天然、重组水蛭素对大鼠随意皮瓣淤血模型的影响研究
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摘要
第一部分天然、重组水蛭素对大鼠随意皮瓣淤血模型VEGF、CD34的影响
目的:探讨天然、重组水蛭素对大鼠随意皮瓣淤血模型成活保护机制作用,以及对皮瓣血管内皮细胞生长因子(VEGF)、CD34的作用是否相同。
方法:1、动物模型建立:Wistar大鼠30只,雌雄不限。大鼠麻醉后,取俯卧位固定于手术台,以8%硫化钠涂于大鼠背部术区进行脱毛,温水清洗脱毛区后用画线笔设计以后正中线为轴,蒂在尾端(以骼棘连线为底)的10cm×3cm的背部超长随意皮瓣。消毒术区,铺无菌手术巾,严格无菌操作。沿设计线切开至背部肌膜,在其浅面将皮肤与肉膜由头端向尾端剥离,保证所形成皮瓣为随意皮瓣,在皮瓣形成后即刻3-0丝线缝合。根据术后注射药物不同,皮瓣随机分为3组(n=30),每组10只。A组在皮下注射5u天然水蛭素; B组皮下注射5u重组水蛭素;C组皮下注射相同剂量生理盐水。每组皮瓣注射部位均为在距皮瓣末端约1.5cm和3cm处,分术后即刻、第3、5天注射达皮下层次。
2、术后对大鼠背部皮瓣进行连续的临床观察并记录。观察实验动物是否出现腹泻,弓背,翘毛,精神萎靡及皮肤溃疡等免疫排斥反应。观察皮瓣颜色、厚度、弹性、渗出、毛发生长、坏死范围及针刺皮瓣出血情况,详细记录。
3、术后第7天,在皮瓣远端约2～3 cm处切取0.5cm×0.5cm大小全层皮肤组织;组织浸泡于4%多聚甲醛液中。固定7天后石蜡包埋,制成4um厚的切片,HE染色。观察皮瓣组织的中性粒细胞浸润、细胞水肿、毛细血管增生、肉芽组织生成等情况。
4、手术后的第3、5、7天分别在皮瓣远端2～3 cm处取约0.5cm×0.5 cm大小的全层组织,测定VEGF、CD34含量,以血管内皮细胞生长因子和CD34作为血管标记行免疫组化染色,计数皮瓣新生血管密度。荧光RT-PCR测定VEGFmRNA表达量。
5、术后第7天,麻醉大鼠后,展平背部,用数码照相机拍摄皮瓣情况,用Image-Pro6.0软件分析系统计算皮瓣坏死面积、成活面积和总面积并计算皮瓣成活率。
结果:1、皮瓣存活情况的大体观察三组大鼠均无死亡,皮瓣术后无感染。术后第7天,三组皮瓣远端不同面积痂壳形成,坏死区域稳定,成活与坏死界限较清楚,天然、重组水蛭素组皮瓣痂壳呈现深浅不一致灰色,浅灰色,部分痂壳较软,能轻易剥落,剥落后可见创面鲜红出血,对照组痂壳呈棕深黑,较硬,不易剥落,局部有溃烂。
2、皮瓣组织学观察术后7天,天然水蛭素组皮瓣表皮完整,无萎缩,轻度角化过度,真皮内及皮下无明显炎症细胞浸润,组织无水肿,真皮下微血管多;重组水蛭素组表皮萎缩不明显,皮下组织轻度水肿,炎症细胞浸润略重,真皮下微血管较多;对照组皮下水肿明显,大量炎症细胞浸润,局部表皮溃疡形成,真皮下仅有少量微血管。
3、VEGF染色阳性的各实验组血管密度值免疫组化着色好,染色效果好,背景干净无污染,阳性细胞显示明确。VEGF主要定位于细胞浆中,VEGF阳性表达的细胞被染成棕色颗粒。术后第3天,VEGF染色阳性血管数目:天然水蛭素组(A)为41.71±3.08,重组水蛭素组(B)为33.05±2.23,对照组(C)为14.71±2.67,各组两两比较P<0.05,其中A组VEGF染色阳性血管数最多,B组次之,C组最少。术后第5天,VEGF染色阳性血管数目:天然水蛭素组(A)为55.24±3.96,重组水蛭素组(B)为41.63±3.97,对照组(C)为8.63±2.59,各组两两比较P<0.05,其中A组VEGF染色阳性血管数最多,B组次之,C组最少。术后第7天,VEGF染色阳性血管数目:天然水蛭素组(A)为40.76±3.69,重组水蛭素组(B)为32.93±2.54,对照组(C)为3.58±2.97,各组两两比较P<0.05,其中A组VEGF染色阳性血管数最多,B组次之,C组最少。在A组,VEGF染色阳性血管数目在术后第5天与第3、7天相比P<0.05,高峰在术后第5天;在B组,VEGF染色阳性血管数目在术后第5天与第3、7天相比P<0.05,高峰在术后第5天;在C组,VEGF染色阳性血管数目在术后第3天与第5、7天两两相比P<0.05,高峰在术后第3天。
4、CD34染色阳性的各实验组血管密度值CD34主要定位于细胞浆中,少量在细胞膜亦有表达,CD34阳性细胞表现为棕黄色至棕褐色颗粒。术后第3天,CD34染色阳性血管数目:天然水蛭素组(A)为56.83±3.19,重组水蛭素组(B)为47.12±2.13,对照组(C)为21.82±2.56,各组两两比较P<0.05,其中A组CD34染色阳性血管数最多,B组次之,C组最少。术后第5天,CD34染色阳性血管数目:天然水蛭素组(A)为70.13±3.85,重组水蛭素组(B)为52.74±3.08,对照组(C)为16.52±2.48,各组两两比较P<0.05,其中A组CD34染色阳性血管数最多,B组次之,C组最少。术后第7天,CD34染色阳性血管数目:天然水蛭素组(A)为55.87±3.75,重组水蛭素组(B)为45.93±3.65,对照组(C)为11.47±2.87,各组两两比较P<0.05,其中A组CD34染色阳性血管数最多,B组次之,C组最少。在A组,CD34染色阳性血管数目在术后第5天与第3、7天相比P<0.05,高峰在术后第5天;在B组,CD34染色阳性血管数目在术后第5天与第3、7天相比P<0.05,高峰在术后第5天;在C组,CD34染色阳性血管数目在术后第3天与第5、7天两两相比P<0.05,高峰在术后第3天。
