神经生长因子对大鼠跨区穿支皮瓣成活的影响

发布时间：2018-03-28 22:19

本文选题：神经生长因子　切入点：跨区皮瓣　出处：《第三军医大学》2017年硕士论文

【摘要】：背景及目的:穿支皮瓣作为创面覆盖、组织修复技术,在临床上发挥着举足轻重的作用。Taylor提出血管体区(angiosome)及Choke Vessels是穿支皮瓣临床运用的解剖学基础,而相邻血管体间choke vessels的开放及“潜在供区”有效循环的建立成为移植后皮瓣远端组织成活的关键。在跨区修复较大创面时,穿支皮瓣术中、术后坏死时有发生,处理棘手。外科延迟技术通过多期手术能够改善远端血管体区在皮瓣正式切取后的血供,有效扩大皮瓣的修复面积。目前多数研究认为其机制是促进相邻血管体区间的choke血管的扩张及侧支循环的建立。这一过程可被某些药物、细胞及其分泌的生化因子加强甚至替代,从而衍生出化学皮瓣延迟(chemical flap delay)。神经营养因子(NGF)是一个多功能多肽分子,可以通过内皮祖细胞酪氨酸激酶受体TrkA途径触发内皮细胞的增殖、迁移,维持血管内皮功能。NGF还直接或者间接调节VEGF表达促进血管生成。此外,内皮组细胞上分布血管生长因子II型受体KDR,VEGF与其结合后发挥血管生成的作用。NGF能够上调KDR的表达,与VEGF有协同作用,此为血管生成的另一途径。然而目前NGF能否通过以上可能的机制影响皮瓣choke区微血管形态变化及潜在供区微循环建立,从而替代外科延迟手术改善大面积皮瓣成活,还不十分清楚。基于以上分析,本研究筛序并建立动物模型,通过显微CT评价NGF对SD大鼠背部跨区供血穿支皮瓣显微血管形态变化的影响,并对其机制进行探讨,为提高临床皮瓣成活提供理论支持。方法:第一部分:利用Micro-CT重建技术观察神经生长因子对大鼠跨区穿支皮瓣血管形态变化的影响成年SD大鼠40只,随机分为实验组和对照组。通过大体解剖筛选合适的大鼠背部跨区供血穿支皮瓣模型。切取面积约3cm×10cm的背部跨区皮瓣。实验组皮下注射NGF溶液(10 nmol·ml-1·kg-1);对照组同法注射0.1M磷酸盐缓冲溶液(1ml/kg)。术后第3、7天氧化铅-明胶溶液血管灌注后,利用显微CT技术,获取血管体三维形态,Matlable软件分析血管容积及总长度等计量参数变化。术后第7天统计皮瓣存活面积。第二部分:神经生长因子对大鼠跨区穿支皮瓣成活初步机制研究成年SD大鼠44只,随机分为实验组和对照组。切取面积约3cm×10cm的背部跨区皮瓣。实验组皮下注射NGF溶液(10 nmol·ml-1·kg-1);对照组同法注射0.1M磷酸盐缓冲溶液(1ml/kg)。术后第3、7天局部取材运用Western Blot技术半定量检测VEGF及CD34蛋白含量;术后第7天免疫组化染色观测血管生长因子受体(KDR)及NGF受体(Trk A)的表达。结果:1.设计以髂腰动脉为单穿支血管蒂的跨三个血管体区的大鼠背部皮瓣模型,具有恒定的坏死面积。结果显示,血管形态学分析示NGF和时间因相互作用增加血管容积(F=33.304,P0.05)及总长度(F=8.493,P=0.01);实验组皮瓣成活面积明显大于对照组(P0.05)。2.NGF明显促进皮瓣微血管VEGF及CD34蛋白分泌。第3天,VEGF表达实验组与对照组无显著统计学差异(P=0.088);实验组CD34表达明显高于对照组(P=0.004);第7天,两种蛋白分子表达,实验组显著均优于对照组(P0.05)。NGF能够诱导皮瓣微血管TrkA的阳性表达。结论:NGF能增加choke区微血管的密度及长度,促进的跨区供血皮瓣的成活面积;其机制可能是NGF促进皮瓣微血管VEGF及CD34蛋白分泌,诱导Trk A受体阳性表达,通过TrkA-VEGF途径,促进choke区微血管生成,维持其内皮功能,最终改善潜在供区微循环血供,影响皮瓣成活。
[Abstract]:Background and purpose: to cover the wounds as perforator flap, tissue repair technique in clinical plays a pivotal role in.Taylor of vascular body region (angiosome) and Choke Vessels are the anatomical basis for clinical application of perforator flap, and the effective circulation of adjacent blood vessels between choke and vessels open "potential donor" became a key of the flap the distal tissue survival after transplantation. To repair a larger wound in the cross, perforator flap surgery, postoperative necrosis occurred, treatment difficult. Surgical delay through surgery can improve the body region in the distal flap formally after removal of the blood supply, effectively expand the repair area of skin flap. Most studies suggest that the the mechanism is to promote the expansion of choke vessel body interval and adjacent blood vessels in the establishment of collateral circulation. This process can be some of the drugs, and the secretion of cytokines and biochemical and even Instead, which are derived from chemical (chemical flap delay) flap delay. Neurotrophic factor (NGF) is a multifunctional polypeptide that can migrate through the tyrosine kinase receptor TrkA pathway of endothelial progenitor cells to trigger the proliferation of endothelial cells, vascular endothelial function, maintain.NGF also directly or indirectly regulate the expression of VEGF to promote angiogenesis. In addition, the distribution of vascular endothelial growth factor receptor type II KDR cells, VEGF and its combination play a role in angiogenesis of.NGF can upregulate the expression of KDR and VEGF have synergistic effect, this is another way of angiogenesis. However, whether NGF could influence mechanism through the above choke flap area micro microcirculation morphological changes and potential for the district established, so as to replace the surgical delay