| Background Human free flap transplantation is more and more extensive application in the repair of severe caused by trauma and tumors of the skin and soft tissue defect, the survival rate of free skin flap is still only 95%. Previous studies have considered many factors that affect the survival of the flap, the flap ischemia-reperfusion injury is an important factor to affect the survival of skin flap. To ensure the free flap survived completely, improve flap survival rate is the primary goal of orthopedic doctor. The researchers found that pharmacological and mechanical intervention can reduce reperfusion injury, but the drug intervention in clinical application is still limited. "Ischemic postconditioning" for long-term after ischemia and reperfusion before a series of mechanical repetition cycle short reperfusion and re occlusion of blood circulation. At present, the protective effect of ischemic postconditioning has been confirmed in the free flap in rabbits, rats; but also no flap related research, action mechanism of protective and post processing is not clear. This study is to deal with model through the establishment of one flap microvascular endothelial cells after hypoxia, analog processing in the body after ischemia, the study of protective effect of ischemic postconditioning, lay the foundation for the research on the mechanism of concrete.Objective Extraction method of reference domestic skin flap dermal microvascular endothelial cells, using the method of enzyme digestion and extraction of sorted human flap microvascular endothelial cells. To determine the best hypoxia treatment after anoxia and reoxygenation after hypoxia treatment time, model.Methods By enzyme digestion combined with magnetic bead sorting method to obtain human dermal microvascular endothelial cells(HDMECs) cultured in vitro, passage to P5, using the VIII factor immunofluorescence and CD309, CD34 and flow cytometry were identified; detection of lactate dehydrogenase(LDH) in hypoxic content changes with time; staining apoptosis rate were calculated by Tunel; Bcl-2 Bax and Caspase-3, and the protein content of activated Caspase-3 expression was detected using the method of Western Blot.Results1. After 4 days of isolation, cells scattered in the colony, surrounding with fibroblast cells, after 7d, the cells showed cobblestone like arrangement; 2. After second times of separation, visible cells arranged closely the nest shaped or concentric appearance, cobblestone were. Second CD31 magnetic activated cell sorting after positive rate > 95%; 3.Two CD31 magnetic bead sorting flow cytometry after identification results: the positive rate of CD309 was(95.4 + 1.34)%; the positive rate of CD34 was(42.5 + 3.23)%; 4.Immunofluorescence detection of LDH in the supernatants of LDH: results and with the increase of hypoxia time, 8h and 0h, 2h, 4h, 6h compared with LDH significantly increase the content of p<0.05, 8h and 12 h have no significant differences p>0.05; 5.Tunel and Western results showed that hypoxia 2min*3s and 5min*3s after treatment(hypoxic postconditioning, H-post C) can play aprotective role;Conclusion1.According to the cell colony morphology and immunofluorescence and flow cytometry analysis of the results obtained, digestive enzyme plus CD31 magnetic bead sorting dermal microvascular endothelial cells(HDECMs) to stable and reliable; 2.According to the lactate dehydrogenase(LDH) with hypoxia time change results, determine the best time to hypoxia; 3. Tunel staining to detect the apoptosis ratio; Western blot(Western Blot, WB) for detection of Bax expression, Bcl-2 and activation of Caspase-3, Caspase-3 protein content, processing model is built in hypoxic human dermal microvascular endothelial cells after 2min*3s and 5min, wherein3 s is the cycle number and duration of treatment after the best.Discussion This study successfully extracted and train HDMECsby using CD31 magnetic bead sorting, identification with factor VIII immunofluorescence, preliminary confirmed HDMECs; at the same time using CD34 and biological characteristics of CD309 streaming antibody detection cell, this can be more convenient to us to know in physiological and pathophysiological characteristics of HDMECS in vitro. According to the analysis of WB and Tunel results we also found that 8h/ 24 h hypoxia reoxygenation, activation of caspase-3/caspase-3, Bax/Bcl-2 and apoptosis ratio is higher than the other group. At the same time 2min*3s and 5min*3s after hypoxia treatment can inhibit cell apoptosis, but at 10min*3s, 20min*3s after hypoxia treatment and can not achieve the ideal of protection. So we speculated on flap microvascular endothelial cells, the appropriate processing time by reducing the activation of Bax/Bcl-2, the ratio of caspase-3/caspase-3, to inhibit cell apoptosis.So we have successfully established ahypoxic postconditioningmodelof human dermal microvascular endothelial cells... |
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