【作者】 王一兵；

【导师】 何剪太；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 目的：烧伤创面愈合是一个复杂而有序的生物学过程,包括炎性反应、细胞增殖、创面重塑几个阶段。关于创面愈合涉及的炎症细胞、修复细胞、炎症介质、生长因子和细胞外基质等成分的研究已有大量报道。诸多现象表明神经因素可能在创面愈合中具有重要作用,已有关于神经生长因子及神经肽对创面愈合影响的报道,但是少有对烧伤创面愈合过程中神经再生过程、再生神经形态学变化规律及其与创面愈合关系的研究。我们通过动物实验观察烧伤创面愈合过程中的神经变化情况,并通过研究消除神经因素及加强神经因素对创伤愈合速度和创面愈合质量的影响,初步确定皮肤神经对烧伤创面愈合的作用；同时观察人体不同时期伤区组织和瘢痕组织中的二、三维形态等神经构筑情况,总结创面愈合过程中神经纤维的变化规律及其可能作用,从而初步探明神经与创面愈合的相互作用,为从神经角度调控创面愈合提供新的依据。方法：1.大鼠神经变化对烧伤创面愈合的影响随机将90只大鼠分为三组：A组(失神经支配+烫伤,30只)B组(富神经支配+烫伤,30只)和C组(烫伤,30只)。将A组大鼠右侧T9-L1脊神经切断制作失神经支配模型；然后在A、B、C组大鼠背部右侧T9-L1节段制作直径2cm的深Ⅱ度烫伤创面。B组大鼠伤后注射NGF 1U/g.d只,连续14天制作富神经支配模型。伤后7、14、21天进行观察,取材。观察创面大体情况,计算创面愈合率及创面愈合时间；普通组织病理学观察炎性细胞、血管、上皮化等情况；以神经丝蛋白NFP作为神经标记物,利用荧光免疫组织化学法检测创面神经再生情况,通过神经容积分数(NVF)进行半定量分析；利用rt-PCR检测VEGFmRNA的表达水平,观察神经变化对血管形成的影响；免疫组织化学法观察Ⅰ、Ⅲ胶原分泌情况,通过胶原容积分数(CVF)进行半定量分析,并计算Ⅰ／Ⅲ型胶原比例的变化,观察创面愈合的质量。2.人烧伤愈合过程中神经的变化将取自人体的伤区组织或瘢痕组织分为三个组：A组(Ⅲ度伤区组织,便于手术取材),伤后1周,2周,3周,4周组织各10例；B组(瘢痕组织),增生期瘢痕(≤6月)、成熟期瘢痕(>6月)各10例。C组为正常对照组,实验组和对照组按5：1标本取自以上患者的供皮区,共12例；改良三色法染色,普通光学显微镜观察组织的病理学改变,并胶原面积／视野面积,计算胶原容积分数(CVF)进行半定量分析；筛选神经标记物,分别应用蛋白基因产物PGP9.5及神经丝蛋白作为神经标记物进行免疫荧光染色。利用效果良好NFP作为神经标记物进行实验；普通荧光显微镜观察神经纤维的平面结构和数量改变,计算神经容积分数(NVF)进行半定量分析；激光扫描共聚焦显微镜观察神经纤维的内部结构和三维立体结构改变。结果：1.大鼠神经变化对烧伤创面愈合的影响创面组织学观察及创面愈合时间：伤后7天,B组炎性细胞浸润较A、C组明显,上皮化情况各组无明显差别；伤后14天,B组炎性细胞浸润情况开始缓解,上皮化快于A、C两组,A组上皮化落后C组。A组痂皮脱落较慢,愈合时间最短21天,最长26天,平均23.5±2.95天,B组痂皮脱落较快,创面愈合时间最短18天,最长20天,平均18.8±1.48天,C组愈合时间最短19天,最长22天,平均20.4±2.86天神经密度比较：伤后各时间点A组切片未见免疫阳性染色区域,B组、C组在伤区基底部可见NFP免疫阳性染色,其形状因界面不同而异,多呈圆点状,少数呈现细小短棒状。伤后14天,B、C组NFP免疫阳性染色区域开始增多,一般为圆点、短棒状,B组NVF高于C组(p<0.05)。伤后21天,B、C组NFP免疫阳性染色区域明显增多,呈圆点、短棒或者豆芽状。B组NVF显著高于C组(p<0.05)。大鼠皮肤神经短小难以三维重建。VEGFmRNA表达情况：A、B、C组VEGFmRNA伤后开始升高,B、C组迅速升高,伤后7天即达高峰,A组升高较缓慢至伤后14天达到高峰。伤后21天各组维持相对较高水平,伤后21天B组VEGFmRNA显著高于C组(p<0.05),A组与C组无显著差异。伤后各时间B组VEGFmRNA表达一直高于其他两组。Ⅰ、Ⅲ型胶原分泌情况及Ⅰ／Ⅲ型胶原比例变化：各组Ⅰ型胶原在伤后持续增加,A组Ⅰ型胶原分泌明显低于C组,B组Ⅰ型胶原明显高于C组；各组Ⅲ型胶原除A组持续升高外,其余两组无显著变化。伤后21天A组Ⅰ／Ⅲ型胶原比例显著低于C组,B组Ⅰ/Ⅲ型胶原比例与C组比较差异统计学意义。2.人烧伤愈合过程中神经的变化伤后胶原比例逐渐增大,到增生期CVF达到最高,成熟期趋于正常。PGP9.5组的灰度值,NVF值显著低于NFP组,PGP9.5染色阳性率低于NFP组(p<0.05)。伤后3周内神经纤维数目低于正常,3-4周后神经数目开始高于正常,并逐渐变长,随着时间延长出现扭曲缠绕或交错排列,分布呈现区域性集中；增生期瘢痕NVF达到最高,神经纤维分布杂乱,形态不规；成熟期瘢痕神经纤维NVF明显降低,接近正常,排列较规整；分层扫描和三维重建可见伤后2周神经稀疏、短小,3周后神经纤维有不规则肿胀和扭曲变形,内部结构不完整并伴有局部断裂、破损甚至崩解,可见明显的皱褶和出芽,并呈现较正常神经纤维更强的荧光信号,在不同层次以及不同角度的切片上观察到的病理改变程度不同。结论：1.在大鼠及人烧伤创面愈合中,神经纤维密度逐渐增高；在人创面重塑期神经纤维密度先增高后降低。再生神经的形态、分布由简单到混乱再到规律,神经在创伤愈合过程中存在重构现象。2.利用激光扫描共聚焦显微镜技术,能分层扫描、并重建人伤区组织及瘢痕组织内神经纤维,观察到细微的形态变化,但是难以对大鼠皮肤神经进行重建。3.大鼠失神经支配伤区组织VEGFmRNA表达量较富神经支配创面组织低、愈合相对变慢,说明神经可以通过影响微血管再生对创面愈合产生影响。4.增强神经因素在创面早期可以促进创面愈合,减轻神经因素在创面重塑期有助于改善重塑质量更多还原

【Abstract】 Objective:Burn wound healing is a complex and sequencing process, which is generally divided into three interrelated/overlapping phases:inflammation, wound tissue formation, and tissue remodeling. The healing process involves interplay of epithelial-stromal-inflammatory cell-cell interactions as well as interactions between cytokines and growth factor, released by structural and immune or inflammatory cells, in order to achieve a normal recovery of the injured tissue.These mechanism have been detailed by many reports.Skin innervation