5、荧光RT-PCR检测各组皮瓣组织VEGFmRNA的表达电泳后各泳道均可见特异性条带,与DNA标记进行比较,产物大小与预期产物大小一致,分别为VEGF164 bp,GAPDH 152 bp。术后第3天, VEGFmRNA的表达:天然水蛭素组(A)为28.69±2.07,重组水蛭素组(B)为23.94±3.12,对照组(C)为19.64±1.92,各组两两比较P<0.05,其中A组VEGFmRNA的表达最多,B组次之,C组最少。术后第5天,VEGFmRNA的表达:天然水蛭素组(A)为33.13±4.07,重组水蛭素组(B)为29.52±2.89,对照组(C)为15.52±3.48,各组两两比较P<0.05,其中A组VEGFmRNA的表达最多,B组次之,C组最少。术后第7天,VEGFmRNA的表达:天然水蛭素组(A)为27.87±2.58,重组水蛭素组(B)为22.93±3.43,对照组(C)为10.47±2.86,各组两两比较P<0.05,其中A组VEGFmRNA的表达最多,B组次之,C组最少。在A组,VEGFmRNA的表达在术后第5天与第3、7天相比P<0.05,高峰在术后第5天;在B组,VEGFmRNA的表达在术后第5天与第3、7天相比P<0.05,高峰在术后第5天;在C组,VEGFmRNA的表达在术后第3天与第5、7天两两相比P<0.05,高峰在术后第3天。
6、皮瓣存活表面积:皮瓣存活表面积:天然水蛭素组(A): 26.64±2.24(cm~2),重组水蛭素组(B): 23.64±2.02(cm~2),对照组(C): 20.71±1.41(cm~2);皮瓣存活率(%):天然水蛭素组(A): 88.87±2.24,重组水蛭素组(B): 79.97±2.02,对照组(C): 69.07±1.42.各组之间两两比较,P<0.05。
结论:天然、重组水蛭素能够促进随意皮瓣VEGF、CD34表达,改善淤血皮瓣的微循环,有利于新生血管的生成,能够提高随意皮瓣的成活率,天然水蛭素效果优于重组水蛭素。
第二部分天然、重组水蛭素对大鼠随意皮瓣淤血模型SOD、MDA、ET影响
目的:探讨天然、重组水蛭素对大鼠随意皮瓣淤血模型存活保护机制作用,以及对皮瓣的丙二醛(MDA)、超氧化物岐化酶(SOD)、内皮素(ET)作用是否相同。
方法:1、动物模型建立:同第一部分。
2、术后对大鼠背部皮瓣进行连续的临床观察并记录。同第一部分。
3、皮瓣组织学的观察,同第一部分。
4、术后第7天测定皮瓣成活率,以及丙二醛(MDA)、超氧化物岐化酶(SOD)、内皮素(ET)含量;其中丙二醛(MDA)的检测采用硫代巴比妥酸法,超氧化物岐化酶(SOD)的检测采用羟胺法,内皮素(ET)的检测采用酶联免疫吸附试验法。
结果:1、皮瓣存活情况的大体观察同第一部分。
2、皮瓣组织学观察同第一部分
3、术后第7天皮瓣组织丙二醛(MDA)、超氧化物岐化酶(SOD)、内皮素(ET)含量:皮瓣组织丙二醛(MDA)含量:天然水蛭素组(A):10.20±3.41(nmol/mgprot),重组水蛭素组(B):22.12±3.84(nmol/mgprot),对照组(C):32.65±3.91(nmol/mgprot);各组之间两两比较,P<0.05。术后第7天皮瓣组织超氧化物岐化酶(SOD)含量:天然水蛭素组(A):72.59±17.48(U/mgprot),重组水蛭素组(B):36.68±8.32 (U/mgprot),对照组(C):27.66±11.34(U/mgprot);各组之间两两比较,P<0.05。术后第7天皮瓣组织内皮素(ET)含量:天然水蛭素组(A):85.67±37.90,重组水蛭素组(B):163.55±43.45,对照组(C):247.56±38.04;各组之间两两比较,P<0.05。用药组(A组和B组)皮瓣成活率比对照组(C组)高,超氧化物岐化酶(SOD)用药组(A组和B组)比对照组(C组)明显增多,丙二醛(MDA)、内皮素(ET)含量则减少。
4、皮瓣存活表面积:同第一部分。
结论:水蛭素能够提高随意皮瓣的成活率、超氧化物岐化酶(SOD)的含量;减低皮瓣的坏死率、丙二醛(MDA)、内皮素(ET)的含量。实验组较对照组有明显差异。水蛭素具有抗血栓、抗凝、抗氧化和抗炎症作用。其中,天然水蛭素效果最明显,重组水蛭素次之。
Objective
To investigate the effect of natural hirudin and recombinant hirudin which are applied locally to the vein congestion on random skin flap in rats models, and their effect on the flap of VEGF and CD34 are the same or not.
