KGF和TGF-β1活性短肽缓释型重构瘢痕真皮替代物RSDS的实验研究

To resolve the difficult problem of the insufficient source of autogenic skin and dermal scaffold for large scar repair, the hypothesis of the reconstitution and recycle of scar tissue was advanced. The project was described as followed: ① KGF and TGF-β1 active peptides were compounded respectively according to the previous analysis of the gene sequence of KGF and TGF-β1; ② To assess and optimize the active peptides, we proceeded in vitro study with epidermal cells and fibroblasts as well as in vivo study with the chronic wound of the rat model of diabetes mellitus; ③ The fresh scar tissue from the patients who underwent the cicatrectomy was harvested and applied for the fabrication of the reconstituted dermal matrix membrane with acelluar process and cross-linked treatment; ④ The reconstituted collagen membrane was complexed with type I collagen from fresh scar tissue after frozen-dry and cross-linking treatment; ⑤ In vitro study was carried out to assess and optimize the biological characteristics of the reconstituted scar dermal substitutes (RSDS) (including reconstituted dermal matrix membrane and reconstituted collagen membrane) as cell supporter; ⑥ In vivo study was carried out to assess and optimize the biological properties and bio-security of RSDS; ⑦ KGF and TGF-β1 active peptides were utilized for reconstituting RSDS, and in vitro study and in vivo study were carried out respectively to assess the effect of KGF and TGF-β1 active peptides on the bioactivity of RSDS. Through the study, such modified RSDS was expected to be a promising alternative for dermal scaffold and a novel drug to promote wound healing, and to provide a new method to repair large scar.

为解决大面积瘢痕修复皮源和真皮支架不足难题，提出瘢痕组织重构再利用的研究思路，拟：①依据前期KGF和TGF-β1序列分析结果，合成KGF和TGF-β1活性短肽；②通过表皮细胞和成纤维细胞离体试验和糖尿病大鼠慢性创面在体试验，检测优化活性短肽；③选取新鲜瘢痕组织，经过脱细胞和交联处理，制备重构真皮基质膜；④选取新鲜瘢痕组织，提取I型胶原蛋白，进行冷冻成膜和交联处理，制作重构胶原蛋白膜；⑤进行重构瘢痕真皮替代物（Reconstituted scar dermal substitutes, RSDS）离体试验，检测作为细胞载体的优越性，进行结构优化；⑥进行RSDS在体试验，检测其生物学性能及应用安全性，进行结构优化；⑦在RSDS制备过程中添加KGF和TGF-β1活性短肽，进行离体和在体试验，检测活性短肽对RSDS生物活性的诱导作用。期望能够获得一种性能优良的RSDS和一种促进创面愈合的新型药物。

背景：目前临床上应用的生长因子和真皮支架材料存在很多缺点，难以满足临床应用需要。.主要内容：进行KGF和TGF-β1的活性短肽的合成、纯化、修饰及免疫荧光标记。用免疫荧光法检测KGF活性短肽和TGF-β1活性短肽的细胞亲和力。用CCK-8法检测KGF活性短肽促进表皮细胞增殖的作用，检测TGF-β1活性短肽促进成纤维细胞增殖的作用。用RT-PCR法和Western-blot法检测表皮细胞上的特异性受体KGFR的表达水平，检测成纤维细胞上的特异性受体的表达水平。进行SD糖尿病大鼠创面愈合试验。制备瘢痕脱细胞真皮基质，进行生物力学性能和组织结构检测，以及离体细胞种植实验和在体动物试验。.重要结果：合成3个KGF活性短肽，能够与表皮细胞相结合，促进表皮细胞增殖，促进SD糖尿病大鼠的难愈性创面愈合。合成1个TGF-β1活性短肽，能够与成纤维细胞相结合，促进成纤维细胞增殖，促进SD糖尿病大鼠的难愈性创面愈合。制备三种ADM，正常皮肤ADM和成熟性瘢痕ADM质地柔软、有弹性，而增生性瘢痕ADM质地和结构稍差，需要进行结构改进。动物试验显示成熟性瘢痕ADM和增生性瘢痕ADM非劣于正常皮肤ADM。.关键数据：CCK-8结果显示与阴性对照组相比，3个KGF活性短肽能够促进表皮细胞增殖，1个TGF-β1活性短肽能够促进成纤维细胞增殖，并呈浓度依赖性，具有统计学差异。RT-PCR和Western-blot结果显示3个KGF活性短肽组中表皮细胞表达特异性受体KGFR增强，1个TGF-β1活性短肽组中成纤维细胞表达特异性受体增强。动物试验结果显示与阴性对照组相比，3个KGF活性短肽和1个TGF-β1活性短肽能够显著地促进创面愈合，具有统计学差异。增生性瘢痕ADM、成熟瘢痕ADM和正常皮肤 ADM的应力-应变参数β值、蠕变斜率和最大拉伸应力之间差异无统计学意义，成熟瘢痕ADM与正常皮肤ADM的应力-应变伸长比、松弛斜率、松弛量、蠕变量之间的差异无统计学意义，而增生性瘢痕ADM与正常皮肤ADM的应力-应变伸长比、松弛斜率、松弛量和蠕变量之间差异有统计学意义。三种ADM组的复合植皮成活率和成活皮肤质量无显著性差异。.科学意义：KGF活性短肽和TGF-β1活性短肽有望应用于促进创面愈合，为生长因子的结构调整和生产方法开拓新的思路；瘢痕脱细胞真皮基质有望作为真皮支架材料应用于临床。