摘要
尽管国内外学者长期以来对增生性瘢痕(hypertrophic scar,HS)进行了大量、不懈的研究,但有关HS的形成机制目前还不清楚,临床上也缺少特别有效的防治手段,造成这种局面的原因之一就是缺少一种理想的HS动物模型。只有人才会自然发生HS,这是HS的特征之一,依据“内因是事物发展的根据,它决定着事物发展的基本趋向”这一自然辩证法则,我们推测HS的形成是人类皮肤的某种或某些内在的因素所决定的,因此通过将人皮肤移植于裸鼠的方法可以建立HS动物模型。本课题研究旨在通过移植人皮肤于裸鼠体表建立HS动物模型,较系统地观察瘢痕形成病理过程,并就瘢痕增生的形成机制进行相关实验研究,主要结果与结论如下:
1.人全厚皮片移植于裸鼠体表后会发生一定程度的排斥反应,移植皮片的表皮及部分真皮逐渐脱落。但由于裸鼠是T细胞免疫缺陷动物,这种排斥反应发生迟,程度较轻,移植人皮片的真皮层部分得以存活,并成为移植皮片干痂脱落后瘢痕增生的基础。我们的结果一方面说明裸鼠体内存在非T细胞依赖的免疫机制,另一方面说明长期的慢性免疫反应可能是瘢痕增生的促进因素。
2.移植人全厚皮片的表皮及部分真皮脱落后,局部可以发生明显的瘢痕增生,该瘢痕无论在大体外观、增生特性还是病理组织学特性上都与人的HS相似。
3.用免疫组织化学的方法研究模型瘢痕中的TGFβ-1的变化规律,发现增生期模型瘢痕中TGF-β1持续高水平表达,而消退期则显著降低。
4.体外细胞培养实验结果表明,模型瘢痕成纤维细胞与人HS成纤维细胞形态上非常相似,细胞增殖及胶原合成活性亦无显著差别。
5.对比模型瘢痕组织以及正常皮肤中胶原酶活性,发现增生期的模型瘢痕胶原酶活性低,表明模型瘢痕中胶原等细胞外基质降解减少。
6.以模型瘢痕组织和正常人皮肤组织及人HS组织的DNA为模板进行PCR,结果发现都可以扩增出人源性特异性基因,说明模型瘢痕组织中肯定存在有人源性DNA。
7.只有移植人的全厚皮片才会发生明显的瘢痕增生,而移植人的刃厚皮片以及大鼠的全厚皮片则根本不发生瘢痕增生,移植人中厚皮片虽然部分可以发生一定程度的瘢痕增生,但增生程度及发生率均明显不如移植人的全厚皮片。这说明人皮肤真皮层是HS发生的决定因素。本发现在人体外证实了只有人皮肤才会发生HS这一现象,并证明了人皮肤真皮层是HS发生的决定因素,而不是表皮层或其他外界环境因素,进一步揭示了HS的发生机制。
上述结果表明,人皮肤移植于裸鼠后会发生一定程度的排斥反应,排斥反应的发生使移植的人皮肤的表皮及部分真皮坏死脱落,但仍有部分真皮存活,存活的这部分人真皮是HS发生的基础。真皮中的人成纤维细胞在局部炎性反应的持续刺激下功能状态发生改变,细胞增殖旺盛,并合成大量的胶原等细胞外基质,从而导致瘢痕的持续增生。该模型瘢痕增生明显,增生持续时间长,瘢痕的大体形态、生长特性、组织学特点等都和人HS相似,具有很好的代表性,可用于瘢痕形成机制及临床防治的相关研究。
结论:
1.通过在裸鼠背部体表移植人全厚皮片的方法可以建立HS动物模型;
2.人皮肤移植于裸鼠体表后可以发生不完全的排斥反应,其表皮及部分真皮受排斥而逐渐脱落,但有部分真皮长期存活;
3.该模型无论在大体外观、生长特性、组织学特点以及遗传学特性等方面都和人的HS相似,且具有很好的可控性,能观察瘢痕发生发展的全过程,是一种较为理想的人HS动物模型;
4.人皮肤真皮是发生HS的内在决定因素,而局部的持续炎性反应则是重要的促进因素。
Hypertrophic scar formation remains the major problem for severely burned patients who survive their injuries. This scarring can result in both cosmetic and functional deformities. One of the major problems in dealing with this complication is the lack of an adequate animal model with which to test various possible therapeutic modalities.