Methods
1. Establishment of animal model:
Thirty Wistar rats, including male and female,were employed to establish animal model of vein congestive flap. All rats were anaesthetized by 10% chloral hydrate (300mg/kg). The hair of dorsal skin was removed with 8% sodium sulfide and the skin was sterilized with betadine. Caudally based dorsal flaps (10 cm×3 cm) in size were raised under sterile conditions. The palpable hip joints were used as anatomical landmarks to define the base of the flap. The flap was dissected and detached from its panniculus carnosus and reattached in the native position with separate sutures. The sites of injection were 1 ~ 3 cm distal to the flaps. The time of injection was immediately after surgery and re-injected at day 3 and day 5 post-operation. The injection spot was at the hypodermic level. The rats were divided randomly into 3 groups (ten in each group). In group A, 5 U of natural hirudin was locally applied to each flap. In groups B, 5 U of recombinant hirudin was locally applied to each flap. In group C, isotonic NaCl was locally applied to each flap as a control group.
2. Make continuous clinical observation and record the back flap of the rats during 7 days after operation. Observe whether there is immunological rejection , such as diarrhea, Camponotus, Alice hair,depression and skin ulcers to the experimental animals. Observe the flap color,thickness, elasticity, exudation,hair growth, necrosis area and acupuncture bleeding situation,then record in detail.
3. Histopathological evaluation: The samples were preserved in 10% formalin solution. Transverse sections were taken at the flap base 3 cm (viable area) or 7 cm (necrosis area) distal to the viable-necrotic area boundaries. The tissue samples were embedded in paraffin blocks; 0.5cm×0.5cm thick sections were cut and stained with haematoxylin and eosin and evaluated with a light microscope.
4. VEGF and CD34 levels were measured on postoperative day 3,5 and 7. VEGF and CD34 were determined by immunohistochemistry. VEGF mRNA expression measured by fluorescence RT-PCR.
5. To determine the skin flap survival rate, photographs of the skin flaps 7 days after surgery were analyzed using the Image-Pro 6.0 software. The flap survival rate was calculated as the percentage of the surviving flap area over the total flap area.
Results
1.General observation of the flap survival All rats survived until the end of the study without infection. Seven days after surgery, the necrotic area was stabilized with clear boundaries between the viable and necrotic areas. In the natural and recombinant hirudin treated groups, parts of the crust shells of the flaps were inconsistent in depth and were gray or light gray in color. These soft scar tissue can be easily peeled off revealing the wound and bleeding underneath. In contrast, the crust shells in the control group were hard with deep dark brown color.and contained local fester which can not be easily peeled off.
2. Histopathological observation of the flaps
In the natural hirudin group, no atrophy was observed. Only mild hyperkeratosis and inflammatory cell infiltration were present in the flap tissue. There was an increase in subcutaneous vessel densities in the treated group. In contrast, subcutaneous flap edema and local ulceration were seen in the control group, with fewer subcutaneous vessels observed. The histopathological features of the recombinant hirudin group were intermediate among the three groups.
3. The VEGF positive vessel density in flap
Immunohistochemical coloring well. Good dyeing effect. Clean background pollution. Positive cells displayed clear. Postoperative day 3, VEGF staining the number of positive vessels: natural hirudin group (A): 41.71±3.08, recombinant hirudin group (B): 33.05±2.23, control group (C): 14.71±2.67. Both treatment groups have significantly higher numbers of VEGF positive vessels compared to control group (P<0.05). The largest number of VEGF positive vessels stained was group A . Group B followed it .Group C was the least. Postoperative day 5, VEGF staining the number of positive vessels: natural hirudin group (A): 55.24±3.96, recombinant hirudin group (B) : 41.63±3.97, control group (C): 8.63±2.59. Both treatment groups have significantly higher numbers of VEGF positive vessels compared to control group (P<0.05). The largest number of VEGF positive vessels stained was group A . Group B followed it. Group C was the least. Postoperative day 7, VEGF staining the number of positive vessels: natural hirudin group (A) : 40.76±3.69, recombinant hirudin group (B) : 32.93±2.54, control group (C): 3.58±2.97. Both treatment groups have significantly higher numbers of VEGF positive vessels compared to control group (P<0.05). The largest number of VEGF positive vessels stained was group A . Group B followed it .Group C was the least. In group A, the 5th day VEGF positive vessels stained compared with the 3th and 7th day ,P <0.05, peak at postoperative day 5; In group B, the 5th day VEGF positive vessels stained compared with the 3th and 7th day P <0.05, peak at postoperative day 5; In the C group, the 5th day VEGF positive vessels stained compared with the 3th and 7th day P <0.05, peak at postoperative day 3.
