重组水蛭素对大鼠撕脱皮瓣存活影响的实验研究

发布时间：2018-05-25 00:01

本文选题：重组水蛭素 + 撕脱皮瓣　；参考：《泸州医学院》2012年硕士论文

【摘要】：目的：本实验通过建立大鼠背部撕脱皮瓣模型,研究重组水蛭素对撕脱皮瓣的保护作用及其机制,并探讨重组水蛭素在临床治疗撕脱皮瓣的应用前景。方法：选用健康SD大鼠108只,雌雄不拘,体重(250±50g),随机分为3组：2个治疗组(A组：重组水蛭素治疗组,B组：低分子肝素治疗组)和1个空白对照组(C组：注射用生理盐水组),每组36只,按40mg/kg体重、1%戊巴比妥钠腹腔注射麻醉,以背部中线为准,蒂位于头侧,设计4cm×6cm大小的长方形随意皮瓣,蒂部行保护性固定后,远端用缝线固定于细铁棒后与精密称量的重物相连,施予均为8kg的牵拉力,时间均为10秒形成撕脱皮瓣模型,远端游离缘稍加修剪后原位缝合。分别于术后即刻、12小时、24小时、48小时于皮瓣蒂部、距蒂部2cm和4cm三等分点皮下均匀注射重组水蛭素(2mg/lml/皮瓣)、低分子肝素(2mg/lml/皮瓣),空白对照组同法给予注射等量生理盐水(1m1/皮瓣)。术后观察皮瓣表面毛发生长情况,淤血、肿胀情况,皮瓣存活面积；分别检测术后12h、1d、3d皮瓣远端组织内P-选择素含量,3d、5d、7d皮瓣远端组织内VEGF含量；高倍镜下观察术后12h、1d、3d、5d、7d皮瓣远端组织病理切片内毛细血管内炎性细胞聚集情况及微血栓形成情况。结果：1.大体观察：术后12、24小时3组皮瓣均出现不同程度的发绀、肿胀,空白对照组较两治疗组发绀、肿胀严重；空白对照组淤滞带界限较清晰,治疗组淤滞带界限不清。术后第5、7天,治疗组皮瓣远端发绀、肿胀逐步消退,存活良好。空白对照组皮瓣远端和周缘仍较肿胀,且呈暗黑色,质地变硬,表面毛发有脱失,未见毛细血管反应,趋向于坏死。术后第14天重组水蛭素组和低分子肝素组存活面积百分比分别为(86.07±1.30)%和(81.46±1.06)%,均比空白对照组(77.03±1.25)%高,有显著性差异(p0.01),重组水蛭素组比低分子肝素组皮瓣成活率高,差异有统计学意义(p0.05)2.组织学观察：术后12h：空白对照组镜下见组织内大片出血区,充血、水肿明显,有大量中性粒细胞浸润；肝素组镜下见组织内片状出血、充血,水肿较明显,有较多中性粒细胞浸润；重组水蛭素组镜下见组织内点状出血,可见充血,水肿,少量中性粒细胞浸润。1d：空白对照组镜下见组织内大片出血区,充血、水肿明显,中性粒细胞呈团块状聚集,并可见早期坏死；肝素组镜下见组织内充血、水肿,中性粒细胞浸润等均较明显；重组水蛭素组镜下见组织内充血、水肿,可见数量不等的中性粒细胞散在存在。3d：空白对照组镜下见组织内大片出血,淤血明显,大量形成炎性肉芽组织,其中可见中性粒细胞散在存在；肝素组镜下见组织内淤血、水肿,有炎性肉芽组织形成,可见中性粒细胞散在分布；重组水蛭素组镜下见组织内散在淤血,出血少见,炎性肉芽组织形成,散在的少量中性粒细胞。5d：空白对照组镜下可见少量片状出血、淤血,可见微血栓形成,炎性肉芽组织水肿；肝素组镜下见少量淤血、水肿,炎性肉芽组织较成熟；重组水蛭素组镜下见少量淤血,炎性肉芽组织成熟。7d：空白对照组镜下见组织内微血栓形成,毛细血管闭塞,新生毛细血管少；肝素组镜下见组织内少量微血栓形成,有新生毛细血管；重组水蛭素组镜下见组织内新生血管增生活跃,肉芽组织成熟。3.P-选择素含量检测(pg／m1)：术后第12小时、1天、3大,重组水蛭素组与低分子肝素组P-选择素含量分别为(129.740±12.302)与(151.584±12.409)(111.030±6.824)与(134.434±8.707)、(101.674±4.429)与(118.308±7.535),其对应空白组为(196.034±18.442)(162.087±14.485)、(132.473±8.142),第12小时、1天、3天重组水蛭素组与低分子肝素组和空白对照组之间差异有统计学意义(P0.05)。4.VEGF含量检测：术后第3、5、7天,重组水蛭素组与低分子肝素组VEGF含量分别为(212.507±15.096)与(158.733±15.311)、(214.010±13.811)与(167.134±11.455)、(218.093±13.464)与(177.484±8.183),其对应空白组为(120.454±7.575)、(130.133±19.159)、(133.800±4.882),第3、5、7天重组水蛭素组与低分子肝素组和空白对照组之间差异有统计学意义(P0.05)。结论：局部注射重组水蛭素能有效抑制P-选择素的生成,减少炎性细胞的聚集,阻断微血栓的形成,改善撕脱皮瓣局部缺血、缺氧状态；减弱内皮细胞的损伤,促进VEGF的表达,刺激新生毛细血管增生,提高撕脱皮瓣的存活率。
[Abstract]:Objective: To study the protective effect of recombinant hirudin on the avulsion flap and its mechanism, and to explore the prospect of recombinant hirudin in the treatment of avulsion flap. Methods: 108 healthy SD rats were selected and divided into 3 groups randomly: 2 groups (group A: 2). The recombinant hirudin group, B group, low molecular weight heparin treatment group and 1 blank control group (group C: saline group), 36 rats in each group, with 40mg/kg weight, 1% pentobarbital sodium intraperitoneal injection, the middle back line as the criterion, the pedicle at the head side, and the design of 4cm x 6cm large rectangular free flap, the pedicle was protected and distal to the distal end. The stitches were attached to the weight of the fine weight after the stitches were fixed to the fine iron rod. The pulling force was given to 8kg. The time was 10 seconds to form a rip flap model, and the distal free margin was sutured after a little pruning. The pedicle of the flap was 12 hours, 24 hours and 48 hours after the operation, and the recombinant hirudin was injected subcutaneously from the pedicle of the pedicle, 2cm and 4cm three. 2mg/lml/ skin flap), low molecular weight heparin (2mg/lml/ flap), blank control group was given equal amount of saline injection (1m1/ flap) with the same method. After the operation, the hair growth, congestion, swelling, and the survival area of the skin flap were observed. The content of P- selectin in the distal tissue of 12h, 1D, 3D flaps after the operation was detected respectively, 3D, 5D, 7d skin flap in the distal tissue of the flap. The volume of inflammatory cells and the formation of microthrombosis in the distal sections of 12h, 1D, 3D, 5D and 7d were observed at high magnification.
