联合运用人工真皮与血管内皮生长因子浓度梯度在促进糖尿病猪模型伤口愈合中的作用

发布时间：2018-03-15 05:20

  本文选题：VEGF　切入点：浓度梯度　出处：《南京大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的糖尿病(Diabetes mellitus,DM)患者的创面是世界范围内最常见的难愈性创面之一。影响这类创面不遵循正常有序的愈合进程的原因可分为内在因素和外在因素两个方面。内在因素包括:糖尿病导致的周围神经或血管病变和糖尿病严重程度。外在因素包括:创面感染,愈伤组织形成和对创面区域的过度压力。多重的异常致病因素相互作用,在糖尿病创面中形成恶性循环。此外,血流灌注的不足以及生长因子水平的下降,加上血管再生的受损使愈合过程更复杂化。血管内皮生长因子(Vascular endothelial growth factor,VEGF)是最重要的促血管生成介质之一。它可作为促内皮细胞的分裂剂和趋化剂,同时诱导血管的通透性。在链脲佐菌素(Streptozotocin,STZ)诱导的糖尿病小鼠创面模型研究中,发现几种生长因子合成的减少,其中就包括VEGF。在糖尿病患者中,低水平的VEGF导致血管密度的降低,肉芽组织形成的减少,最终延迟创面的愈合。足够水平的VEGF可以通过多种活性刺激创面的愈合,包括胶原沉积,血管生成和再上皮化。单独使用VEGF或与其他治疗方法联合,已被证明是糖尿病创面治疗的有效方法。人工真皮(Artificial dermal,AD)已经在人类和动物模型全层皮肤缺损创面的治疗中显示出巨大的潜力。本实验中使用的人工真皮由两层结构组成:上层为加强型的硅胶膜,下层为来源于猪肌腱的无末端胶原蛋白海绵层。它能有效诱导新生真皮样肉芽组织的形成,从而有效的改善创面的愈后。在我们之前的体外研究中,已经证明,一定浓度的VEGF可以促进血管内皮细胞的增殖和移行,而VEGF的浓度梯度模型可以促进血管内皮细胞的迁移。因此,在本研究中,我们尝试通过联合运用人工真皮和均一浓度的VEGF或VEGF浓度梯度来促进糖尿病猪模型的创面愈合。充分利用人工真皮和VEGF在改善创面愈合过程的优势。将人工真皮作为一个持续的VEGF给药系统。与此同时,比较不同均一浓度的VEGF和VEGF浓度梯度之间在疗效上的差异。材料与方法实验材料:将人工真皮切割成1cm×1cm的正方形碎片供测试其吸收性和缓释效率。剩余人工真皮被切割成其他两种形状以用于随后的动物模型手术:1型为直径18mm的圆,并准备浸入不同浓度的VEGF。2型为三个同心圆(H,M,L)。H:直径6mm。M:内径6mm,外径12mm。L:内径12mm,外径18mm。基于之前的体外研究,将型的同心圆分别浸泡于三种浓度的VEGF中。高浓度(H):100ng/ml。中等浓度(M):25ng/ml。低浓度(L):5ng/ml。从而建立VEGF内高外低的浓度梯度。实验方法1.挑选体重20-25公斤的健康实验用小型母猪只,以125mg/kg的剂量静脉内推注链脲佐菌素(STZ)建立糖尿病动物模型。其中两只猪用于评估愈合率。另外四只用于随后的样本采集。2.在全麻下,每只猪的脊柱两侧制备36个全层皮肤缺损创面(直径18mm)。将这些创面随机分为6组:空白对照组(C,仅PBS,n=6),人工真皮组(AD,仅AD,n=6),低浓度组(AD+L,AD+5ng/mlVEGF,n=6),中浓度组(AD+M,AD+25ng/ml VEGF,n=6),高浓度组(AD+H,AD+100ng/mlVEGF,n=6),浓度梯度组(AD+G,AD+VEGF浓度梯度,n=6)。对照组以外的所有创面表面覆盖人工真皮膜片,与创面边缘缝合固定,并在外层打包固定。3.术后第7、14、21天计算伤口愈合率。在各时间对每个伤口进行拍摄,并通过图像分析软件进行分析。愈合率=[(原始创面面积大小-未愈合面积)/原始面积]×100%。4.将石蜡包埋的组织切成5μm厚的切片(HE)染色在100倍放大倍数下分析从创面中心部的肉芽组织。用anti-von Willebrand factor抗体对连续切片进行免疫标记,以检测肉芽组织的新血管形成水平。术后第21天的样本用anti-VEGF polyclonal抗体进行免疫标记。在400倍放大倍数下,定量VEGF蛋白特异性染色区域。VEGF蛋白特异性染色面积的百分比=VEGF蛋白阳性面积/总组织面积×100%。结果1.AD的吸收率为0.3ml/cm2。缓释效率前4天(第1天:24.96±2.38ng/ml,第2天:22.19±1.27ng/ml,第 3 天:20.20±1.62ng/ml,第 4 天:10.30±1.79ng/ml)。饱和 AD 连续释放VEGF,从第5天起,缓释效率明显降低(第5天:5.24±0.93ng/ml,第6天:1.80±0.34ng/ml),在第 7 天接近 0。2.动物模型维持持续高血糖状态(血糖在300至500mg/dl之间)稳定一周后,糖尿病猪模型成功建立。3.术后第7天,各组愈合率差异无统计学意义(P0.05)。AD+M组AD+H组和AD+G组在第14天,AD+H组和AD+G组在第21天的愈合率均明显高于对照组(P0.05)。4.术后第7天所有治疗组肉芽组织形成速度明显快于对照组。AD+H组和AD+G组中发现较厚的肉芽组织,与对照组相比高出2倍以上。但两组之间比较差异无统计学意义(P0.05)。在整个实验过程中,AD+H组和AD+G组的肉芽组织厚度均显著高于对照组(P0.05)。5.第7天,AD+H组(21.7±2.0每视野)和AD+G(19.6±1.6每视野)的血管密度显著高于对照组(4.2±1.0每视野)(P0.05)。AD组显示血管数量较少(5.0±1.1每视野)。在第14天和第21天,所有组的新血管形成水平不断增加。在实验中,AD+H组和AD+G组诱导血管生成数量最明显。6.与对照组相比,所有治疗组的VEGF蛋白特异性染色面积百分比均比较高。特别是AD+H组和AD+G组,显著高于对照组(P0.05),分别高于对照组3倍和2倍。结论人工真皮产品与足够浓度的VEGF组合具有促进糖尿病创面愈合的显著能力。其中,联合使用人工真皮和特定VEGF浓度梯度,使用较少的剂量,但达到相似的效果。这种组合可以促进新生血管发生,增加上皮化,最终加速糖尿病性创面的直接愈合,或为二期皮肤移植做好准备。这个治疗方法可能为是糖尿病创面治疗的提供一个新的思路。
[Abstract]:ObjectiveDiabetes (Diabetes mellitus, DM) in patients with refractory wound wound is one of the most common in the world. The reasons influencing the wound does not follow the normal and orderly healing process can be divided into two aspects of internal and external factors. The internal factors include: diabetes caused by peripheral nerve or vascular disease and diabetes mellitus degree. The external factors include: wound infection, excessive pressure on the callus formation and wound area. The abnormal interaction of multiple pathogenic factors, forming a vicious spiral in diabetic wound. In addition, insufficient blood perfusion and decreased the level of growth factor, coupled with impaired vascular regeneration so that the healing process is more complicated. Vascular endothelial growth factor (Vascular endothelial growth factor, VEGF) is one of the most