4. The CD34 positive vessel density in flap
Postoperative day 3, CD34 staining the number of positive vessels: natural hirudin group (A): 56.83±3.19, recombinant hirudin group (B): 47.12±2.13, control group (C):21.82±2.56. Both treatment groups have significantly higher numbers of CD34 positive vessels compared to control group (P<0.05). The largest number of CD34 positive vessels stained was group A. Group B followed it. Group C was the least. Postoperative day 5, CD34 staining the number of positive vessels: natural hirudin group (A):70.13±3.85, recombinant hirudin group (B): 52.74±3.08, control group (C):16.52±2.48. Both treatment groups have significantly higher numbers of CD34 positive vessels compared to control group (P<0.05). The largest number of CD34 positive vessels stained was group A .Group B followed it. Group C was the least. Postoperative day 7, CD34 staining the number of positive vessels: natural hirudin group (A):55.87±3.75, recombinant hirudin group (B):45.93±3.65, control group (C):11.47±2.87.Both treatment groups have significantly higher numbers of CD34 positive vessels compared to control group (P<0.05). The largest number of CD34 positive vessels stained was group A . Group B followed it. Group C was the least. In group A, the 5th day CD34 positive vessels stained compared with the 3th and 7th day P <0.05, peak at postoperative day 5; In the B group, the 5th day CD34 positive vessels stained compared with the 3th and 7th day P <0.05, peak at postoperative day 5; In the C group, the 5th day CD34 positive vessels stained compared with the 3th and 7th day P <0.05, peak at postoperative day 3.
5. Fluorescent RT-PCR to detect the expression of flap tissue VEGFmRNA
Postoperative day 3, VEGFmRNA expresses:natural hirudin group (A):28.69±2.07, recombinant hirudin group (B) : 23.94±3.12, control group (C):19.64±1.92. Both treatment groups have significantly higher numbers of VEGFmRNA compared to control group (P<0.05). The largest number of VEGFmRNA expresses was group A, Group B followed it. Group C was the least. Postoperative day 5, VEGFmRNA expresses:natural hirudin group (A):33.13±4.07, recombinant hirudin group (B):29.52±2.89, control group (C):15.52±3.48. Both treatment groups have significantly higher numbers of VEGFmRNA compared to control group (P<0.05). The largest number of VEGFmRNA expresses was group A. Group B followed it. Group C was the least. Postoperative day 7, VEGFmRNA expresses:natural hirudin group (A):27.87±2.58, recombinant hirudin group (B):22.93±3.43, control group (C):10.47±2.86. Both treatment groups have significantly higher numbers of VEGFmRNA compared to control (P<0.05). The largest number of VEGFmRNA expresses was group A. Group B followed it. Group C was the least. In group A, the 5th day VEGFmRNA expresses compared with the 3th and 7th day P <0.05, peak at postoperative day 5; In group B, the 5th day VEGFmRNA expresses compared with the 3th and 7th day P <0.05, peak at postoperative day 5; In group C, the 5th day VEGFmRNA expresses compared with the 3th and 7th day P <0.05, peak at postoperative day 3.
6. Flap survival
Surface area of flap survival: natural hirudin group (A): 26.64±2.24 (cm~2), recombinant hirudin group (B): 23.64±2.02 (cm~2), control group (C): 20.71±1.41 (cm~2); flap survival rate (%): natural hirudin group (A): 88.87±2.24, recombinant hirudin group (B):79.97±2.02, control group (C):69.07±1.42. Pairwise comparison between three groups, P <0.05.
Conclusion
VEGF and CD34 expression in random skin flap can be increased by natural hirudin and recombinant hirudin which are conducive to angiogenesis and can improve the survival rate of random skin flap. Natural hirudin is superior than recombinant hirudin.
OBJECTIVE
To investigate the effect of natural hirudin and recombinant hirudin which are applied locally to the vein congestion on random skin flap in rats models. And the flap of SOD, MDA and ET expression are the same or not.
METHODS
1,Animal model: It is the same with the first.
2. Make continuous clinical observation and record the back flap of the rats during 7 days after operation. It is the same with the first.
3. Histopathological evaluation: It is the same with the first.
4. SOD, ET and MDA levels were measured after surgery. In the light microscope, the flap cell morphological changes were observed.Flap survival 7 days after surgery was calculated. MDA was detected through thiobarbituric acid method. SOD was detected using hydroxylamine method. ET was detected by enzyme linked immunosorbent assay method.
Results
1.General observation of the flap survival It is the same with the first.
2.Histopathological observation of the flaps It is the same with the first.
3. MDA, SOD, and ET levels in the flaps Postoperative day 7, MDA content of the flap: Natural hirudin group (A): 10.20±3.41 (nmol / mgprot), Recombinant hirudin group (B): 22.12±3.84 (nmol / mgprot), Control group (C) : 32.65±3.91 (nmol / mgprot); MDA is significant different from each other (P <0.05). Postoperative day 7, SOD levels of the flap: natural hirudin group (A): 72.59±17.48 (U / mgprot), recombinant hirudin group (B): 36.68±8.32 (U / mgprot), the control group (C) : 27.66±11.34 (U / mgprot); SOD is significant different from each other (P <0.05). ET levels of the flap: Natural hirudin group (A): 85.67±37.90, Recombinant hirudin group (B): 163.55±43.45, Control group (C): 247.56±38.04; ET is significant different from each other (P <0.05).
4. Flap survival
It is the same with the first.
CONCLUSIONS
Hirudin can improve the flap survival rate and SOD content; reduce the rate of flap necrosis, MDA, and ET levels. The experimental group were significantly different from the control group. Natural hirudin demonstrated more pronounced effects than recombinant hirudin.
