Fleischmajer R, Kühn K, Sato Y, MacDonald E D, Perlish J S, Pan T C, Chu M L, Kishiro Y, Oohashi T, Bernier S M, Yamada Y, Ninomiya Y
Department of Dermatology, Mount Sinai School of Medicine, New York, NY 10029, U.S.A.
J Invest Dermatol. 1997 Oct;109(4):527-33. doi: 10.1111/1523-1747.ep12336696.
Temporal and spatial expression of alpha1 (IV), alpha2 (IV), alpha3 (IV), alpha4 (IV), alpha5 (IV), and alpha6 (IV) collagen chains was studied during the formation of the basal lamina in an "in vitro" skin model. A sequential study was performed at 7-d and 14-d cultures (lamina densa absent) and at 28-, 36-, and 56-d cultures (lamina densa present). Expression of beta1, beta4, alpha1, alpha2, alpha3, alpha5, alpha6 integrin subunits and co-localization with collagen IV was studied by regular and laser confocal indirect immunofluorescence microscopy. mRNA expression of alpha2 (IV) and alpha6 (IV) chains was estimated by northern blots. The earliest expression of alpha1 (IV) and alpha2 (IV) collagen chains was noted in 7-d cultures restricted to basal keratinocytes. At 14-d cultures, alpha1 (IV) and alpha2 (IV) chains were noted in basal keratinocytes and as a broad band (10 microm) in the adjacent dermis. At this stage 80% of the alpha2 (IV) mRNA was expressed in the dermis and 20% in the epidermis. At 28-, 36-, and 56-d cultures the alpha1 (IV) and alpha2 (IV) chains were present in a linear distribution at the epidermo-dermal junction and in the upper dermis. The alpha6 (IV) collagen chains were expressed much later at 36-d cultures and the alpha5 (IV) at 56 d, both mostly in a linear distribution but also in the adjacent dermis. Alpha6 (IV) mRNA was demonstrated in the dermis of 36-d cultures. There was co-localization of collagen IV and beta1 integrin subunits in 14-d cultures at the matrix site of keratinocytes. Functional perturbation studies with AIIB2 monoclonal antibody (anti-beta1 subunits) and competitive inhibition with a collagen cyanogen bromide digestion derived fragment (CB3[IV]) that contains the collagen IV ligand for alpha1beta1, alpha2beta1 integrins, altered the pattern of collagen IV deposition.
在一个“体外”皮肤模型的基膜形成过程中，研究了α1(IV)、α2(IV)、α3(IV)、α4(IV)、α5(IV)和α6(IV)胶原链的时空表达。在7天和14天培养物（无致密板）以及28天、36天和56天培养物（有致密板）中进行了顺序研究。通过常规和激光共聚焦间接免疫荧光显微镜研究了β1、β4、α1、α2、α3、α5、α6整合素亚基的表达及其与IV型胶原的共定位。通过Northern印迹法估计α2(IV)和α6(IV)链的mRNA表达。最早在7天培养物中观察到α1(IV)和α2(IV)胶原链的表达，局限于基底角质形成细胞。在14天培养物中，在基底角质形成细胞以及相邻真皮中的一条宽带（10微米）中观察到α1(IV)和α2(IV)链。在此阶段，80%的α2(IV)mRNA在真皮中表达，20%在表皮中表达。在28天、36天和56天培养物中，α1(IV)和α2(IV)链呈线性分布于表皮-真皮交界处和真皮上层。α6(IV)胶原链在36天培养物中表达较晚，α5(IV)在56天表达，两者大多呈线性分布，但也存在于相邻真皮中。在36天培养物的真皮中证实有α6(IV)mRNA。在14天培养物中，在角质形成细胞的基质部位发现IV型胶原和β1整合素亚基的共定位。用AIIB2单克隆抗体（抗β1亚基）进行功能干扰研究，并用含有α1β1、α2β1整合素的IV型胶原配体的溴化氰消化衍生片段（CB3[IV]）进行竞争性抑制，改变了IV型胶原的沉积模式。