important angiogenic medium. It can be used as split agent to promote endothelial cell and chemotactic agent with When the induction of vascular permeability in streptozotocin (Streptozotocin, STZ) on diabetic mice model induced by wound, found several growth factor synthesis reduction, including VEGF. in diabetic patients, low level of VEGF leads to a decrease in vascular density, reduce the formation of granulation tissue, eventually delayed wound healing. Adequate levels of VEGF can stimulate the wound healing through a variety of activities, including collagen deposition and angiogenesis and reepithelialization. Using VEGF or other therapy alone, has been proved to be an effective method of treatment of diabetic wound. Artificial dermis (Artificial dermal, AD) have shown great potential in the treatment of human and animal model of full-thickness skin defect wound. Used in the experiment of artificial dermis is composed of two layers: the upper layer is to strengthen the silicone membrane type, the lower is derived from porcine tendon free At the end of the collagen sponge layer. It can effectively induce the formation of new true dermoid granulation tissue, so as to effectively improve the wound after healing in vitro. Our previous study has shown that in a certain concentration of VEGF can promote the migration and proliferation of vascular endothelial cells, and the concentration gradient model of VEGF can promote the migration of vascular endothelial cells. Therefore, in this study, we try to through a combination of artificial dermis and uniform concentration of VEGF or VEGF concentration gradient to promote wound healing in diabetic porcine model. Make full use of artificial leather and VEGF advantage in improving the wound healing process. The artificial dermis as a continuous VEGF administration system. At the same time. The effect of the differences between homogeneous VEGF concentration and VEGF concentration gradient. Materials and methods: the experimental materials will be cut into square pieces of artificial dermis 1cm x 1cm test The absorption and release efficiency. The remaining artificial dermis was cut into two other shapes for subsequent surgery animal model: type 1 18mm diameter circle, and prepare immersed in different concentrations of VEGF.2 type three concentric circles (H, M, L).H: diameter 6mm.M: inner diameter 6mm, diameter 12mm.L diameter: 12mm 18mm., based on in vitro studies before the outer diameter, VEGF concentric type were immersed in three concentrations. High concentration (H): 100ng/ml. (M): moderate concentration of low concentration of 25ng/ml. (L): 5ng/ml. to establish a high concentration gradient of low VEGF. 1. experimental methods to select healthy experimental weight 20-25 kg with a small infusion of sows, streptozotocin to intravenous 125mg/kg (STZ) in the establishment of animal models of diabetes mellitus. The two pigs used to evaluate the healing rate. Another four samples for subsequent.2. under general anesthesia, each pig spinal column on both sides of the preparation of 36 full-thickness skin Skin wound defect (18mm in diameter). The wounds were randomly divided into 6 groups: control group (C, PBS only, n=6 group (AD), artificial leather, AD, n=6), low concentration group (AD+L, AD+5ng/mlVEGF, n=6), middle dose group (AD+M, AD+25ng/ml, VEGF, n=6) the high concentration group (AD+H, AD+100ng/mlVEGF, n=6), the concentration gradient group (AD+G, AD+VEGF concentration gradient, n=6). The control group all wound covering the surface of artificial dermis diaphragm, suture and wound edges, and in outer packing fixed.3. 