| Objective: To observe the changes of cell apoptosis and the expression of Caspase-3 gene in different timepoint during ischemia-reperfusion injury in rat skin flaps by means of RT-PCR technique and immunohistochemial technique and application of exogenous zinc gluconate induced MT formation in vivo,on the basis of rat abdominal skin flap model of ischemia-reperfusion injury.To investigate the effects of MT on expression of apoptosis gene Caspase-3 in ischemia-reperfusion injury in rat skin flaps and to expoud the mechanism of protiecting flap about MT on ischemia-reperfusion injury in the genetic level for proveding new ideas and theories that can Prevent various tissue flaps in the ischemia-reperfusion injury occurring and heighten the survival rate in clinical skin flap.Method:1 Laboratory animal: 120 clean healthy male Wister rats were weighted and ordered.2 The rats were divided into three groups randomly:(1) the control group,(2) group A (Ischemia reperfusion group),(3) group B (Ischemia reperfusion group that induced MT),each group included 40 rats. each group was divided into 4 small groups accoring to five time points,each of the five groups consisted of 8 rats.3 The preparation of skin flap: The rats were anesthetized with 2% sodium pentobarbital (40mg/kg) by intraperitoneal injection,their limbs were fixed and their hairs on abdomen were sheared,the skin of abdomen was sterilized by 75% alcohol after the hairs were cut down. A right low abdominal island skin flap,which size was 6cm×3cm,was created according to the method of Petry JJ's. There were no other treatments after creating the skin flap according to the method above in the control group.In group A,after the skin flap formed according to the method above,the proximal end of the point that the superficial epigastric artery arised from femoral artery,was occluded by a vascular clamp.confirmed the femoral artery and superficial epigastric artery completely blocked Under the operating microscope,sutued the flap,and the vascular clamp was taken out in another operation after 8 hours. In group B,24 hours before surgery,to tube feeding rats zinc gluconate 140mg/Kg,and the rest of the operation as the A group of experimental.4 To draw the materials from the skin flaps: in the A and B group,Full-thickness of the skin flap which size was 1cm×0.5cm was taken at the moment when the skin flap was created,1 hours,12 hours,24 hours after ischemia-reperfusion. In the control group,The same sample of skin flap was taken respectively at the moment when the skin flap was created,9 hours,20 hours,32 hours after operation. One part of the obtain skin flap was conserved in 4% paraformaldehyde liquor , in order to dyeing using method of immunohistochemistry. The other part of the obtain skin flap (100mg) was conserved in Ultra-low temperature freezera and produced single cell suspension,in order to detected the the expression of Caspase-3 mRNA by RT-PCR technique.5 The method of detection and the observation target: (1) HE staining:observed under light microscope for histopathological,Nuclei is blue,cytoplasm is pink,muscle fibers and collagen fibers is red.(2) According to the specification of the test kit , dyed the paraffin section by immunohistochemistry,the pigmenting and the location of Caspase-3 were observed with a light microscope. The immunohisto-chemical scores (IHS) was calculated by using image analysis system.(3)The expression of Caspase-3 mRNA was detected by RT-PCR technique. 6. Analysis of results: Different kinds of results were dealed with by using SPSS13.0 software.Results:1 The Histopathologic changes of the skin flap when it was in ischemia-reperfusion:Most of the cell body in group A shrinked,nuclear condensated and eosinophilic cytoplasm deeply stained,which becomes more observe in 24 hours after reperfusion,meanwhile,the Morphological structure of the control group was normal,no such changes were observed.however, the abnormal cells in group B were decreased more dramatically than Group A.they are damaged lesser,cytoplasm mildly stained,as well as nuclear condensation,nucleolus disappearance were rarely noticed.2 The results of immunohistochemistry stained:The Caspase-3 positive cells were distributed in cytoplasm of epidermal basal cells,fibroblasts,hair follicle,sweat glands,sebaceous glands and the vascular cells,which was consistent with the distribution of apoptosis cell. Staining mainly in the cytoplasm,brownish yellow or brown. Little scattered Caspase-3 positive cells were observed in the control Group. small size,light staining and weak expression.The expression of Caspase-3 positive cells in group A were inclined to increase gradually with prolonged ischemia reperfusion,up to the peak after 24 hours ischemia reperfusion.You can see the cells were connecting into line flakes with deepest staining and strongest expression.while Group B was significantly decreased by contrast with group A.There were statistical differences on the expression among control group,Group A and Group B.(P<0.05).The immunohistochemical staining marked: the results showed: As time goes on, the positive cells in group A would be increasing progressively with significant difference compared with control group,which would be up to peak after 24 hours reperfusion. However group B would increase gradually,there was no significant difference between the adjacent time points(P>0.05).while the difference on expression of posive cells between group A and group B were obvious.(P<0.01)which explained that metallothionein restrained the expression of Caspase-3 activity after application of zinc gluconate.3 The expression of Caspase-3 mRNA with RT-PCR technique: According to PCR-Marker,the the amplification of Caspase-3 gene fragment was about 349 bp.Internal referenceβ-Actin gene fragment was about 576 bp consistent with the target fragment size , which were deemed as specificity expansion.There are a small amount of Caspase-3 mRNA expression in the control group. in group A , Caspase-3 mRNA expression significantly increased after the skin flap lifting as well as 1 hours , 6 hours ischemia-reperfusion.As time goes on , Caspase-3 mRNA expression significantly increased,reached peak after 24 hours then gradually decreased growth.(compared with the adjacent time points,P<0.05).In group B,with time prolonged the expression of Caspase-3 mRNA activity increased,but the growth has been decreased compared with group A. (P<0.05).Conclusion:1 Caspase-3 activity is increased within the tissues after skin flap is in ischemia- reperfusion. Which suggests that Caspase-3 is involved in the regulation of apoptosis process in the skin flap ischemia reperfusion.2 After zinc applied , the Caspase-3 activity is decreased in ischemia-reperfusion. Which furtherly demonstrate that Caspase-3 is involved in the flap tissue cell apoptosis as well as restrain the Caspase-3activity to improve ischemic Reperfusion injury.3 Zinc can be induced to produce metallothionein,which can decrease the acivitity by regulating Caspase-3 to protect the flap ischemia-reperfusion injury in rats. |
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