plays important role in physiological wound repair, as observed during diabetic neuropathies or paraplegia, where ulcers do not repair successfully. The reason is still unknown and an explanation might be that neuropeptides or nerve growth factors may work. Few works have delineated the process of reinnervation, morphology changes of regenerated nerve fibers and impact of skin innervation on burns wound healing.Abolishment or reinforcement of skin innervation were performed in a animal experiment to observe the changes of wound healing and its healing quality, by which the role of skin innervation in burn wound healing.Meanwhile, innervation of burn wound tissue and scars were observed. With these outcomes we try to verify the rule of reinnervation during burn wound healing and throw some new light to regulate wound healing.Methods:1.Impact of innervation changes of rat on wound healingNinety wistar rats were divided into 3 groups randomly, A A(scalds+denervation,n=30),B(scalds+NGF,n=30),C(scals,n=30).Sample s of wound tissue were harvested on 7,14,21 days post burns(DPB).establishment of denervated and over-innervated rat model: the right spinal nerves from T9 to L1 were cut off in 30 rats 10 days before scalds.The denervated skin area was marked. Then each rat received one deep scald wound with diameter of 2 cm the right side of dorsal skin within the segment from T9 to L1.Each rat in group B received nerve growth fact of 1u daily via intramuscular injection for 14 days.The wound appearance,wound healing rate(WBR), wound duration were compared on 7,14,21 DPB. Histopatholgy of inflammatory cell infiltration, angiogenesis, epithelization were observed.Immunofluorescence techniques were performed to determine the NFP immune-positive nerve fibers, and nerve fiber volume fraction(NVF) were calculated for semi-quantity analysis;immunohistochemitry techniques were performed to determine the expression of collagenⅠand collagenⅢ; collagenⅠ/Ⅲratios were compared respectively. Rt-PCR technique were applied to determine the expression of VEGF mRNA,a representative marker for vessels.2.Changes of innervation during burn wound healing of human.The experiment subjects were divided into three groups:A,B,C three groups.Group A (full thickness burns wound tissues) 10 samples of 1,2,3 and 4 weeks after injury, respectively.Group B for hypertrophic scars,10 samples of proliferative hypertrophic scars(<six months) and mature hypertrophic scars(>six months), respectively.Group C for the normal controls total of 10 cases were from donor sites of patients correspondingly.Each specimen was cut into three plots.One was made into a 10% neutral formalin-fixed paraffin section and stained by the modified trichrome staining method, followed by pathological observation with ordinary optical microscope.The two others were made into frozen sections after liquid nitrogen fixation and then stained by immunofluorescent staining method using anti-neurofilament protein monoclonal antibody. One of the frozen sections was 10μm thick, then ordinary fluorescent