The Study On The Variance Disciplinary Of Correlated Apoptosis Gene Bax In Rat Flaps With Ischemia-reperfusion Injury

Objective: To observe the expressive location and quantity of the relative poptosis gene Bax in rat flaps with ischemia–reperfusion injury through the method of immunohistochemistry and reverse transcriptase PCR; to observe the apoptosis conditions and changing regulation of the rat flaps with ischemia–reperfusion injury through the method of TUNEL ; research the relativation between the above. And then to reveal the relations and changing regulation between the relative Apoptosis gene Bax and apoptotic cell in rat flaps with ischemia–reperfusion injury. Thereby to find a new protection route for therat flaps with ischemia–reperfusion injury.Method: 1.laboratory animal: 50 with irrespective of paroecious and healthy Wister rats were weighed and ordered. 2. The creation method of flap:The rats were anesthetized with 3﹪sodium pentobarbital (0.1～0.13ml/100mg) by intraperitoneal injection ,and there was another anesthetized every one hour and 0.05ml. their limbs were fixed and their hairs on abdmon were sheared,the abdmon was sterilized by 75﹪alcohol after the hairs were cut down.A right low abdominal island flap ,which size was 3㎝×6㎝,was created according to the method of Petry JJ's. 3. The rats were divided into two section randomly. The first section was control group, which included 25 rats. There were no other treatments after creating the flap in this group. The second section was ischemia-reperfusion group(IR group), which included 25 rats: After creating the flap rat,The distally of the point that the superficial epigastric artery arised from the femoral artery was ligated with 5-0 suture silk. The proximal end of the point was occluded by a vascular clamp. The flap was sewed up with 3-0 suture silk. The vascular clamp was taken out in another operation after 8 hours.Five groups according the time of after taking out vascular clamp were divided into 0,1,6,12,24 hour group. 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 control group and testing group. Part of the obtained flap was conserved in 4﹪paraformaldehyde in order to commit paraffin section and dyeing of immunohistochemistry . Part of the obtained flap was conserved in a -80℃freezer after freezing in LIN in order to detect with RT-PCR. 5. The observation marker: (1) According to the specification of the kit directions, adopted to the method of TUNEL, by means of the percentage of average masccline cell as the index of apoptosis cell(AI). (2)To detect the expression of Bax protein: It will be obstructed endogenous POD and repaired antigen after brought the slice to deparaffinage and water. To detect the expression of Bax protein thought the method of SP,DAB coloration;According to the pill's color of palm and brown in cell membrane or endochylema as the masculine of Bax protein . the result be expressed by the express index of the BAX proteinum masccline: adopt to computer image analysis system: the chip be enlarged under 400 and 200 times light microscope. random select 5 view through image analysator, detect the quantity of masccline cell, (express by A) Grade 0~1%=0,1~10%=1,10~50%=2,50~80%=3,80~100%=4; detect the quantity of masccline cell, detect the coloration intension of the masccline cell (express by B) grade 0 (Negative),1(weakly positive),2(masculine),3(strength masculine)HIS= A×B (3)Bax mRNA express detection,after abstract total RNA, adopt reverse transcription polymerase chain reaction, Through the analysis of PCR production electrophoresis, take RT-PCR production into sepharose do electrophoresis;The test result were printed by usuing UVP gelatum image imaging system and performed by usuing Gel-Pro Analyzer Version . The relative amount of Bax mRNA were expression with the value of Bax strap gray scale .It was sandardizated strap gray scale ofβ-actin. 6.Analysis of results:All the test data were expression with mean±standard deviation(X±S),dealed with by usuing T analysis of SAS software, significance analysised by usuing the contrast statistics significance with the test P<0.05. Results: 1.The observation result of TUNEL cell: From this test ,in the rat flaps of normal architecture ,that is point to the moment when the flap was created ,we can find odds and ends TUNEL cells in slice of immunohistochemistr with a light microscope.IN the experimental group, with prolonging the time of ischemic and reperfusion, The TUNEL cell population were increased rapidly,but it's speed was degraded slowly. In control group we can find the same result ,but it's speed was slowly than in the experimental group.we haven't observed the period time that recovered to normal. And there were statistical significance between the control group and experimental group at every period time(P<0.05).So we can draw a conclusion:The cell apoptosis is an important injure factor for ischemia-reperfusion . 2. The expression of Bax protein:From this test, we couldn't observed the expression of Bax protein in the normal flap that was created at the moment.In experimental group,With prolonging time of ischemia-reperfusion injure,the expression of Bax protien was increased suddenly,and achieved to peak after reperfusing 6 hour,from this time it was decreased slowly.And in control group,we can find the expression of bax protein was decreased slowly than in experimental group . In this test ,we couldn't observed the period time that recovered to normal .There was statistical significance between control group and experimental group after reperfusing the period time of 0,1,6,12,24 hour. Another expression was observed that there was an tendency dependablity between Bax protein and cell apoptosis. From this result we can draw a conclusion :The apoptosis related gen Bax can control the process of apoptosis in architecture flaps. 3.The expression of Bax mRNA: From this test, we can observed that Bax mRNA can chang into specific DNA frag after amplification by RT-PCR.The genic frag length is 407bp.In experimental group,with the time lengthened that expression level of Bax genetic transcription raised up gradually, and reached up to peak after ischemia- reperfusion injury 6 hour. From this time it will decreased gradually ,but we couldn't find the time that recoved to normal.In control group,we can find the expression of Bax genetic transcription decresed slowly than in the experimental group with the time prolonged,and not find the period time of recoved too normal. The expression of both Bax mRNA and Bax proteinum were coincidenced in rat skin flaps. Quibus we could draw a conclusion: From some kind of degree ,the related gene Bax could control the progress of apoptosis in rat skin flaps.Conclusion: Through observed the change rule of apoptosis related gene Bax in rat flaps with ischemia–reperfusion injury ,we can conclude some results:(1)The quantity of apoptosis was increased obviously in rat flaps with ischemia-reperfusion . With prolonging the time and decreasing contaminate agent, the quantity of apoptosis was increased in both groups.but in control group,it's degree of apoptosis was lower than in the experimental group.So we could conclusion that the apoptosis was a injure factor for ischemia-reperfusion of flaps. (2) The expression of the apoptosis gene Bax and the transcript Bax-mRNA by usuing the method of immunohistochemistry and reverse transcriptase PCR :With the ischemia-reperfusion happened in rat flaps, the expression of apoptosis gene Bax was increased significantly ,and reach to the peak after ischemia-reperfusio 6 hours,then recovered slowly.this phenomenon was identical with apoptotrule rule. it can provide a correlated A/W for clinical practice on protecting and curing the flaps ischemia-reperfusion injure.Thereby to find a new protection route for the the rat flaps with ischemia–reperfusion injury.