Imai K, Senoo H
Department of Anatomy, Akita University School of Medicine, Japan.
Anat Rec. 1998 Apr;250(4):430-7. doi: 10.1002/(SICI)1097-0185(199804)250:4<430::AID-AR6>3.0.CO;2-0.
Hepatic stellate cells lie in the perisinusoidal space in a three-dimensionally distributed extracellular matrix (ECM). This three-dimensional structure of the ECM regulates the proliferation, morphology, and functions of the stellate cell. To investigate how the three-dimensional structure of ECM regulates behavior of the cells, we cultured stellate cells two- or three-dimensionally and examined the morphology of the cells in both cases as well as the localization of cell-surface adhesion molecules specific for the ECM.
Isolated rat stellate cells and human stellate cells were cultured in Dulbecco's modified Eagle's medium. Rat stellate cells were cultured in non-coated polystyrene culture dishes, or on or in type I collagen gels. The morphology of cell-ECM adhesion was examined under transmission and scanning electron microscopes. Localization of integrin alpha2 and integrin beta1 in human stellate cells was examined by immunoelectron microscopy. Immunostaining was performed with a mouse monoclonal anti-human integrin alpha2 or integrin beta1 antibody and goat anti-mouse IgG coupled with 10-nm immunogold.
Hepatic stellate cells cultured in polystyrene dishes spread well. However, the cells cultured on or in the type I collagen gel became slender. The cells extended long cellular processes onto or into the gel. The cellular processes were entangled three-dimensionally with the type I collagen fibers and directly adhered to these fibers. The cells inoculated in type I collagen gels formed a large number of adhesive structures that resembled focal adhesions. These adhesive structures were distributed not only on the lower side but also on the upper side of both the cell bodies and cellular processes. Moreover, each adhesive area formed a face but not a point. Integrin alpha2 and integrin beta1 were detected on the surfaces of cell bodies, cellular processes, and microprojections.
The cells cultured in type I collagen gel develop a three-dimensional adhesive structure.
肝星状细胞位于肝血窦周隙中，处于三维分布的细胞外基质（ECM）中。这种细胞外基质的三维结构调节星状细胞的增殖、形态和功能。为了研究细胞外基质的三维结构如何调节细胞行为，我们对星状细胞进行二维或三维培养，并观察两种情况下细胞的形态以及细胞表面特异性黏附分子在细胞外基质中的定位。
分离的大鼠星状细胞和人星状细胞在杜氏改良 Eagle 培养基中培养。大鼠星状细胞在未包被的聚苯乙烯培养皿中培养，或在 I 型胶原凝胶上或凝胶内培养。在透射电子显微镜和扫描电子显微镜下观察细胞 - 细胞外基质黏附的形态。通过免疫电子显微镜检测人星状细胞中整合素α2 和整合素β1 的定位。用小鼠抗人整合素α2 或整合素β1 单克隆抗体和与 10 纳米免疫金偶联的山羊抗小鼠 IgG 进行免疫染色。
在聚苯乙烯培养皿中培养的肝星状细胞铺展良好。然而，在 I 型胶原凝胶上或凝胶内培养的细胞变得细长。细胞在凝胶上或凝胶内伸出长的细胞突起。这些细胞突起与 I 型胶原纤维三维缠绕并直接附着于这些纤维。接种在 I 型胶原凝胶中的细胞形成大量类似黏着斑的黏附结构。这些黏附结构不仅分布在细胞体和细胞突起的下侧，也分布在上侧。此外，每个黏附区域形成一个面而非一个点。在细胞体、细胞突起和微突起表面检测到整合素α2 和整合素β1。
在 I 型胶原凝胶中培养的细胞形成三维黏附结构。
