Regulation of morphology and gene expression in cultured hepatic stellate cells by three-dimensional structure of extracellular matrix

在响应细胞外基质（ECM）成分，特别是间质胶原蛋白作为基质，培养的肝星状细胞（HSC）表现出显着的变化，在其形态，并延长了许多类似于在体内结构的细胞过程。我们的研究结果表明，在培养的HSC中，突起延伸的诱导依赖于细胞表面整合素与间质胶原的结合、细胞内信号传导、微管相关蛋白2（MAP 2）以及最终的微管重组。ECM成分也影响增殖能力和胶原合成和分泌活性，除了细胞形态，在培养的HSC。通过原位酶谱、明胶酶谱和逆转录-聚合酶链反应（RT-PCR）分析培养的HSC中基质金属蛋白酶（MMP）的表达，提示HSC组分对MMP-1、MMP-2、MMP-13和膜型MMP（MT 1-MMP）的表达具有调节作用， 关于我们 以及一些MMPs被加工成活化形式。因此，HSC周围的ECM组分似乎在转录和/或翻译阶段或在翻译后加工中控制基底膜组分（包括IV型胶原和层粘连蛋白）或基质组分（如I型或III型胶原）的重组。由于当HSC在I型胶原包被的培养皿中培养时没有发生突起伸长，因此HSC似乎识别I型胶原的二级或三级结构。然而，尚不清楚细胞如何识别I型胶原蛋白的二级或三级结构。我们将HSC分别在聚苯乙烯表面、I型胶原包被表面、I型胶原凝胶表面、I型胶原凝胶中培养，然后用RT-PCR和差异显示技术检测不同ECM成分的mRNA差异表达。通过鉴定I型胶原凝胶培养的HSC中表达的特异性mRNA，可以阐明HSC识别I型胶原的二级或三级结构，并被诱导表达特异性mRNA并改变其形态和功能的机制。少

In response to extracellular matrix (ECM) components, particularly, to interstitial collagen used as a substratum, cultured hepatic stellate cells (HSCs) exhibited a marked change in their morphology, and elongated many cellular processes resembling in vivo structure. The results from our study indicated that the induction of process elongation in cultured HSCs was dependent on cell surface integrin-binding to interstitial collagen, intracellular signaling, microtubule-associated protein 2 (MAP2), and finally microtubule reorganization. ECM components affected also proliferative ability and collagen synthesis and excretion activity, in addition to cell morphology, in cultured HSCs. Matrix metalloproteinase (MMP) expression in cultured HSCs was analyzed by in situ zymography, gelatin zymography, and reverse transcription- polymerase chain reaction (RT-PCR), suggesting the regulatory role of HSC components on the expression of MMP-1, MMP-2, MMP-13, and membrane type of MMP (MT1-MMP), as … More well as the processing of some MMPs to activated form. Thus, ECM components surrounding HSCs appear to control reorganization of basal membrane components including type TV collagen and laminin or stromal components such as type I or type III collagen at transcriptional and/or translational stages or at posttranslational processing. Since no process elongation was occurred when HSCs were cultured in type I-collagen coated dishes, HSCs seems to recognize a secondary or tertiary structure of type I collagen. However, it is not known how cells recognize the secondary or tertiary structure of type I collagen. We cultured HSCs on polystyrene surface, on type I collagen-coated surface, on type I collagen gel, or in type I collagen get, and then examined the differential expression of mRNA species depending on ECM components by using RT-PCR and differential display method. The identification of specific mRNAs expressed in HSCs cultured using type I collagen gel may leads us to clarify the mechanism by which HSCs recognize the secondary or tertiary structure of type I collagen, and are induced to express the specific mRNAs and to alter their morphology and function. Less