目的:探讨天然、重组水蛭素对大鼠随意皮瓣淤血模型成活保护机制作用,以及对皮瓣血管内皮细胞生长因子(VEGF)、CD34的作用是否相同。
方法:1、动物模型建立:Wistar大鼠30只,雌雄不限。大鼠麻醉后,取俯卧位固定于手术台,以8%硫化钠涂于大鼠背部术区进行脱毛,温水清洗脱毛区后用画线笔设计以后正中线为轴,蒂在尾端(以骼棘连线为底)的10cm×3cm的背部超长随意皮瓣。消毒术区,铺无菌手术巾,严格无菌操作。沿设计线切开至背部肌膜,在其浅面将皮肤与肉膜由头端向尾端剥离,保证所形成皮瓣为随意皮瓣,在皮瓣形成后即刻3-0丝线缝合。根据术后注射药物不同,皮瓣随机分为3组(n=30),每组10只。A组在皮下注射5u天然水蛭素; B组皮下注射5u重组水蛭素;C组皮下注射相同剂量生理盐水。每组皮瓣注射部位均为在距皮瓣末端约1.5cm和3cm处,分术后即刻、第3、5天注射达皮下层次。
2、术后对大鼠背部皮瓣进行连续的临床观察并记录。观察实验动物是否出现腹泻,弓背,翘毛,精神萎靡及皮肤溃疡等免疫排斥反应。观察皮瓣颜色、厚度、弹性、渗出、毛发生长、坏死范围及针刺皮瓣出血情况,详细记录。
3、术后第7天,在皮瓣远端约2～3 cm处切取0.5cm×0.5cm大小全层皮肤组织;组织浸泡于4%多聚甲醛液中。固定7天后石蜡包埋,制成4um厚的切片,HE染色。观察皮瓣组织的中性粒细胞浸润、细胞水肿、毛细血管增生、肉芽组织生成等情况。
4、手术后的第3、5、7天分别在皮瓣远端2～3 cm处取约0.5cm×0.5 cm大小的全层组织,测定VEGF、CD34含量,以血管内皮细胞生长因子和CD34作为血管标记行免疫组化染色,计数皮瓣新生血管密度。荧光RT-PCR测定VEGFmRNA表达量。
5、术后第7天,麻醉大鼠后,展平背部,用数码照相机拍摄皮瓣情况,用Image-Pro6.0软件分析系统计算皮瓣坏死面积、成活面积和总面积并计算皮瓣成活率。
结果:1、皮瓣存活情况的大体观察三组大鼠均无死亡,皮瓣术后无感染。术后第7天,三组皮瓣远端不同面积痂壳形成,坏死区域稳定,成活与坏死界限较清楚,天然、重组水蛭素组皮瓣痂壳呈现深浅不一致灰色,浅灰色,部分痂壳较软,能轻易剥落,剥落后可见创面鲜红出血,对照组痂壳呈棕深黑,较硬,不易剥落,局部有溃烂。
2、皮瓣组织学观察术后7天,天然水蛭素组皮瓣表皮完整,无萎缩,轻度角化过度,真皮内及皮下无明显炎症细胞浸润,组织无水肿,真皮下微血管多;重组水蛭素组表皮萎缩不明显,皮下组织轻度水肿,炎症细胞浸润略重,真皮下微血管较多;对照组皮下水肿明显,大量炎症细胞浸润,局部表皮溃疡形成,真皮下仅有少量微血管。
3、VEGF染色阳性的各实验组血管密度值免疫组化着色好,染色效果好,背景干净无污染,阳性细胞显示明确。VEGF主要定位于细胞浆中,VEGF阳性表达的细胞被染成棕色颗粒。术后第3天,VEGF染色阳性血管数目:天然水蛭素组(A)为41.71±3.08,重组水蛭素组(B)为33.05±2.23,对照组(C)为14.71±2.67,各组两两比较P<0.05,其中A组VEGF染色阳性血管数最多,B组次之,C组最少。术后第5天,VEGF染色阳性血管数目:天然水蛭素组(A)为55.24±3.96,重组水蛭素组(B)为41.63±3.97,对照组(C)为8.63±2.59,各组两两比较P<0.05,其中A组VEGF染色阳性血管数最多,B组次之,C组最少。术后第7天,VEGF染色阳性血管数目:天然水蛭素组(A)为40.76±3.69,重组水蛭素组(B)为32.93±2.54,对照组(C)为3.58±2.97,各组两两比较P<0.05,其中A组VEGF染色阳性血管数最多,B组次之,C组最少。在A组,VEGF染色阳性血管数目在术后第5天与第3、7天相比P<0.05,高峰在术后第5天;在B组,VEGF染色阳性血管数目在术后第5天与第3、7天相比P<0.05,高峰在术后第5天;在C组,VEGF染色阳性血管数目在术后第3天与第5、7天两两相比P<0.05,高峰在术后第3天。
4、CD34染色阳性的各实验组血管密度值CD34主要定位于细胞浆中,少量在细胞膜亦有表达,CD34阳性细胞表现为棕黄色至棕褐色颗粒。术后第3天,CD34染色阳性血管数目:天然水蛭素组(A)为56.83±3.19,重组水蛭素组(B)为47.12±2.13,对照组(C)为21.82±2.56,各组两两比较P<0.05,其中A组CD34染色阳性血管数最多,B组次之,C组最少。术后第5天,CD34染色阳性血管数目:天然水蛭素组(A)为70.13±3.85,重组水蛭素组(B)为52.74±3.08,对照组(C)为16.52±2.48,各组两两比较P<0.05,其中A组CD34染色阳性血管数最多,B组次之,C组最少。术后第7天,CD34染色阳性血管数目:天然水蛭素组(A)为55.87±3.75,重组水蛭素组(B)为45.93±3.65,对照组(C)为11.47±2.87,各组两两比较P<0.05,其中A组CD34染色阳性血管数最多,B组次之,C组最少。在A组,CD34染色阳性血管数目在术后第5天与第3、7天相比P<0.05,高峰在术后第5天;在B组,CD34染色阳性血管数目在术后第5天与第3、7天相比P<0.05,高峰在术后第5天;在C组,CD34染色阳性血管数目在术后第3天与第5、7天两两相比P<0.05,高峰在术后第3天。
