| PTD-FNK protein is an artificial protein with an inhibiting effect on cell death.It is formed by the fusion of FNK protein and protein transduction domain(PTD).It can enter cells at a great speed.And this protein has been shown to have a protective effect against apoptosis caused by various causes.So far,although the function of PTD-FNK protein has been widely studied,there has been no report on the study of PTD-FNK protein used to protect abnormal apoptosis of mouse Leydig cells(TM3).The objective of this study was to detect the protective effect of different concentrations of PTD-FNK protein on TM3 abnormal apoptosis induced by ethane dimethan sulphonate(EDS).This study will provide experimental basis for further understanding of the physiological function of PTD-FNK protein,and accelerate the clinical application of this protein in male reproductive regulation.The specific research content is as follows:1)TM3 cells were cultured in vitro and their purity was determined with immunofluorescence staining of 3β-HSD cells.TM3 cells in the cell incubator(37 0C,5%CO2)grew well,well-shaped and were upon the logarithmic growth phase during 12-36 h.After staining,a large number of blue granules appeared in the cells,indicating that TM3 cells were of high purity.2)Selected TM3 cells with better growth status were digested with trypsin and passaged.When growing to an about 80%fusion degree,TM3 cells were preincubated for 0.5 h with different concentrations(100,50,5,0.5 and 0.05 nmol/L)of PTD-FNK protein before being induced with apoptosis inducer EDS(750 μ g/mL)for 24 h.The viability of TM3 cells in each group was detected by enhanced CCK-8 assay.The effects of PTD-FNK protein on the apoptosis and necrosis of TM3 cells were evaluated under fluorescence microscopy using Hoechst 33342 and PI double staining.The activity of TM3 cells in the treated groups was higher than that of the negative control group.0.5 nmol/L(P<0.01),5 nmol/L and 0.05nmol/L(P<0.05)PTD-FNK protein significantly increased the activity of TM3 cells,but 50 nmol/L and 100nmol/L PTD-FNK protein showed no significant improvement on the parameter(P>0.05).The cell necrosis was not obvious in each group.There were a lot of strong blue fluorescence and red fluorescence in EDS negative control group,pointing to the apoptosis and necrosis of its TM3 cells being the most.Compared with the negative control group,0.5(P<0.01),50,5 and 0.05(P<0.05)nmol/L PTD-FNK protein significantly reduced the apoptosis of TM3 cells.100 nmol/L PTD-FNK protein group showed no significant difference compared with the control group(P>0.05).The apoptosis rate of TM3 cells was the lowest in the blank control group without addition of PTD-FNK protein and EDS.3)The effect of PTD-FNK protein on the activity of Caspase-3 and Caspase-9 in TM3 cells was assessed with microplate reader.Compared with the negative control group,0.05,5(P<0.05)and 0.5(P<0.01)nmol/L PTD-FNK protein significantly downgraded Caspase-3 activity of TM3 cells induced by EDS(750μg/mL).Different concentrations of PTD-FNK protein didn’t demonstrate significant effects on Caspase-9 activity of TM3 cells treated with EDS.It suggested that the inhibition of PTD-FNK protein from TM3 cell apoptosis may not be related to the reduction of Caspase-9 activity.In summary,the PTD-FNK protein may decrease abnormal apoptosis of TM3 and ultimately promote cell viability via attenuating Caspase-3 activity of TM3 cells in vitro induced by EDS. |
