To compare the effects of two different cryoprotectants on human desmoglein 1 (Dsg 1), and to provide experimental basis for the optimization of cryoprotectant. Methods Five donated thin spl it-thickness skin grafts were used, and the experiment was conducted within 4 hours after skin grafts harvest. The skin grafts were divided into 3 groups: group A (n=2) in which skin grafts were immersed in 0.5 mol/L trehalose/DMSO; group B (n=2) in which skin grafts were immersed in DMSO/propanediol; group C (n=1) in which fresh skin graft received no further treatment. Groups A and Bwere stored in — 196℃ l iquid nitrogen for 7 and 21 days, respectively, and then underwent experiment. mmunohistochemistry staining observation was performed on each group, RT-PCR method was used to detect the expression of Dsg 1 in skin. Results The immunohistochemistry staining showed that the protein in groups A and B was stained brown-yellow and distributed evenly 7 days after cryopreservation; the expression signal of epidermal basal cell was similar to that of group C; absorbance (A) value of groups A, B and C was 0.285 ± 0.006, 0.284 ± 0.004 and 0.287 ± 0.008, respectively, suggesting there was no significant difference between groups A and B and group C (P gt; 0.05). At 21 days after cryopreservation, the expression of positive cells in group B decreased; no obvious decrease was observed in group A, A value of groups A and B was 0.282 ± 0.004 and 0.275 ± 0.005, respectively, indicating there was a significant difference between group B and groups A and C (P lt; 0.05). RT-PCR detection showed that A value of groups A and B at 7 days after cryopreservation was 0.810 ± 0.012 and 0.803 ± 0.008, respectively; A value of groups A and B at 21 days after cryopreservation was 0.806 ± 0.008 and 0.782 ± 0.013, respectively; and the A value of group C was 0.814 ± 0.012, indicating there was significant difference between group B and groups A and C at 21 days after ryopreservation (P lt; 0.05), and no significant differences among groups were noted at other time points (P gt; 0.05).Conclusion Trehalose/DMSO is better than traditional cryoprotectant DMSO/propanediol in protecting Dsg 1 of human skin.
To compare the effect of trehalose with that of different traditional cryoprotectants on human skin and to detect the new protection mechanism of trehalose in hypothermia. Methods The skins to be cryopreserved were first treated with DMSO/Propyleneglycol (D/P group), trehalose/DMSO (T/D group), DMSO/ serumfree keratinocyte medium(D/K group), DMEM (DMEM group), respectively, so as to be compared with fresh skin (control grouop). Then the histological structure of skin of different groups was observed and analyzed by pathological technology (SP immunohistochemistry, DAB staining). Furthermore, the influence of trehalose on α-actinin at gene level with RT-PCR was investigated. The viabil ity of skin in 5 respective groups was evaluated by using succinate dehydrogenase (SDH). The experiments were carried out 14 days after cryopreservation. Results The results of immunohistochemistry showed that A values of control group, T/D group, D/P group, D/K group and DMEM group were 27.50 ± 7.92, 18.40 ± 5.81, 13.10 ± 5.11, 11.50 ± 4.54 and 5.30 ± 2.14, respectively. There was no significant difference between control group and T/D group (P gt; 0.05), but control group was significantly different from the other groups (P lt; 0.05). The results of PCR studies showed that A values of control group, T/D group, D/P group, D/K group and DMEM group were 0.816 ± 0.134, 0.723 ± 0.245, 0.564 ± 0.265, 0.245 ± 0.071 and 0.148 ± 0.048, respectively. Control group was not significantly different from T/D group and D/P group (P gt; 0.05), but was significantly different from D/K group and DMEM group (P lt; 0.05). The results of SDH showed that A valuse of control group, T/D group, D/P group, D/K group and DMEM group were 18.2 ± 3.7, 12.3 ± 3.6, 10.2 ± 2.4, 7.3 ± 2.1 and 5.7 ± 1.5, respectively. There was no significant difference between control group and T/D group (P gt; 0.05), while control group was significantly different from the other groups (P lt; 0.05). Conclusion The results suggest that cryopreservation protocol-trehalose/DMSO is better than the traditional cryoprotectant for ryopreservation on α-actinin of human skin.
ObjectiveTo determine if the cryoprotectant solution supplementation with Vitamin C can improve the protective effect of human ovarian tissue cryopreservation by anti-apoptosis. MethodsHuman ovarian cortical tissues were collected from nine patients treated between March 2012 and April 2013. The cortical tissue pieces obtained from each patient were divided into two groups:control (conventional slow freezing) and trial group (slow freezing supplementation with Vitamin C). The preservation effects in the two groups were compared by histology using light microscope and apoptosis assessed by TUNEL assay. ResultsThe proportion of morphologically normal primordial follicles in the trial group was higher than that in the control group (P<0.05). The proportion of apoptotic primordial follicles and stromal cells in the trial group was lower than that in the control group (P<0.05). ConclusionCryoprotectant solutions supplementation with Vitamin C can improve the preservation of primordial follicles and stromal cells. It might be a method worth to try in order to improve the protective effect of human ovarian tissue cryopreservation by inhibiting of apoptosis.
ObjectiveTo determine if the anti-apoptosis agent can improve the protective effect of human ovarian tissue with novel vitrification method. MethodsHuman ovarian cortical tissues were collected from ten patients treated between October 2012 and August 2013. The samples obtained from each patient were divided into two groups:control (the novel immersed vitrification) and experimental group (the novel immersed vitrification supplemented with vitamin C). The preservation effects in the two groups were compared by ultrastructure using electronic microscopy, and apoptosis was assessed by terminal deoxynucleotidyl transferase-medicated dUTP nick-end labeling (TUNEL) staining. ResultsThe proportion of normal ultrastructure of oocytes and granulosa cells in the experimental group was higher than that in the control group (P<0.05). The proportion of TUNEL-positive primordial follicles in the experimental group was 21.6% (49/227) and the proportion of TUNEL-positive primordial follicles in the control group was 38.6% (91/236), with a significant difference between the two groups (P<0.001). The proportion of TUNEL-positive stromal cells in the experimental group was (21.33±3.41)% and the proportion of TUNEL-positive stromal cells in the control group was (33.46±3.09)%, also with a significant difference between the two groups (P<0.001). ConclusionAnti-apoptosis agents can improve the preservation of primordial follicles and stromal cells by inhibiting of apoptosis. It may be a method worthy of trying in order to improve the protective effect of human ovarian tissue vitrification.