5、荧光RT-PCR检测各组皮瓣组织VEGFmRNA的表达电泳后各泳道均可见特异性条带,与DNA标记进行比较,产物大小与预期产物大小一致,分别为VEGF164 bp,GAPDH 152 bp。术后第3天, VEGFmRNA的表达:天然水蛭素组(A)为28.69±2.07,重组水蛭素组(B)为23.94±3.12,对照组(C)为19.64±1.92,各组两两比较P<0.05,其中A组VEGFmRNA的表达最多,B组次之,C组最少。术后第5天,VEGFmRNA的表达:天然水蛭素组(A)为33.13±4.07,重组水蛭素组(B)为29.52±2.89,对照组(C)为15.52±3.48,各组两两比较P<0.05,其中A组VEGFmRNA的表达最多,B组次之,C组最少。术后第7天,VEGFmRNA的表达:天然水蛭素组(A)为27.87±2.58,重组水蛭素组(B)为22.93±3.43,对照组(C)为10.47±2.86,各组两两比较P<0.05,其中A组VEGFmRNA的表达最多,B组次之,C组最少。在A组,VEGFmRNA的表达在术后第5天与第3、7天相比P<0.05,高峰在术后第5天;在B组,VEGFmRNA的表达在术后第5天与第3、7天相比P<0.05,高峰在术后第5天;在C组,VEGFmRNA的表达在术后第3天与第5、7天两两相比P<0.05,高峰在术后第3天。
6、皮瓣存活表面积:皮瓣存活表面积:天然水蛭素组(A): 26.64±2.24(cm~2),重组水蛭素组(B): 23.64±2.02(cm~2),对照组(C): 20.71±1.41(cm~2);皮瓣存活率(%):天然水蛭素组(A): 88.87±2.24,重组水蛭素组(B): 79.97±2.02,对照组(C): 69.07±1.42.各组之间两两比较,P<0.05。
结论:天然、重组水蛭素能够促进随意皮瓣VEGF、CD34表达,改善淤血皮瓣的微循环,有利于新生血管的生成,能够提高随意皮瓣的成活率,天然水蛭素效果优于重组水蛭素。
第二部分天然、重组水蛭素对大鼠随意皮瓣淤血模型SOD、MDA、ET影响
目的:探讨天然、重组水蛭素对大鼠随意皮瓣淤血模型存活保护机制作用,以及对皮瓣的丙二醛(MDA)、超氧化物岐化酶(SOD)、内皮素(ET)作用是否相同。
方法:1、动物模型建立:同第一部分。
2、术后对大鼠背部皮瓣进行连续的临床观察并记录。同第一部分。
3、皮瓣组织学的观察,同第一部分。
4、术后第7天测定皮瓣成活率,以及丙二醛(MDA)、超氧化物岐化酶(SOD)、内皮素(ET)含量;其中丙二醛(MDA)的检测采用硫代巴比妥酸法,超氧化物岐化酶(SOD)的检测采用羟胺法,内皮素(ET)的检测采用酶联免疫吸附试验法。
结果:1、皮瓣存活情况的大体观察同第一部分。
2、皮瓣组织学观察同第一部分
3、术后第7天皮瓣组织丙二醛(MDA)、超氧化物岐化酶(SOD)、内皮素(ET)含量:皮瓣组织丙二醛(MDA)含量:天然水蛭素组(A):10.20±3.41(nmol/mgprot),重组水蛭素组(B):22.12±3.84(nmol/mgprot),对照组(C):32.65±3.91(nmol/mgprot);各组之间两两比较,P<0.05。术后第7天皮瓣组织超氧化物岐化酶(SOD)含量:天然水蛭素组(A):72.59±17.48(U/mgprot),重组水蛭素组(B):36.68±8.32 (U/mgprot),对照组(C):27.66±11.34(U/mgprot);各组之间两两比较,P<0.05。术后第7天皮瓣组织内皮素(ET)含量:天然水蛭素组(A):85.67±37.90,重组水蛭素组(B):163.55±43.45,对照组(C):247.56±38.04;各组之间两两比较,P<0.05。用药组(A组和B组)皮瓣成活率比对照组(C组)高,超氧化物岐化酶(SOD)用药组(A组和B组)比对照组(C组)明显增多,丙二醛(MDA)、内皮素(ET)含量则减少。
4、皮瓣存活表面积:同第一部分。
结论:水蛭素能够提高随意皮瓣的成活率、超氧化物岐化酶(SOD)的含量;减低皮瓣的坏死率、丙二醛(MDA)、内皮素(ET)的含量。实验组较对照组有明显差异。水蛭素具有抗血栓、抗凝、抗氧化和抗炎症作用。其中,天然水蛭素效果最明显,重组水蛭素次之。
Objective
To investigate the effect of natural hirudin and recombinant hirudin which are applied locally to the vein congestion on random skin flap in rats models, and their effect on the flap of VEGF and CD34 are the same or not.
