Effects Of Ischemia-reperfusion Injury On Cell Apoptosis And The Expression Of Fas Gene In Skin Flap

Objective: To observe the morphological changes of flap tissues with ischemia-reperfusion injury (ischemia reperfusion injury IRI);To study the changing regulation of cell apoptosis of flap tissues with ischemia-reperfusion injury compared to Non-ischemia-reperfusion injury; To study the expression of Fas protein of flap tissues in different time point with ischemia-reperfusion injury compared to Non-ischemia-reperfusion injury; To study the expression of relative Fas gene of flap tissues in different time point with ischemia-reperfusion injury compared to Non-ischemia-reperfusion injury.So as to elucidate the role of Fas genes in the ischemic reperfusion of flaps, and discuss the relationship of apoptotic gene Fas with cell apoptosis in rat flaps with ischemia–reperfusion injury, in order provide a new thinking and theoretical basis to improve skin flap survival rate in the treatment of ischemia–reperfusion injury of flaps in the genic level.Method:1 Experimental animal: 50 male and healthy Wister rats were weighted and ordered.2 The animal model formation: The belly hair of rats were depilated by 8% sodium sulfide three days before operation(Area is 4 cm x 10cm). They were washed by warm water and dry naturely. The rats were anesthetized with 3% sodium pentobarbital (40mg/kg) by intraperitoneal injection, and there was another anesthetized every three hour(20mg/kg). The rats were lay down by supine position, fixed and sterilized by 75% alcohol after successful anesthesia, formed an about 3㎝ x 6㎝ island flap unber the right lower quadrant abdomen taking the abdominal wall shallow blood vessel as the pedicle.3 Experimental animals grouping: The rats were divided into two groups randomly,Non- IR group, which included 25 rats. There were no other treatments after creating the flap in this group. Time points to form after the selection of flap formation 8,9,20,14,32h and divided into five groups ,each group was five rats, compared with experimental group. IR group which included 25 rats: After creating the rat flap, The proximal Femoral Artery beside which inferior epigastic artery sending was occluded by a microvessel clamp. The flap was sutured with 3-0 suture silk by the certain of the blood stream was blocked under the observation of operating microscope. The vascular clamp was taken out in another operation after 8 hours. Reperfusion was successful by which the blood flow was restored under the observation of operating microscope. Five groups were divided according the time of after taking out vascular clamp 0,1,6,12,24 hours. There are five rats in every group.4 To draw the materials from the flaps: The full-thickness of the flap which size was about 1㎝×0.5㎝ was taken respectively at the time in Non - IR group and IR group. Part of the obtained flap was conserved in paraformaldehyde in order to commit paraffin section and dyeing of immunohistochemistry to detect with apoptotic cells and Fas. Part of the obtained flap was conserved in a refrigerator after freezing in liquid nitrogen in order to detect with RT-PCR.5 Testing methods and observation index:5.1 Paraffin for HE dyed, structure change of tissues were observed.5.2 The apoptotic cells in situ end labeling (TUNEL method) test: According to the specification of the test kit, the paraffin section of flaps were labeled the fragment of DNA in situ to detect with apoptotic cells by TUNEL method. By means of the percentage of average masculine cell as the index of apoptotic cell( AI=the count of apoptotic cells/ the count of the total cells×100%).5.3 Fas gene protein expression detection:According to the specific- ation of the test kit, dyed the paraffin section by immunohistochemistry of SP method, the pigmenting and the location of Fas were observed with a light microscope. The average gray values of positive cells per unit area were analysed by using of computer image analysis system. The Fas expression level was shown by the taken of their average means of the slice respectively5.4 Fas mRNA express detection: after abstracted total RNA, adopt reverse transcription polymerase chain reaction, Through the analysis of PCR production electrophoresis.analysed objective electrophoresis corresponding bands by using of gel image analysis system (wuhan tongji medical university HMIAS - 2000 type). The actin electrophoresis strip as a reference, the result was demonstrated by the integral absorbency of both.6.Analysis of results:All the test data were expressed with mean±standard deviation(X±S),dealed with by using SPSS16 software, significance analysised by usuing the contrast statistics significance with the test P<0.05.Results:1 HE dyed observation: IR group compared with the Non-IR group subcutaneous edema were more apparently, skin layers structure were disorder, loose, gathered a lot of neutrophils, adhered to the microvascular endothelial cells and capillaries were damaged, integrity were destroyed ,there were large amount of leukocyte exuded into the tissue clearance, partial red blood cells accumulated in capillaries and around tiny vein.2 The cell apoptosis was detected by TUNEL method: Application method of skin flap of tissue TUNEL apoptotic cells were confirmed by IR, found that groups and test the-all happened were confirmed by IR group cell apoptosis, positive cells located mainly in the epidermis, subcutaneous tissue accidentally visible in positive cells scattered. IR group was higher than the-IR group (P < 0.05)3 The expression of Fas protein: This experiment observed in the control group , Fas were weak positive expression in the flap.The difference of average gray values among each time point was no statistically significant.; The Fas protein expression sharp increase with ischemia-reperfusion were extended, and reach peak after reperfusion for 12 hours,Fas positive cells connected into linear flake, dyed the deepest, expressed the strongest in the one of IR12h; IR24h expressed weakness . There was statistical significant between Non-IR group and IR group by SPSS statistical detection (P < 0.05). 4 The expression of Fas mRNA: The flap tissues samples were augumented by RT-PCR ,and it got a production, The change of difference time point was no statistically significant(P > 0.05). In the IR group the mRNA level transcripted and expressed by Fas gene was increasing in trend with ischemia-reperfusion were extended, and reach peak after reperfusion for 12 hours. IR24h group expressed weakness compared with IR12h group. There was statistical significant between Non-IR group and IR group (P < 0.05).Conclusion:1 It may accelerate cell apoptosis after the flap with ischemia-reperfusion injury.2 Fas protein expression increase dramatically as same as the mRNA transcripted by Fas gene when the flap with ischemia-reperfusion injury.3. Cell apoptosis has relevant modify function and relative apoptotic gene Fas participate in the process of cell apoptosis in the ischemia-reperfusion injury of flap.