The Protective Effect And Mechanism Of Trehalose In Sternum Cryopreservation

EXPERIMENT1:Effect of Trehalose on Cell Viabilities Following Cryopreservation SternumObjective The aim of this study was to examine whether trehalose has protective effect on sternum tissue and cell viabilities after cryopreservation by investigating the activity of ALP and incorporation of 3H-TdR.Methods The sternums were removed from Sprague-Dawley(SD) rats and immersed immediately in the freezing medium of low-potassium dextran (LPD) solution containing with 10% dimethyl sulfoxide (DMSO) (Group I) and containing with 10% DMSO+0.15mol·L-1 trehalose (GroupⅡ) respectively. A frozen pipe containing a 1-cm-long sternum was filled with the frozen solution and cooled in the process of cooling apparatus freezers (1℃/min) to-80℃for cryopreservation. Fresh tissue and four groups after different periods of preservation (1d,15d,30d,60d,120d)were randomly selected. Then the specimens were cultured and detected ALP activity in supernatant and 3H-TdR incorporation of sternum tissue.Results After different periods of preservation (1d,15d,30d,60d,120 d), ALP activity in supernatant in groupⅡ(95.14%～71.85%) were higher than that in group I (85.63%～69.66%) obviously,and the average rate of 3H-TdR incorporation in groupⅡ(98.86%～81.02%) were higher than that in groupⅠ(94.45%～74.46%) obviously. The protective function lasted for 120 days.Conclusions LPD solution containing with 10% DMSO and 0.10 mol·L-1 trehalose could protect the sternums viability after 120 days cryopreservation and its protective effect was better than that with 10% DMSO only. The measurement of the activity of ALP and incorporation of 3H-TdR is a reliable index as a marker of sternum viability. EXPERIMENT 2:Choice of the Most Effective Concentration of Trehalose in the Sternum Cryopreservative SolutionObjective To detect the most effective concentration of trehalose in the sternum cryopreservative solution.Methods The sternums were removed from Sprague-Dawley(SD) rats and immersed immediately in the freezing medium of low-potassium dextran (LPD) solution and 10% dimethyl sulfoxide (DMSO) containing with 0.05mol·L-1 trehalose (GroupⅠ),0.10mo l·L-1 trehalose (GroupⅡ),0.20mol·L-1 trehalose (GroupⅢ) and 0.30mol·L-1 trehalose (Group IV) respectively. A frozen pipe containing a 1-cm-long sternum was filled with the frozen solution and cooled in the process of cooling apparatus freezers (1℃/min) to-80℃for cryopreservation. Fresh tissue and four groups after cryopreservation (120 days) were randomly selected. Then the specimens were cultured and detected ALP activity in supernatant and 3H-TdR incorporation of sternum tissue.Results The ALP activity in supernatant and 3H-TdR incorporation in sternum of group III was the highest among four groups after preservation (120 days).Conclusions The most effective concentration of trehalose is 0.2 mol·L-1 in the sternum cryopreservative solution. The measurement of the activity of ALP and incorporation of 3H-TdR. is a reliable index as a marker of sternum viability.EXPERIMENT 3; Effect of Trehalose on Bcl-2 mRNA and Bax mRNA Expression Following Cryopreservation SternumObjective The aim of this study was to examine whether trehalose has protective effect on the sternum after cryopreservation (120 days) by investigating Bcl-2 and Bax gene expression.Methods The sternums were removed from Sprague-Dawley (SD) rats and immersed immediately in the freezing medium of low-potassium dextran (LPD) solution only (GroupⅠ), containing with 10% dimethyl sulfoxide (DMSO) (GroupⅡ), containing with 0.20mol·L-1 trehalose (GroupⅢ), and containing with 10% DMSO and 0.20mol·L-1 trehalose (Group IV) respectively. A frozen pipe containing a 1-cm-long sternum was filled with the frozen solution and cooled in the process of cooling apparatus freezers (1℃/min) to-80℃in liquid nitrogen. Fresh tissue and four groups after cryopreservation (120 days) were randomly selected. We observed Bcl-2 and Bax gene expression by reverse transcription-polymerase chain reaction (RT-PCR).Results In the cryopreservation groups, Bcl-2 gene expression in GroupⅢwas higher than in GroupⅠand GroupⅡ(p<0.01). The highest expression of Bcl-2 was Group IV, which was similar to fresh sternums and there were no significant differences between Group IV and the group of fresh sternums(p>0.05). Bax gene expression in Group III was lower than in Group I and Group II (p<0.01). The lowest expression of Bax was Group IV, which was similar to fresh sternums and there were no significant differences between Group IV and the group of fresh sternums(p>0.05).Conclusions These results may indicate that trehalose has a protective effect on the sternum cell after cryopreservation (120 days) by enhancing Bcl-2 gene expression and inhibiting Bax gene expression. It may also suggest that the concomitant use of trehalose and DMSO has a synergistic effect.