, and partial cells showed long fusiform.When intervented with high concentration above 24 h, cells' number were few,with atypia growth,and the morphology was incomplete, and cells were suspended, and the apoptosis rates were high.2. MTT assay's results : Compared with the control group, the OD values of each experimental group were significant difference(P<0.05). With the increase of concentration and duration of Co2+, the OD values of cells reduced gradually, and the proliferation of cells decreased gradually.3. RT-PCR assay's results:(1)At same time, the m RNA expression of Bax increased and had a significant difference(P<0.05). The m RNA expression of Bcl-2 decreased and had a significant difference(P<0.05).(2) At same concentration, the mRNA expression of Bax increased and had no significant difference(P>0.05). The m RNA expression of Bcl-2 decreased and had a significant difference(P<0.05). With the increase of concentration and duration of Co2+, the m RNA expression of Bax increased gradually, and the m RNA expression of Bcl-2 decreased gradually, and the ratio of Bax/Bcl-2 increased gradually, which promoted the occurrence of apoptosis.Conclusions:1. Co2+ had no toxicity on renal tubular epithelial cells at low concentration(<7.5?M) during the observation time(12h-48h), but it had mild toxicity at high concentration(?7.5?M) after cultivation for 24 h.2. With the increase of concentration and duration, Co2+ promoted the m RNA expression of Bax and inhibited the m RNA expression of Bcl-2, which caused the apoptosis of human renal tubular epithelial cells.3. Co2+ had a certain effect on the growth, proliferation and apoptosis of human renal tubular epithelial cells, which was positively correlated with the concentration and the duration, but its bicompatibility needed further clinical observation and experimental verification.