Results: 1. gross observation: 12,24 hours after operation, 3 groups of skin flaps were cyanosis to different degrees, swelling, the blank control group was cyanosis and severe swelling compared with the two treatment group; the limit of Stasis Zone in the blank control group was clearer and the Stasis Zone of the treatment group was not clear. After the operation on day 5,7, the distal cyanosis, swelling gradually subsided, and the survival was good. The distal and peripheral skin of the skin flap were still swollen, dark black, the texture became hard, the surface hair was lost and no capillary reaction was found. The survival area percentage of the recombinant hirudin group and low molecular weight heparin group was (86.07 + 1.30)% and (81.46 + 1.06)%, respectively, fourteenth days after the operation, which were all higher than that in the blank control group (77.03 + 1.25)%. The survival rate of the flaps in the recombinant hirudin group was higher than that of the low molecular weight heparin group (P0.01). The difference was statistically significant (P0.05) 2. histological observation: after the operation, 12h: the blank control group showed massive hemorrhage area under the microscope, hyperemia, edema obviously, and a large number of neutrophils infiltration. The heparin group was seen under the heparin group the bleeding, congestion and edema were obvious under the heparin group. In the recombinant hirudin group, there was a lot of neutrophils infiltration, and the recombinant hirudin group showed punctate hemorrhage in the tissue under the microscope. There was congestion, edema, and a small amount of neutrophils infiltrating.1d. The blank control group was seen under the microscope, with massive hemorrhage area, congestive, edema, neutrophils aggregation, and early necrosis. Edema, neutrophils infiltration and so on were obvious. The recombinant hirudin group showed hyperemia, edema, and quantity of neutrophils scattered in the presence of.3d: in the blank control group, large hemorrhage in the tissue was seen under the microscope, the blood was obvious, and a large number of inflammatory granulation tissues were formed, and the neutrophils were found in the presence of neutrophils; the heparin group was under the microscope. There was blood, edema, and inflammatory granulation tissue in the tissue, and the neutrophils were scattered in the tissue, and the recombinant hirudin group was found to be scattered in the tissues and scattered in the tissues. The hemorrhage was rare, the inflammatory granulation tissue was formed, and a small amount of neutrophils scattered in.5d were found in the blank control group. In the heparin group, a small amount of congestion, edema, and inflammatory granulation tissue were more mature in the heparin group. The recombinant hirudin group showed a small amount of blood stasis and the inflammatory granulation tissue matured.7d. The microthrombus was formed in the blank control group, and the capillary was obliterated and the newborn capillaries were few. In the recombinant hirudin group, the recombinant hirudin group was found to be active in neovascularization and to detect the content of.3.P- selectin in granulation tissue (PG / M1): Twelfth hours, 1 days, 3, and P- selectin in the recombinant hirudin group and low molecular weight heparin group (129.740 + 12.302) and (151.584 + 12.409) (111.030 + 6.824) and 134.434 + 8.707), (101.674 + 4.429) and (118.308 + 7.535), the corresponding blank group was (196.034 + 18.442) (162.087 + 14.485), (132.473 + 8.142), Twelfth hours, 1 days, the difference between the recombinant hirudin group and the low molecular weight heparin group and the blank control group was statistically significant (P0.05).4.VEGF content detection: after 3,5,7 day after operation, the recombinant hirudin group and the low level The content of VEGF in the molecular heparin group was (212.507 + 15.096) and (158.733 + 15.311), (214.010 + 13.811) and (167.134 + 11.455), (218.093 + 13.464) and (177.484 + 8.183), and the corresponding blank group was (120.454 + 11.455). The difference between the recombinant hirudin group and the low molecular weight heparin group and the blank control group was poor. P0.05. Conclusion: local injection of recombinant hirudin can effectively inhibit the formation of P- selectin, reduce the accumulation of inflammatory cells, block the formation of microthrombus, improve local ischemia and hypoxia, weaken the damage of endothelial cells, promote the expression of VEGF, stimulate the proliferation of newborn capillaries, and increase the skin flap of the capillaries. The survival rate.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R641;R-332

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