We had attempted to establish an animal model of hypertrophic scarring by transplanting full-thickness human skin grafts onto the backs of nude mice and then subjecting the grafts to an episode of burn. We found, accidentally, that obvious local hypertrophic scars developed in mice not subjected to burn insults after the epidermis and upper portions of the dermis of the human skin grafts were shed. Based on this finding, we further investigated the feasibility of establishing an animal model of hypertrophic scarring by transplanting human skin grafts onto nude mice without burning. Main results and conclusions are as below:
1. After transplantation, the epidermis and upper portions of the dermis of the human skin grafts began to stiffen, change from pink to brownish yellow and then to black with cocked edges, and fall off by degrees. After the epidermis and upper portions of the dermis of the human skin grafts were shed, local scars developed. 54 of 60 nude mice (90.0 percent) developed significant, persistent hypertrophic scars. Scar hypertrophy reached a peak approximately 5 months after transplantation, with an average maximal thickness of 3.20 mm (maximum, 5.10 mm). On average, scar hypertrophy persisted for 135 days. The dermis layer of the scars thickened significantly and the demarcation between the papillary and reticular layers of dermis was obscure; collagen fibers were dense, with derangements in collagen bundles, which were horizontally arranged in the profound dermis and nodular or swirled in the superficial dermis. Cells and microvessels increased within the dermis and karyomitosis was obvious. These scars were extremely similar to human hypertrophyic scar both in gross appearance and histology.
2. Tissues from various stages of model sccar were harvested to examine TGF-β1 by immunohistochemical method. Results show that TGF-β1 were highly expressed in the scar tissues 1 and 3 months later after human skin graft transplantation, whereas the expression was obviously declined in the scar tissues 6 months later.
3. Fibroblasts cultured from model scar tissue are extremely similar in morphology to those from human hypertrophic scar, and the cellular proliferation (by MTT method) and collagen synthesis ability(by ~3H-proline uptaken) between them are of no significant difference.
4. Comprared with collagenase activity in human nommal skin, collagenase activity was significantly reduced in model scar tissue, which showed that the degradation of ollagen was deficient as in human hypertrophic scar.
5. Special fragment of human HLA-A gene could be detected by PCR both in model scar tissue and human hypertrophic scar or skin tissue, but the production was lower in model scar tissue. The result shows that there was human resourced DNA in model scar tissue, while human was not the only resource.
6. Obvious scar proliferation only developed in group under full-thickness human skin graft transplantation, not in groups under epidermal human skin graft transplantation nor full-thickness rat skin graft transplantation. So it's the human skin itself, not other surroundings such as cytokines, growth factors or wound liquids, determined whether scar proliferation would happen.
7. Because nude mice are immunodefective of T cells, the rejection against transplanted human skin graft is weak and incomplete, so the profound dermis of weaker antigenicty can survive the rejection after transplantation and might be the base of later scar proliferation.
Considered together, the animal model of hypertrophic scarring can be established by transplanting human skin grafts onto nude mice, and this model can reproduce hypertrophic scars readily. The hypertrophic scars can persist for a long time and possess the genetic and histologic properties of human hypertrophic scars, and they are also controllable and comparable to human hypertrophic scars, thus making possible the observation of the entire process of hypertrophic scar formation. As a result, it will facilitate further research on the pathogenesis of hypertrophic scarring and provide an important tool for clinical and laboratory study of this pathologic condition.
引文
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