發(fā)布時(shí)間：2018-03-15 05:20

  本文選題：VEGF　切入點(diǎn)：濃度梯度　出處：《南京大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的糖尿病(Diabetes mellitus,DM)患者的創(chuàng)面是世界范圍內(nèi)最常見(jiàn)的難愈性創(chuàng)面之一。影響這類(lèi)創(chuàng)面不遵循正常有序的愈合進(jìn)程的原因可分為內(nèi)在因素和外在因素兩個(gè)方面。內(nèi)在因素包括:糖尿病導(dǎo)致的周?chē)窠?jīng)或血管病變和糖尿病嚴(yán)重程度。外在因素包括:創(chuàng)面感染,愈傷組織形成和對(duì)創(chuàng)面區(qū)域的過(guò)度壓力。多重的異常致病因素相互作用,在糖尿病創(chuàng)面中形成惡性循環(huán)。此外,血流灌注的不足以及生長(zhǎng)因子水平的下降,加上血管再生的受損使愈合過(guò)程更復(fù)雜化。血管內(nèi)皮生長(zhǎng)因子(Vascular endothelial growth factor,VEGF)是最重要的促血管生成介質(zhì)之一。它可作為促內(nèi)皮細(xì)胞的分裂劑和趨化劑,同時(shí)誘導(dǎo)血管的通透性。在鏈脲佐菌素(Streptozotocin,STZ)誘導(dǎo)的糖尿病小鼠創(chuàng)面模型研究中,發(fā)現(xiàn)幾種生長(zhǎng)因子合成的減少,其中就包括VEGF。在糖尿病患者中,低水平的VEGF導(dǎo)致血管密度的降低,肉芽組織形成的減少,最終延遲創(chuàng)面的愈合。足夠水平的VEGF可以通過(guò)多種活性刺激創(chuàng)面的愈合,包括膠原沉積,血管生成和再上皮化。單獨(dú)使用VEGF或與其他治療方法聯(lián)合,已被證明是糖尿病創(chuàng)面治療的有效方法。人工真皮(Artificial dermal,AD)已經(jīng)在人類(lèi)和動(dòng)物模型全層皮膚缺損創(chuàng)面的治療中顯示出巨大的潛力。本實(shí)驗(yàn)中使用的人工真皮由兩層結(jié)構(gòu)組成:上層為加強(qiáng)型的硅膠膜,下層為來(lái)源于豬肌腱的無(wú)末端膠原蛋白海綿層。它能有效誘導(dǎo)新生真皮樣肉芽組織的形成,從而有效的改善創(chuàng)面的愈后。在我們之前的體外研究中,已經(jīng)證明,一定濃度的VEGF可以促進(jìn)血管內(nèi)皮細(xì)胞的增殖和移行,而VEGF的濃度梯度模型可以促進(jìn)血管內(nèi)皮細(xì)胞的遷移。因此,在本研究中,我們嘗試通過(guò)聯(lián)合運(yùn)用人工真皮和均一濃度的VEGF或VEGF濃度梯度來(lái)促進(jìn)糖尿病豬模型的創(chuàng)面愈合。充分利用人工真皮和VEGF在改善創(chuàng)面愈合過(guò)程的優(yōu)勢(shì)。將人工真皮作為一個(gè)持續(xù)的VEGF給藥系統(tǒng)。與此同時(shí),比較不同均一濃度的VEGF和VEGF濃度梯度之間在療效上的差異。材料與方法實(shí)驗(yàn)材料:將人工真皮切割成1cm×1cm的正方形碎片供測(cè)試其吸收性和緩釋效率。剩余人工真皮被切割成其他兩種形狀以用于隨后的動(dòng)物模型手術(shù):1型為直徑18mm的圓,并準(zhǔn)備浸入不同濃度的VEGF。2型為三個(gè)同心圓(H,M,L)。H:直徑6mm。M:內(nèi)徑6mm,外徑12mm。L:內(nèi)徑12mm,外徑18mm�；谥暗捏w外研究,將型的同心圓分別浸泡于三種濃度的VEGF中。高濃度(H):100ng/ml。中等濃度(M):25ng/ml。低濃度(L):5ng/ml。從而建立VEGF內(nèi)高外低的濃度梯度。實(shí)驗(yàn)方法1.挑選體重20-25公斤的健康實(shí)驗(yàn)用小型母豬只,以125mg/kg的劑量靜脈內(nèi)推注鏈脲佐菌素(STZ)建立糖尿病動(dòng)物模型。其中兩只豬用于評(píng)估愈合率。另外四只用于隨后的樣本采集。2.在全麻下,每只豬的脊柱兩側(cè)制備36個(gè)全層皮膚缺損創(chuàng)面(直徑18mm)。