Reports of biochemistry & molecular biology · 2018
The Effect of Caffeic Acid on Spermatogonial Stem Cell-type A Cryopreservation.
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Sayed Mahdi Nasiri, Azarbani Farideh, Pirnia Afshin, Abbaszadeh Abolfazl, Gholami Mohammadreza
Аннотация
BACKGROUND: Cancer treatment methods can lead to male infertility .in this regard, cryopreservation of spermatogonial stem cells (SSC) and cell-to-person transplantation after the course of treatment to resolve the problem of infertility is a good one. The cryopreservation of SSC is an important process as it can help on the return of spermatogenesis. However, during this process, the stem cells often become damaged which degrades their value for experiments and treatments. Caffeic acid (CA) is an antioxidant that has been shown to increase the viability of cells under stress. The aim of this study was to investigate the effect of CA has on spermatogonial stem cell (SSC) cryopreservation.
METHODS: Spermatogonial stem cells isolated from the testes of Balb/c mice pups were cultured in laminincoated dishes, purified using CD90.1 microbeads, then cryopreserved in vitrification media supplemented with 10 µM CA either through a slow or rapid freezing process. After thawing, cell viability was evaluated. Expression of Bax, Fas, Bcl-2 and P53 genes was determined by real-time PCR. Gel electrophoresis was used to confirm the results of the real-time PCR.
RESULTS: The viability of the SSCs that were rapidly frozen and treated with CA was observed to be significantly reduced compared to the control group (p < 0.003). The viability SSCs that received CA and underwent the slow freezing treatment was significantly reduced compared to controls (p < 0.002). The expression levels of BAX, BCL-2, and Fas in the rapid freeze-thaw group didn't significantly change. However, the levels of P53 expression were shown to increase. In the group of SSCs that underwent the slow freezing process, the BAX gene expression levels increased, while the levels of BCL-2 gene expression decreased. No significant changes in the level of Fas and P53 expression were detected. When comparing the groups that received CA treatment, SSCs that were rapidly frozen showed an up-regulation of Fas and P53 expression and a down-regulation of Bcl-2 and Bax expression.
CONCLUSION: Caffeic acid may protect intact SCCs during the cryopreservation process through stimulating the induction of apoptosis in injured SSCs. Supplementing the vitrification media with CA has a superior effect on the preservation of SSCs.
METHODS: Spermatogonial stem cells isolated from the testes of Balb/c mice pups were cultured in laminincoated dishes, purified using CD90.1 microbeads, then cryopreserved in vitrification media supplemented with 10 µM CA either through a slow or rapid freezing process. After thawing, cell viability was evaluated. Expression of Bax, Fas, Bcl-2 and P53 genes was determined by real-time PCR. Gel electrophoresis was used to confirm the results of the real-time PCR.
RESULTS: The viability of the SSCs that were rapidly frozen and treated with CA was observed to be significantly reduced compared to the control group (p < 0.003). The viability SSCs that received CA and underwent the slow freezing treatment was significantly reduced compared to controls (p < 0.002). The expression levels of BAX, BCL-2, and Fas in the rapid freeze-thaw group didn't significantly change. However, the levels of P53 expression were shown to increase. In the group of SSCs that underwent the slow freezing process, the BAX gene expression levels increased, while the levels of BCL-2 gene expression decreased. No significant changes in the level of Fas and P53 expression were detected. When comparing the groups that received CA treatment, SSCs that were rapidly frozen showed an up-regulation of Fas and P53 expression and a down-regulation of Bcl-2 and Bax expression.
CONCLUSION: Caffeic acid may protect intact SCCs during the cryopreservation process through stimulating the induction of apoptosis in injured SSCs. Supplementing the vitrification media with CA has a superior effect on the preservation of SSCs.