| Objective :(1)To investigate the influence of Simvastatin on apoptosis of marrow mesenchymal stem cells induced by TNF-αin vitro culture. (2)To investigate the influence of Simvastatin on the activity of caspase-3 of marrow mesenchymal stem cells induced by TNF-αin vitro culture.Methods : The cultured and isolated mouse marrow mesenchymal stem cells were depurated by taking density gradient centrifugation and repeatedly adherent cytopheresis in vitro. The MSCs of passage 4 were adopted as research objects. (1)The apoptosis rate of marrow mesenchymal stem cells from normal control, TNF-αstimulus group and Simvastatin pretreatment group ,double dyed by AnnexinV/ PI, were detected by flow cytometer. Whether the difference has the statistic meaning was decided by comparing the apoptosis rate between normal control and TNF-αstimulus group ,among different concentration TNF-αstimulus subgroups, between Simvastatin pretreatment group and 0.5ng/ml TNF-αstimulus subgroup, between normal control and Simvastatin pretreatment group and among different concentration Simvastatin pretreatment subgroups.(2)caspase-3 detection kit was adopted.The optical density (OD) values of normal control ,TNF-αstimulus group and Simvastatin pretreatment group were measured by ELIASA at 405nm. Whether the difference has the statistics meaning was decided by comparing the OD values between normal control and TNF-αstimulus group ,among different concentration TNF-αstimulus subgroups, between Simvastatin pretreatment group and 0.5ng/ml TNF-αstimulus subgroup, between normal control and Simvastatin pretreatment group and among different concentration Simvastatin pretreatment subgroups. The above-mentioned data was calculated statistically (SPASS version13.0).To determine the statistical significance of numerical data, comparisons were performed by ANOVA. Significance was accepted when p< 0.05.Results:(1)The following are the percentages of the apoptosis rate in each group: 10.21±1.46% in normal control; 30.27±2.71% in 0.05ng/ml TNF-αstimulus subgroup, 35.38±2.00% in 0.5ng/ml TNF-αstimulus subgroup, 41.8±2.53% in 5ng/ml TNF-αstimulus subgroup;23.6±2.94% in 1.0×10-7M Simvastatin pretreatment subgroup, 17.18±2.52% in 1.0×10-6 M Simvastatin pretreatment subgroup, 22.68±2.03% in 1.0×10-5M Simvastatin pretreatment subgroup. (2)The following are the OD values measured by ELIASA in each group: 0.112±0.013 in normal control; 0.466±0.042 in 0.05ng/ml TNF-αstimulus subgroup, 0.692±0.041 in 0.5ng/ml TNF-αstimulus subgroup, 0.829±0.040 in 5ng/ml TNF-αstimulus subgroup; 0.348±0.027 in 1.0×10-7M Simvastatin pretreatment subgroup, 0.275±0.021 in 1.0×10-6 M Simvastatin pretreatment subgroup, 0.350±0.018 in 1.0×10-5M Simvastatin pretreatment subgroup. Analyzed by statistics, the difference of the apoptosis rates and OD values have the statistics meaning between normal control and TNF-αstimulus group (p<0.05) ; the difference of the apoptosis rates and OD values have the statistics meaning among different concentration TNF-αstimulus subgroups ( p<0.05) ; the difference of the apoptosis rates and OD values have the statistics meaning between Simvastatin pretreatment group and 0.5ng/ml TNF-αstimulus subgroup ( p<0.05), the difference of the apoptosis rates and OD values have the statistics meaning between normal control and Simvastatin pretreatment group( p<0.05) ; the difference of the apoptosis rates and OD values have the statistics meaning between 1.0×10-7 M Simvastatin pretreatment subgroup and 1.0×10-6M Simvastatin pretreatment subgroup( p<0.05), the difference of the apoptosis rates and OD values have the statistics meaning between 1.0×10-6M Simvastatin pretreatment subgroup and 1.0×10-5M Simvastatin pretreatment subgroup ( p<0.05),while the difference of the apoptosis rates and OD values have none statistics meaning between 1.0×10-7M Simvastatin pretreatment subgroup and 1.0×10-5M Simvastatin pretreatment subgroup( p>0.05) .Conclusion:(1)TNF-αcan induce apoptosis of marrow mesenchymal stem cells in vitro culture in a dose-dependent increment . (2)Simvastatin can partially inhibit apoptosis of marrow mesenchymal stem cells induced by TNF-α. (3)Comparing to the concentration of 1.0×10-5 M and 1.0×10-7 M, the concentration of 1.0×10-6 M Simvastatin is an ideal one chosen to pretreat MSCs. (4)TNF-αprobably induces apoptosis of marrow mesenchymal stem cells partially by activating caspase-3 in vitro culture. (5)Suppression of apoptosis of marrow mesenchymal stem cells,as by Simvastatin, is probably caused by inhibiting activation of caspase-3 induced by TNF-αin vitro culture. |
PDF Full Text Request