microscope was employed to observe the plane structure and the quantity of the nerve fibers.The other was 30μm thick, then the laser scanning confocal microscope was employed to observe the inner and tridimensional structure of the nerve fibers. Results:1.Impact of innervation changes of rat on wound healingWound pathology and wound healing duration:there were no difference of epithelization on 7 DPB among gourp A, B,C,and yet more inflammatory cells infiltrated in group B.The crusts of group A adhered to wound more tightly and dropped off slower than those of group B、C. The wound healing duration of group A,B,C were 23.5±2.95,18.8±1.48, 20.4±2.86 respectively.Innervation:no NFP immunoreactive nerve fibers were found in group A.NFP immunoreactive nerve fibers, with spot or cosh appearance, were found in lower part of wound tissue in group B,C on 7 DPB.Nerve fiber of group B and C,looking like spot or sprout, were increasing on 14 DPB,which further improved on 21 DBP.The NVF on 14 DBP or 21 DBP of group B is higher that of group C(p<0.05)Expression of VEGF mRNA:VEGF mRNA, increased post burns, of group A increase slowly within 14 DPB and then decreased, that of group B increased more rapidly, peaked within 7 DPB and decreased slowly. VEGF mRNA of group B is higher than that of control(p<0.05). VEGF mRNA of these three group expressed more highly than that of normal (p<0.05).CollagenⅠand collagenⅢsynthesis and collagenⅠ/Ⅲratios: collagenⅠsynthesized increasingly post burns.CollagenⅠgroup B were higher than that of group A, which is lower than that of control.There was no significant difference of collagenⅢamong group A, B and C, with an increasing trend in group A.CollagenⅠ/Ⅲratios of group A were lower than those of group C(p<0.05).2.Changes of innervation during burn wound healing of human.The CVF increased during the process of wound healing.The peak CVF was observed in scars during proliferative stage,while that of scars during maturation stage tended to normal level.Gray value and NVF of PGP9.5 positive nerve fibers were lower than those of NFP.The regenerated nerve fibers 2 WPB were sparse, short and small,which was significant lower the normal control.The skin reinnervation improved during the wound healing process and came to peak NVF in proliferative stage.Disintegration and fragmentation were observed frequently in samples from proliferative stage,which seldom occurred during mature stage.Conclusions:1.The remodeling of skin nerve fibers regenerated comprise a sequential process with increasing number, distortion shape during wound healing and then disintegrated, fractured and come to normal2.Three dimension structure of nerve fibers can get with LSCM and remodel were found during the reinnervation post burns. 3.VEGF mRNA decreases with delayed wound healing when denervated, and increases with improved wound healing while over-innervated, which means innervation has an important impact on burn wound healing.4.Over innervation would accelerate wound healing and lower inneration may improve tissue remodeling.更多还原