Methods
1. Establishment of animal model:
Thirty Wistar rats, including male and female,were employed to establish animal model of vein congestive flap. All rats were anaesthetized by 10% chloral hydrate (300mg/kg). The hair of dorsal skin was removed with 8% sodium sulfide and the skin was sterilized with betadine. Caudally based dorsal flaps (10 cm×3 cm) in size were raised under sterile conditions. The palpable hip joints were used as anatomical landmarks to define the base of the flap. The flap was dissected and detached from its panniculus carnosus and reattached in the native position with separate sutures. The sites of injection were 1 ~ 3 cm distal to the flaps. The time of injection was immediately after surgery and re-injected at day 3 and day 5 post-operation. The injection spot was at the hypodermic level. The rats were divided randomly into 3 groups (ten in each group). In group A, 5 U of natural hirudin was locally applied to each flap. In groups B, 5 U of recombinant hirudin was locally applied to each flap. In group C, isotonic NaCl was locally applied to each flap as a control group.
2. Make continuous clinical observation and record the back flap of the rats during 7 days after operation. Observe whether there is immunological rejection , such as diarrhea, Camponotus, Alice hair,depression and skin ulcers to the experimental animals. Observe the flap color,thickness, elasticity, exudation,hair growth, necrosis area and acupuncture bleeding situation,then record in detail.
3. Histopathological evaluation: The samples were preserved in 10% formalin solution. Transverse sections were taken at the flap base 3 cm (viable area) or 7 cm (necrosis area) distal to the viable-necrotic area boundaries. The tissue samples were embedded in paraffin blocks; 0.5cm×0.5cm thick sections were cut and stained with haematoxylin and eosin and evaluated with a light microscope.
4. VEGF and CD34 levels were measured on postoperative day 3,5 and 7. VEGF and CD34 were determined by immunohistochemistry. VEGF mRNA expression measured by fluorescence RT-PCR.
5. To determine the skin flap survival rate, photographs of the skin flaps 7 days after surgery were analyzed using the Image-Pro 6.0 software. The flap survival rate was calculated as the percentage of the surviving flap area over the total flap area.
Results
1.General observation of the flap survival All rats survived until the end of the study without infection. Seven days after surgery, the necrotic area was stabilized with clear boundaries between the viable and necrotic areas. In the natural and recombinant hirudin treated groups, parts of the crust shells of the flaps were inconsistent in depth and were gray or light gray in color. These soft scar tissue can be easily peeled off revealing the wound and bleeding underneath. In contrast, the crust shells in the control group were hard with deep dark brown color.and contained local fester which can not be easily peeled off.
2. Histopathological observation of the flaps
In the natural hirudin group, no atrophy was observed. Only mild hyperkeratosis and inflammatory cell infiltration were present in the flap tissue. There was an increase in subcutaneous vessel densities in the treated group. In contrast, subcutaneous flap edema and local ulceration were seen in the control group, with fewer subcutaneous vessels observed. The histopathological features of the recombinant hirudin group were intermediate among the three groups.
3. The VEGF positive vessel density in flap
Immunohistochemical coloring well. Good dyeing effect. Clean background pollution. Positive cells displayed clear. Postoperative day 3, VEGF staining the number of positive vessels: natural hirudin group (A): 41.71±3.08, recombinant hirudin group (B): 33.05±2.23, control group (C): 14.71±2.67. Both treatment groups have significantly higher numbers of VEGF positive vessels compared to control group (P<0.05). The largest number of VEGF positive vessels stained was group A . Group B followed it .Group C was the least. Postoperative day 5, VEGF staining the number of positive vessels: natural hirudin group (A): 55.24±3.96, recombinant hirudin group (B) : 41.63±3.97, control group (C): 8.63±2.59. Both treatment groups have significantly higher numbers of VEGF positive vessels compared to control group (P<0.05). The largest number of VEGF positive vessels stained was group A . Group B followed it. Group C was the least. Postoperative day 7, VEGF staining the number of positive vessels: natural hirudin group (A) : 40.76±3.69, recombinant hirudin group (B) : 32.93±2.54, control group (C): 3.58±2.97. Both treatment groups have significantly higher numbers of VEGF positive vessels compared to control group (P<0.05). The largest number of VEGF positive vessels stained was group A . Group B followed it .Group C was the least. In group A, the 5th day VEGF positive vessels stained compared with the 3th and 7th day ,P <0.05, peak at postoperative day 5; In group B, the 5th day VEGF positive vessels stained compared with the 3th and 7th day P <0.05, peak at postoperative day 5; In the C group, the 5th day VEGF positive vessels stained compared with the 3th and 7th day P <0.05, peak at postoperative day 3.
