The Dynamic Changes Of Alveolar Apoptosis In Cryopreservation By Piglets Lung Which Perfused By The Lung Protective Solution

Objective To observe the dynamic changes of alveolar apoptosis in cryopreservation by separation piglets lung which perfused by organic preservation solution, and the relation of alveolar apoptosis with lung tissue injury.Methods The whole lungs which separated from BAMA little pigs were made cold ischemia models, and to take the lung tissues after cryopreservation of Oh,8h,12h and 24h. To quantitative detect dry weight/wet weight ratio, also to observe the submicrostructure and the morphologic change of the tissues and cells used by transmission electron microscopy and light microscopy for examing whether the apoptosis cell appeared in the lung tissue. To quantitative detect the expression level of caspase-3 by PCR. To measure theexpression level of apoptotic in lung tissues by Tunel method. To measure the death rate of lung cell by HE staining. From the death rate and the apoptosis rate, the necrotic rate can be calculated.Result The donor lung’s W/D were increasing as preservative period was prolonging.The W/D of the donor lung after saved 8h、12h 24h were significantly higher than after saved Oh(P<0.05).The death rate of HE staining was increasing as preservative period was prolonging. The death rate of the donor lung after saved 8h、12h 24h were significantly higher than after saved Oh(P<0.05). The apoptotic rate of the donor lung by Tunel method increased firsty then fall down as preservative period was prolonging. After saved 12h, the apoptotic rate to be the maximum value. After saved 8h,12h,24h, the apoptotic rate was significant higher than after saved Oh (P<0.05). Also the difference of the apoptotic rate after saved 8h,12h,24h were statistically significant (P<0.05). But the difference of the apoptotic rate after saved 12h, 24h were not statistically significant(P>0.05). The expression level of Caspase-3 in the lung cells increased firstly then fall down and was increased gradually after saved 8h(F=11.16), Also the expression level was droped gradually after saved 12h. The donor lung tissues began to apoptosis after saved 8h by transmission electron microscopy. The ultra microstructure change obviously after saved 12h, we can see plenty of early and middle stage apoptotic cells. The apoptotic rate after saved 24h was not obvious than after saved 12h, but we can see plenty of end stage apoptotic cells.Conclusion 1.The apoptotic rate of the donor lung in cryopreservation was increased as preservative period was prolonging which perfused by organic preservation solution, also the apoptotic rate to be the maximum value after saved 12h.2. After saved 24h, the donor lung’s W/D had a significant relationship with cells death. The cells apoptosis had a significant relationship with lung ischemic damage after saved 0-8h. The cells necrosis had a significant relationship with lung injury after saved 12-24h.3. Caspase-3 RNA reverse transcription, real-time PCR combined with Tunel method which was more comprehensive and objective evaluation method of cells apoptosis can response the whole apoptosis situation of the early, middle and end period.