Damage Effect Of Graphene Oxide On Corneal Epithelium Cells In Rats

Graphene is known to the thinnest material at present,it is a honeycomb two-dimensional carbon nano materials which composed of monolayer of carbon atoms with sp2 hybrid orbitals,graphene oxide is one of the types of graphene derivatives.In recent years,with widely production and applications of graphene and its derivatives,many points of views have been forced on their bio-safety.This study used the standard weight of female Wistar mice and obtained corneal epithelial tissue,the primery Corneal epithelial cells were successfully cultured.The cell viability was measured by MTT colorimetric assay,and got the growth curve of normal cells.The cells which grow to the logarithmic phase were divided into two groups including normal control group and treatment group(concentration of 5 ?g/m L?10 ?g/m L?20 ?g/mL?50 ?g/m L?100 ?g/mL graphene oxide).Control group had no treatment,treatment group were treated with five concentrations of graphene oxide and three different time(24 h?48 h?72 h),the cell viability was measured by MTT;the cellular morphology was observed by microscopy,and HE staining was used to detect the distribution of graphene oxide in the cells.The cell state distribution after exposure with graphene oxide was detected by flow cytometry.The cell injury way further determined by DAPI / PI double staining as well as Trypan blue staining,furthermore,the cell cycle was detected.The results showed that the normal adherent corneal epithelial cells had alar shape with the increasing of the cell density,cell morphology gradually showed spindle shape.Cell nuclei displayed round or oval shape,and the “S” growth curve were obtained in normal cells;the logarithmic growth phase cells were obtained after being cultured 26 h and the cell density reached to 80% after being cultured 43 h.When cells was treated with different concentrations of graphene oxide,the cell states becomed very poor,the swollen or aggregated shapes was appeared in nucleus,the partial pseudopod appears fibrous form.Flow cytometry results revealed that the proportion of the normal cells decreased after the cells being treated with different concentrations of graphene oxide,and the percentage of breakage and necrotic cells significantly increased.HE staining showed graphene oxide could into the corneal epithelium cells and remained around the nucleus,which may play an important role in this damage.DAPI / PI double staining and Trypan blue staining showed that the cell viability decreased,which was caused through cell death,not apoptosis.The cell cycle showed the phase retardation in S and G2/M cells and a large number of cell debris was appeared.Based on the above results,abnormal cell morphology,damage and even death were detected after exposure to 5 ?g/mL-100 ?g/m L graphene oxide,These results indicated that the graphene oxide exist potential dangers for rat corneal epithelium cells.