Effect of laminin 5 on the formation of the lamina densa in vitro and in vivo

这项研究包括发展支持组织，例如IV型胶原，表皮细胞可以优先附着在上面，以及使用层粘连蛋白5，最好位于表皮-真皮交界处，从而促进致密层的形成。我们试图通过将角质形成细胞种植在真皮替代物上，收缩含有成纤维细胞的I型胶原凝胶，并在含层粘连蛋白5的培养液中继续培养一周，以0，1，5和20mug/ml的浓度继续培养一周。培养2周后，在电子显微镜下观察表皮-真皮连接。在没有层粘连蛋白5的情况下，很少发现可识别的板层密度。与对照组相比，在层粘连蛋白5(1，5，20µg/ml)的存在下，致密层常沿皮肤的表皮-真皮交界处出现。通过测量在表皮-真皮交界处单位长度中发现的致密层的总长度，证实了实验的形态计量学数据。结果表明，在含5µg/ml层粘连蛋白5和20µg/ml层粘连蛋白的等效物皮肤中，致密层的发育时间约为对照组的3倍，免疫电子显微镜下可见致密层中有层粘连蛋白5的存在。取标本，用电子显微镜进行评价。移植后第3天，补充层粘连蛋白5的移植物的致密层比对照移植物的致密层更加连续。补充层粘连蛋白5可促进致密层的形成，提高角质形成细胞的覆盖率。因此，外源性的层粘连蛋白5的存在浓度足以形成致密层并促进角质形成细胞的覆盖率。

This study includes the development of supporting tissues, collagen IV for example, on which epidermal cells can adhere preferencially, and administering laminin 5, preferably located on the epidermal-dermal junction, thereby promoting the lamina densa formation.We tried to form the skin equivalents by seeding keratinocytes on the dermal equivalents, contracted collagen I gels containing fibroblasts, and continued to culture for additional one week in the culture medium containing laminin 5 at the concentration of 0,1,5, and 20mug/ml. After 2 weeks culture, the epidermal-dermal junction was examined by electronmicroscopy.The recognizable laminae densae were rarely detected in the absence of laminin 5. By contrast to the control culture, the laminae densae were frequently observed along the epidermal-dermal junction of the skin equivalents cultured in the presence of laminin 5 (1,5,20mug/ml). Morphometrical data of the experiments were confirmed by measuring the total length of the lamina densa found in the unit length of the epidermal-dermal junction. The data indicated that the lamina densa was developed approximately 3 times as long as that of control in the skin equivalents cultured in the presence of laminin 5 at the concentration of 5mug/ml, and 6 times at 20mug/ml. By immuno-electronmicroscopy, laminin 5 was detected at the lamina densa in the skin equivalents.Keratinocytes sheets were grafted to the panniculus carnosus of the nude mice. The samples were taken and assessed by the electronmicroscopy. At day 3 after grafting, the lamina densa of grafts supplemented with laminin 5 were more continuous than that found in the control transplantation. The supplement of laminin 5 promoted the formation of the lamina densa resulting in the improvement of Keratinocytes coverage.In conclusion, exogenous laminin 5 being present at a sufficient concentration allowed to form the lamina densa and promote the keratinocytes coverage.