| Objective: The flap transplantation or transfer surgery has been widelyused in a number of surgical operation. However, local microcirculation aftersurgery, especially venous circulatory disturbance which caused venous crisishas become into serious complications of flap transplantation, easily causesflap necrosis, and resulting in operation failure. Cell apoptosis andproliferation is regarded as symbol of tissue necrosis and survival, while theexpressions of apoptosis and value-added gene are the symbolic characteristicsof proliferation apoptosis.This experiment are using molecular biology method.By studying of different ways and times in gene expression changes ofapoptosis gene caspase-3,proliferation gene ki67, to evaluate the curativeeffect of continuous pressure suction method for flap silting hemorrhagicnecrosis.From the viewpoint of molecular biology, it can more effectivelyvalidate this method in preventing and curing flap silting hemorrhagic necrosis,in order to successful prevent and cure the silting hemorrhagic necrosis afterflap transplantation or flap transference in clinic. It promote the survival oftissue flap transplantation, and also provide new methods and theoreticalbasis.Methods:1Experiment animals:40healthy and clean New Zealand rabbits. Half ofmale and female, body weight2.5-3kg.2Grouping of experiment animals: There are two congestion flaps can bemade in each rabbit. Using random number allocation method,80flaps can bedivided into4groups, the proximal drainage group, the distal drainage group,the recanalization of vein group and the control group. Each group has20flaps and12of them for scanning,8for drawing materials. 3The formation of rabbit abdomen vein congestion flap model: To depilatewhite rabbit’s belly hair by use8%sodium sulfide three days before operation.The surgery area is from bilateral inguinal upward to xiphoid levels, hair ofboth sides removal to axillary line range. Warm water to clean the abdomen,dry naturally. Use gauze and flexible net to bandage the abdomen of rabbit toprevent the skin injured by the cage. The rabbits were anesthetized byXylazine and Ketamine which are mixed by the ratio of1:1, muscle injectionin accordance with0.2ml/kg. According to the anesthetic, another anesthetized0.1ml/kg per hour can be used. The rabbit were supine fixed on rabbitplatform, and sterilized by0.5%iodophor after successful anethesia. Alongshallow vein of lower abdomen, two rectangualr flaps, measuring12×4cm2,were symmetrically formed. Then do different processing to vascular based onfour kinds of groups.4Flap tissues’ drawing: In remote area of flap,1.0×1.0cm2skin were cut atsix different time points of0hour,4hours,8hours,24hours,3days and7daysafter operation. Tissues were cut and immersion into liquid nitrogenimmediately.5Testing methods and observation index:5.1Take pictures and analyze the pixels of them by Photoshop, to evaluatethe necrotic area of flaps in different groups.5.2Use Polymerase Chain Reaction to test flap tissues’s gene of Caspase-3and Ki67.6Analysis of results: All the data were expressed with mean±standarddeviation（x±s）,dealed with by SPSS17.0software, significance analysis byusing the contrast statistics significance with the test P<0.05.Results:1General observation:Every groups’ flap swelling at four hours after operation, especially thecontrol group, and the degree of change was positively correlated with thetime.The flap swelling severity at eight hours and the dark area of flap is gradually increased.24hours later, flaps of four groups swelling further increased, the controlgroup’s flap is more serious than other groups which the dark purple colorspread about1/3distal area, pressed fluctuations, and dark brown crusts canbe seen on the surface of flaps. Flaps of proximal drainage group, distaldrainage group and the recanalization of vein group were lighter swelling andless area of the color changed than control group. The drainage fluid of distaldrainage group is about2ml, more than proximal drainage group.Flaps in every group reduced swelling at three days after operation, andthe range of color changed area is becoming clear. Experimental groups werelighter swelling and less color changed area of flap than the control group. Thedistal drainage group and recanalization of vein groups’ pathological changeswere less than the proximal drainage group. There are no significantdifference between distal drainage group and recanalization of vein group.The swelling of flaps in different groups almost disappeared, and the darkpurple area of flaps were obvious in each group. Hard and clear scars can beseen on the flaps.2Pixels analysis by Photoshop CS5.0The rabbit were supine fixed on rabbit platform and exposed abdomen.Take abdomen pictures above the rabbit abdominal plane. To calculated thepixels of flap necrosis’s area in all range of flap by Photoshop CS5.0.According to the formula: Flap necrosis rate=pixels of flap necrosis’sarea÷total pixels of flap×100%. Statistical comparison every groups of flap,there was significant different between experimental group and control group（P<0.05）. And no obvious different between Distal drainage group andRecanalization group（P>0.05）. Distal drainage group and Recanalizationgroup are different from Proximal drainage group（P<0.05）.3Test result of RT-PCR3.1Use RT-PCR to detect gene expression changes of apoptosis genecaspase-3of different groups in different times(0hour4hours8hours3days7days).0hour after surgery （63.00±2.81），（63.42±5.36）， （65.32±4.73），（68.12±4.66）.4hours（70.41±7.60），（59.84±7.15），（65.73±7.08），（79.70±8.83）.8hours（89.82±5.31），（76.91±8.04），（81.76±5.84），（106.14±11.50）.24hours（162.80±8.32），（149.09±10.60），（156.73±15.76），（189.92±7.76）.3days（171.57±6.70），（155.75±6.44），（163.11±7.19），（204.52±10.22）.7days（163.22±8.65），（153.60±8.55），（157.03±9.60），（163.10±6.74）. There were significant differents betweeneach experimental groups and control group（p<0.05）in the time of4hours to3days. And no differents in every groups of flap at7days afteroperation. Distaldrainage group and Recanalization group are different from Proximal drainagegroup（P<0.05）.3.2The expression of proliferation gene ki67in different groups at0hour,4hours,8hours,24hours,3days and7days.0hour55.21±7.50），（56.84±7.00），（55.94±8.03），（58.83±10.15）.4hours （135.58±7.55），（151.37±5.69），（146.62±8.10），（109.47±7.20）.8hours（146.14±8.14），（173.48±4.49），（168.60±5.57），（127.15±8.72）.24hours（148.68±9.44），（181.29±8.63），（175.59±8.80），（123.27±24.75）.3days（149.86±7.55），（171.80±7.55），（165.05±6.95），（127.52±9.13）.7days（126.07±10.30），（131.84±13.44），（128.81±16.10），（127.34±12.71）. There were significantdifferents between each experimental groups and control group（P<0.05）in thetime of4hours to3days. And no differents in every groups of flap at7daysafteroperation. The expression of apoptosis gene caspase-3and proliferationgene ki67were gradually increasing and get the top in3days after surgery.Conclusion:1Apoptosis gene caspase-3and proliferation gene ki67, get involved inthe process of venous congestion flap organization cell apoptosis andproliferation.2Negative pressure drainage has a significant effect on preventivetreatment of flap venous congestion after flap transplantation or transference.It can relieve the swelling symptom of Venous congestion flap, and alsofacilitate the live rate of flap organization.3In the distal of flap where placed negative pressure drainage to process the pressure suction treatment,it places the near end that can less effectivelyrelieve the hinder of the further side of venous congestion flap’smicrocirculatory system, decrease the blood and other necrotic material’sdeposit under the flap skin,helping to promote tissue get growth and healingat far end, improve survival rate of venous congestion flap that shown onvenous congestion. |
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