將這些創(chuàng)面隨機(jī)分為6組:空白對(duì)照組(C,僅PBS,n=6),人工真皮組(AD,僅AD,n=6),低濃度組(AD+L,AD+5ng/mlVEGF,n=6),中濃度組(AD+M,AD+25ng/ml VEGF,n=6),高濃度組(AD+H,AD+100ng/mlVEGF,n=6),濃度梯度組(AD+G,AD+VEGF濃度梯度,n=6)。對(duì)照組以外的所有創(chuàng)面表面覆蓋人工真皮膜片,與創(chuàng)面邊緣縫合固定,并在外層打包固定。3.術(shù)后第7、14、21天計(jì)算傷口愈合率。在各時(shí)間對(duì)每個(gè)傷口進(jìn)行拍攝,并通過(guò)圖像分析軟件進(jìn)行分析。愈合率=[(原始創(chuàng)面面積大小-未愈合面積)/原始面積]×100%。4.將石蠟包埋的組織切成5μm厚的切片(HE)染色在100倍放大倍數(shù)下分析從創(chuàng)面中心部的肉芽組織。用anti-von Willebrand factor抗體對(duì)連續(xù)切片進(jìn)行免疫標(biāo)記,以檢測(cè)肉芽組織的新血管形成水平。術(shù)后第21天的樣本用anti-VEGF polyclonal抗體進(jìn)行免疫標(biāo)記。在400倍放大倍數(shù)下,定量VEGF蛋白特異性染色區(qū)域。VEGF蛋白特異性染色面積的百分比=VEGF蛋白陽(yáng)性面積/總組織面積×100%。結(jié)果1.AD的吸收率為0.3ml/cm2。緩釋效率前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)。飽和 AD 連續(xù)釋放VEGF,從第5天起,緩釋效率明顯降低(第5天:5.24±0.93ng/ml,第6天:1.80±0.34ng/ml),在第 7 天接近 0。2.動(dòng)物模型維持持續(xù)高血糖狀態(tài)(血糖在300至500mg/dl之間)穩(wěn)定一周后,糖尿病豬模型成功建立。3.術(shù)后第7天,各組愈合率差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。AD+M組AD+H組和AD+G組在第14天,AD+H組和AD+G組在第21天的愈合率均明顯高于對(duì)照組(P0.05)。4.術(shù)后第7天所有治療組肉芽組織形成速度明顯快于對(duì)照組。AD+H組和AD+G組中發(fā)現(xiàn)較厚的肉芽組織,與對(duì)照組相比高出2倍以上。但兩組之間比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。在整個(gè)實(shí)驗(yàn)過(guò)程中,AD+H組和AD+G組的肉芽組織厚度均顯著高于對(duì)照組(P0.05)。5.第7天,AD+H組(21.7±2.0每視野)和AD+G(19.6±1.6每視野)的血管密度顯著高于對(duì)照組(4.2±1.0每視野)(P0.05)。AD組顯示血管數(shù)量較少(5.0±1.1每視野)。在第14天和第21天,所有組的新血管形成水平不斷增加。在實(shí)驗(yàn)中,AD+H組和AD+G組誘導(dǎo)血管生成數(shù)量最明顯。6.與對(duì)照組相比,所有治療組的VEGF蛋白特異性染色面積百分比均比較高。特別是AD+H組和AD+G組,顯著高于對(duì)照組(P0.05),分別高于對(duì)照組3倍和2倍。結(jié)論人工真皮產(chǎn)品與足夠濃度的VEGF組合具有促進(jìn)糖尿病創(chuàng)面愈合的顯著能力。其中,聯(lián)合使用人工真皮和特定VEGF濃度梯度,使用較少的劑量,但達(dá)到相似的效果。這種組合可以促進(jìn)新生血管發(fā)生,增加上皮化,最終加速糖尿病性創(chuàng)面的直接愈合,或?yàn)槎谄つw移植做好準(zhǔn)備。這個(gè)治療方法可能為是糖尿病創(chuàng)面治療的提供一個(gè)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.

【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R587.2;R641
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本文編號(hào)：1614605