4. The CD34 positive vessel density in flap
Postoperative day 3, CD34 staining the number of positive vessels: natural hirudin group (A): 56.83±3.19, recombinant hirudin group (B): 47.12±2.13, control group (C):21.82±2.56. Both treatment groups have significantly higher numbers of CD34 positive vessels compared to control group (P<0.05). The largest number of CD34 positive vessels stained was group A. Group B followed it. Group C was the least. Postoperative day 5, CD34 staining the number of positive vessels: natural hirudin group (A):70.13±3.85, recombinant hirudin group (B): 52.74±3.08, control group (C):16.52±2.48. Both treatment groups have significantly higher numbers of CD34 positive vessels compared to control group (P<0.05). The largest number of CD34 positive vessels stained was group A .Group B followed it. Group C was the least. Postoperative day 7, CD34 staining the number of positive vessels: natural hirudin group (A):55.87±3.75, recombinant hirudin group (B):45.93±3.65, control group (C):11.47±2.87.Both treatment groups have significantly higher numbers of CD34 positive vessels compared to control group (P<0.05). The largest number of CD34 positive vessels stained was group A . Group B followed it. Group C was the least. In group A, the 5th day CD34 positive vessels stained compared with the 3th and 7th day P <0.05, peak at postoperative day 5; In the B group, the 5th day CD34 positive vessels stained compared with the 3th and 7th day P <0.05, peak at postoperative day 5; In the C group, the 5th day CD34 positive vessels stained compared with the 3th and 7th day P <0.05, peak at postoperative day 3.
5. Fluorescent RT-PCR to detect the expression of flap tissue VEGFmRNA
Postoperative day 3, VEGFmRNA expresses:natural hirudin group (A):28.69±2.07, recombinant hirudin group (B) : 23.94±3.12, control group (C):19.64±1.92. Both treatment groups have significantly higher numbers of VEGFmRNA compared to control group (P<0.05). The largest number of VEGFmRNA expresses was group A, Group B followed it. Group C was the least. Postoperative day 5, VEGFmRNA expresses:natural hirudin group (A):33.13±4.07, recombinant hirudin group (B):29.52±2.89, control group (C):15.52±3.48. Both treatment groups have significantly higher numbers of VEGFmRNA compared to control group (P<0.05). The largest number of VEGFmRNA expresses was group A. Group B followed it. Group C was the least. Postoperative day 7, VEGFmRNA expresses:natural hirudin group (A):27.87±2.58, recombinant hirudin group (B):22.93±3.43, control group (C):10.47±2.86. Both treatment groups have significantly higher numbers of VEGFmRNA compared to control (P<0.05). The largest number of VEGFmRNA expresses was group A. Group B followed it. Group C was the least. In group A, the 5th day VEGFmRNA expresses compared with the 3th and 7th day P <0.05, peak at postoperative day 5; In group B, the 5th day VEGFmRNA expresses compared with the 3th and 7th day P <0.05, peak at postoperative day 5; In group C, the 5th day VEGFmRNA expresses compared with the 3th and 7th day P <0.05, peak at postoperative day 3.
6. Flap survival
Surface area of flap survival: natural hirudin group (A): 26.64±2.24 (cm~2), recombinant hirudin group (B): 23.64±2.02 (cm~2), control group (C): 20.71±1.41 (cm~2); flap survival rate (%): natural hirudin group (A): 88.87±2.24, recombinant hirudin group (B):79.97±2.02, control group (C):69.07±1.42. Pairwise comparison between three groups, P <0.05.
Conclusion
VEGF and CD34 expression in random skin flap can be increased by natural hirudin and recombinant hirudin which are conducive to angiogenesis and can improve the survival rate of random skin flap. Natural hirudin is superior than recombinant hirudin.
OBJECTIVE
To investigate the effect of natural hirudin and recombinant hirudin which are applied locally to the vein congestion on random skin flap in rats models. And the flap of SOD, MDA and ET expression are the same or not.
METHODS
1,Animal model: It is the same with the first.
2. Make continuous clinical observation and record the back flap of the rats during 7 days after operation. It is the same with the first.
3. Histopathological evaluation: It is the same with the first.
4. SOD, ET and MDA levels were measured after surgery. In the light microscope, the flap cell morphological changes were observed.Flap survival 7 days after surgery was calculated. MDA was detected through thiobarbituric acid method. SOD was detected using hydroxylamine method. ET was detected by enzyme linked immunosorbent assay method.
Results
1.General observation of the flap survival It is the same with the first.
2.Histopathological observation of the flaps It is the same with the first.
3. MDA, SOD, and ET levels in the flaps Postoperative day 7, MDA content of the flap: Natural hirudin group (A): 10.20±3.41 (nmol / mgprot), Recombinant hirudin group (B): 22.12±3.84 (nmol / mgprot), Control group (C) : 32.65±3.91 (nmol / mgprot); MDA is significant different from each other (P <0.05). Postoperative day 7, SOD levels of the flap: natural hirudin group (A): 72.59±17.48 (U / mgprot), recombinant hirudin group (B): 36.68±8.32 (U / mgprot), the control group (C) : 27.66±11.34 (U / mgprot); SOD is significant different from each other (P <0.05). ET levels of the flap: Natural hirudin group (A): 85.67±37.90, Recombinant hirudin group (B): 163.55±43.45, Control group (C): 247.56±38.04; ET is significant different from each other (P <0.05).
4. Flap survival
It is the same with the first.
CONCLUSIONS
Hirudin can improve the flap survival rate and SOD content; reduce the rate of flap necrosis, MDA, and ET levels. The experimental group were significantly different from the control group. Natural hirudin demonstrated more pronounced effects than recombinant hirudin.
引文
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