7,14,21 days after operation. In the calculation of the rate of wound healing time of each wound shooting, and through image analysis software were analyzed. The healing rate of n (the original wound size - not healing area) / original area] * 100%.4. paraffin embedded tissue was cut into 5 m thick slices (HE) staining at 100 times magnification from the analysis of the center of the wound granulation tissue. Anti-von Willebrand factor antibody Immunogold labeling was performed on serial sections, the detection of new blood vessels formation of granulation tissue. After twenty-first days of the samples by anti-VEGF polyclonal antibodies by immune markers. In 400 times magnification, specific VEGF protein staining region specific.VEGF protein staining area percentage of =VEGF protein positive area / total tissue area X the results of 100%. 1.AD absorption rate of 0.3ml/cm2. release efficiency before 4 days (first days, second days: 24.96 + 2.38ng/ml, 22.19 + 1.27ng/ml, 20.20 + 1.62ng/ml, third days, fourth days: 10.30 + 1.79ng/ml). The saturated AD continuous release of VEGF from the fifth day, release efficiency decreased significantly (fifth days: 5.24. 0.93ng/ml, Sixth days: 1.80 + 0.34ng/ml), in seventh days close to the animal model of 0.2. to maintain sustained high blood glucose (blood sugar in the state between 300 and 500mg/dl) stable after a week, seventh days to establish diabetic porcine model successfully after.3., healing rate There was no statistically significant difference (P0.05).AD+M group, AD+H group and AD+G group on the fourteenth day, AD+H group and AD+G group in the healing rate of twenty-first days was significantly higher than that of the control group (P0.05.4.) seventh days after the formation of granulation tissue in all treatment groups significantly faster than the control of thicker granulation tissue was found in group.AD+H and group AD+G in the group, compared with the control group higher than 2 times. But there was no significant difference between the two groups (P0.05). During the whole experiment, the granulation tissue thickness of AD+H group and AD+G group were significantly higher than those in control group (P0.05).5. seventh days, AD+H group (21.7 + 2 per field) and AD+G (19.6 + 1.6 per field) of the vascular density was significantly higher than the control group (4.2 + 1 per field) (P0.05).AD group showed fewer blood vessels (5 + 1.1 per field). On the fourteenth day and the twenty-first day, all groups to form new blood vessels to increase the level. In the experiment, AD+H group and AD+G group the number of induced angiogenesis. .6. obviously compared with the control group, the treatment group all VEGF protein specific staining area percentage were relatively high. Especially the AD+H group and AD+G group was significantly higher than the control group (P0.05), were higher than those in the control group 3 times and 2 times. Conclusion artificial leather products with sufficient concentrations of VEGF combined with the promotion ability of diabetes significantly wound healing. Among them, the combined use of artificial dermis and the specific VEGF concentration gradient, with fewer doses, but achieve a similar effect. This combination can promote angiogenesis, increase epithelialization and direct healing eventually accelerate the diabetic wound, or prepare for a two skin graft. This treatment may be diabetic wound therapy provides a new idea.

【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R641
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本文编号：1614605