体外培养表皮干细胞复合高分子支架原位修复深度烧伤创面的研究

Study on in vitro cultured epidermal stem cell combined with polymer scaffold and the in situ repair of burn wounds

目的探讨表皮干细胞联合成纤维细胞-丝素蛋白纳米纤维活性支架体内培养,对Ⅲ度烧伤创面的修复和再生作用。方法 (1)表皮干细胞的培养和表征:采用快速贴壁法分离和培养表皮干细胞(Epidermal Stem Cell,ESC)。将表皮干细胞分别在经Ⅳ型胶原蛋白修饰的和未经修饰的培养瓶中,或通过悬浮法培养,研究表皮干细胞的生长特性;以βl整合素和细胞角蛋白CK19免疫荧光染色实验考察细胞表型。(2)活性支架的体外构建和对大鼠Ⅲ度创面的修复作用研究:体外构建成纤维细胞-丝素蛋白纳米纤维活性支架;采取同体对照法,在20只Sprague-Dawley大鼠(SD大鼠)背部制作两个Ⅲ度切痂创面。左侧创面采用体外培养的自体表皮干细胞联合成纤维细胞-丝素蛋白纳米纤维活性支架移植入创面,作为组织工程移植物组;右侧创面采用凡士林纱布敷料覆盖,作为凡士林纱布敷料组。考察组织工程移植物对大鼠Ⅲ度创面的愈合作用。结果以快速贴壁法能够有效地分离得到表皮干细胞,细胞在经Ⅳ型胶原蛋白修饰的培养瓶中生长10d后融合,数目达到5.1×105/cm2。免疫荧光实验表明细胞表面抗原呈βl整合素和角蛋白免疫CK19成阳性,证明分离得到的细胞为表皮干细胞。成纤维细胞能够在丝素蛋白纳米纤维中扩增并分泌细胞外基质,14d后与丝素蛋白纳米纤维形成活性支架。对大鼠Ⅲ度烧伤创面的修复实验表明,组织工程移植物组的创面在第14天和第22天的平均愈合效率为66%和93%,高于凡士林纱布敷料组(32%和69%),P<0.05。组织工程移植物组的创面平均愈合天数为21d,低于凡士林纱布敷料组(31d),P<0.05。结论通过Ⅳ型胶原蛋白黏附法,能够分离得到表皮干细胞,并且其在Ⅳ型胶原蛋白表面修饰的培养瓶中的生长活力较高。大鼠Ⅲ度创面的修复实验表明,组织工程移植物,即表皮干细胞联合成纤维细胞-丝素蛋白纳米纤维支架,能够修复Ⅲ度创面,再生皮肤表真皮结构完整;并且与凡士林纱布敷料相比,能够提高创面的愈合效率,减少创面的愈合时间。

Objective To exploit wound repair and regeneration function by means of epidermal stem cell （ ESC）, together with active scaffold of silk fibroin nanofiber. Methods （ 1 ） Culture and characterization of epidermal stem cell. Fast adherence method was adopted to isolate and culture ESC. ESC was cultured in type Ⅳ collagen superficially modified or non-modified culture plates, or cuhured in suspension. ESC proliferation character was studied. Cell type was studied by immunofluorescence experiment of cell superficial antigen, β1 integrin and cytokeratin, CK19. （2） In vitro construction of active scaffold and study on its healing effect of rat full-thickness burn wound. Active fibrnblast-silk fibroin nanofiber scaffold （SFNS） was in vitro constructed. By applying homobody control method, two full-thickness burn wound were excised on the back of 20 Sprague-Dawley rats （ SD rats）. On the left wound, in vitro cultured ESC together with active fibroblast-SFNS was transplanted, set as test group （tissue engineering transplant group）. On the right wound, paraffin bandage dressing was adopted, set as the control （paraffin bandage dressing group）. Healing effect of tissue engineering transplant on rat 11 -thickness burn wound was studied. Results Fast adherence method could effectively isolate ESC. Cells were proliferated into confluence after 10 days＇ culture in type Ⅳ collagen modified culture flask, at a number of 5.1×10^5/cm^2. Immunofluorescence experiment indicated that β1 integrin and CK19 were positive on cell surface, which confirmed ESC phenotype. Fibroblast was proliferated and secreted extracellular matrix in SFNS, and transformed into active scaffold together with SFNS after 14 days. Experiment on healing effect of rat full-thickness burn wound indicated that healing efficiency of wounds in tissue engineering transplant group was 66% and 93%, respectively for 14 days and 22 days, higher than in paraffin bandage dressing group （32% and 69% ）, P 〈0.05. Wound healing time in tissue engineering transplant group was 21 days, lower than in paraffin bandage dressing group （31 days）, P 〈 0.05. Conclusions By type Ⅳ collagen adhering method, ESC could be isolated and proliferated in type Ⅳ collagen modified culture flask with high activity. Healing experiment of rat full-thickness burn wound indicated that, tissue engineering transplant, active fibroblast-SFNS, could heal third-degree burn wounds, and regenerate epidermal and dermal structure of skin. Moreover, compared with paraffin bandage dressing, it could improve wound healing efficiency and lower wound healing time.