surgery to improve the large area of flap survival, is not very clear. Based on the above analysis, this study screen order and establish an animal model by Effect of micro CT NGF evaluation of SD rat dorsal perforator flap extra-territorial blood supply forms microscopic changes, and to explore its mechanism, to provide theoretical support to improve clinical flap survival. Methods: the first part: the effect of using Micro-CT reconstruction technique to observe the nerve growth factor on vascular morphological changes of flap inter zone perforator in adult rat SD rats 40 rats were randomly divided into experimental group and control group. The gross anatomy of the rat back cross blood screening perforator flap model. The appropriate cut area is about 3cm * 10cm back cross flap. The experimental group was injected NGF solution (10 nmol - ml-1 - kg-1); the control group received phosphate buffer solution (0.1M 1ml/kg). 3,7 days after operation of lead oxide gelatin solution after vascular perfusion, using micro CT technology to obtain vascular 3D measurement parameters, analysis of the changes of blood volume and the total length of the Matlable software after operation. Seventh days of statistical flap survival area. The second part: the nerve growth factor on the inter zone perforator flap survival in rats preliminary study on the mechanism of 44 adult SD rats, were randomly divided into experimental group and control group. The cut area is about 3cm * 10cm back cross flap. The experimental group was injected NGF solution (10 nmol - ml-1 - kg-1); control group with injection of 0.1M phosphate buffer solution (1ml/kg). 3,7 days after operation using Western Blot technology based local semi quantitative detection of VEGF and CD34 protein in seventh days after operation; immunohistochemical staining was used to observe the vascular endothelial growth factor receptor (KDR) and NGF receptor (Trk A) expression. Results: 1. in order to design the iliolumbar arteries for the back flap in rats model of cross three vascular body region single perforator pedicled, with constant necrosis area. The results showed that the vascular morphological analysis showed that NGF and the interaction time due to increased vascular volume (F=33.304, P0.05) and F (total length =8.493, P=0.01); the experimental group flap survival area was significantly higher than the control group (P0.05.2.NGF) significantly promote flap microvascular VEGF and secretion of CD34 protein. The expression of VEGF in third days, there was no statistically significant difference between the experimental group and the control group (P=0.088); the expression of CD34 in experimental group was significantly higher than the control group (P=0.004); the seventh day, the expression of two protein molecules, the experimental group was significantly better than the control group (P0.05) were positive expression of.NGF can induce microvascular flap TrkA. Conclusion: NGF can increase the density and length of choke region of microvessels, the reverse flow flap to promote the survival area; the mechanism may be that NGF can promote flap microvascular VEGF and secretion of CD34 protein induced. The positive expression of Trk A receptor by TrkA-VEGF pathway, promote the formation of choke microvascular endothelial function, maintain, and ultimately improve the blood microcirculation of potential donor for effect of flap survival.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R622

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