毛乳头细胞促进组织工程皮肤血管化的实验研究

EXPERIMENTAL STUDY ON DERMAL PAPILLARY CELLS IMPROVING VASCULARIZATION OF TISSUE ENGINEERED SKIN SUBSTITUTES

目的探讨毛乳头细胞对表皮干细胞与同种异体脱细胞真皮基质构建的组织工程皮肤血管化的影响。方法取门诊包皮环切术患者皮肤,用于表皮细胞培养;取孕19、20周引产胎儿头部皮肤,用于毛乳头细胞培养;患者及家属均知情同意。以中性蛋白酶联合胰蛋白酶消化、分离表皮细胞,IV型胶原黏附法分选表皮于细胞;以Ⅰ型胶原酶消化法分离毛乳头,常规成纤维细胞培养液培养。以同种异体脱细胞真皮基质为支架,在真皮基质的真皮乳头侧种植毛乳头细胞,基底膜侧种植表皮干细胞构建实验组组织工程皮肤替代物;对照组组织工程皮肤替代物不种植毛乳头细胞。取6～8周龄BALB/C-nu裸鼠60只,随机分成实验组和对照组(n=30);实验动物背部制造1 cm×1 cm大小全层皮肤缺损模型,将两组组织工程皮肤替代物移植修复创面,2周后观察移植皮肤成活率。术后2、4周,取移植皮肤替代物标本,行HE及免疫组织化学染色,观察移植皮肤替代物CD31表达水平,并计算两组标本血管密度。结果Ⅳ型胶原分选后表皮细胞同时表达角蛋白19、β_1整合素,提示为表皮干细胞。由毛乳头培养出的细胞表达α平滑肌肌动蛋白,提示为毛乳头细胞。动物移植术后2周,实验组移植皮肤成活率为93.3%(28/30),对照组为80.0%(24/30),比较差异有统计学意义(χ~2=2.31,P=-0.00)。HE染色观察示实验组表皮层达12层,表皮细胞体积较大;对照组表皮层达4～6层,表皮细胞体积较小。实验组术后2、4周,血管密度分别为(38.56±2.49)个/mm2和(49.12±2.39)个/mm^2,对照组分别为(25.16±3.73)个/mm^2和(36.26±3.24)个/mm^2,两组比较差异均有统计学意义(P<0.01)。结论毛乳头细胞可促进移植皮肤替代物血管形成,利于表皮层重建,提高组织工程皮肤移植成活率。

Objective To investigate the impact of dermal papillary cells on vascularization of tissue engineered skin substitutes consisting of epidermal stem cells and allogeneic acellular dormal matrix. Methods Human foreskins from routine circumcisions were collected to separate epidermalc cells by using dispase with trypsogen. Collagen Type IV was used to isolate epidermal stem cells from the second and third passage keratinocytes. Dermal papilla was isolated by the digestion method of collagenase I from fetus scalp and cultured in routine fibroblast medium. Tissue engineered skin substitutes were reconstructed by seeding epidermal stem cells on the papillary side of acellular dermis with （the experimental group） or without （the control group） seeding dermal papillary cells on the reticular side. The two kinds of composite skin substitutes were employed to cover skin defects on the back of the nude mice （n=30）. The grafting survival rate was recorded 2 weeks after grafting. HE staining and immunohistochemistry method were employed to determine the expression of CD31 and calculate the microvessel density at 2 and 4 weeks after grafting. Results Those adhesion cells by collagen type IV coexpressed Keratin 19 and β 1 integrin, indicating that the cells were epidermal stem cells. The cultivated dermal papilla cells were identified by expressing high levels of α-smooth muscle actin. The grafting survival rate was significantly higher in experiment group （93.3%）, than that in control group （80.0%）. HE staining showed that the epithel ial layer in the experimental group was 10-layered with large epithel ial cells in the grafted composite skin at 2 weeks, and that the epithel ial layer in control group was 4-6 layered with small epithel ial cells. At 2 and 4 weeks after grafting, the microvessel density was （38.56 ± 2.49） and （49.12 ± 2.39） per square mill imeter in the experimental group and was （25.16 ± 3.73） and （36.26 ± 3.24） per square mill imeter in the control group respectively, showing significant differences between 2 groups （P 〈 0.01）. Conclusion Addition of dermal papillary cells to the tissue engineered skin substitutes can enhance vascularization, which promotes epidermis formation and improves the grafting survival rate.