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

体外培养的肝星状细胞(HSCs)对细胞外基质(ECM)成分，特别是对间质胶原的作用，表现出明显的形态变化，并拉长了许多类似体内结构的细胞突起。我们的研究结果表明，HSCs突起延长的诱导依赖于细胞表面整合素与间质胶原的结合、细胞内信号转导、微管相关蛋白2(MAP2)，最后是微管重组。除了细胞形态外，细胞外基质成分还影响培养的HSCs的增殖能力、胶原合成和排泄活性。用原位酶谱、明胶酶谱和逆转录-聚合酶链式反应分析培养的肝星状细胞基质金属蛋白酶的表达，提示肝星状细胞成分对基质金属蛋白酶-1、基质金属蛋白酶-2、基质金属蛋白酶-13的表达以及基质金属蛋白酶的膜类型(MT1-…)具有调节作用更多的是将一些MMPs处理成激活表。因此，HSC周围的ECM成分似乎控制着转录和/或翻译阶段或翻译后加工阶段的基底膜成分，包括TV型胶原和层粘连蛋白或间质成分，如I型或III型胶原的重组。由于在I型胶原涂层的培养皿中培养的HSCs没有发生突起延长，HSCs似乎识别I型胶原的二级或三级结构。然而，目前尚不清楚细胞如何识别I型胶原的二级或三级结构。我们在聚苯乙烯表面、I型胶原涂层表面、I型胶原凝胶或I型胶原GET上培养HSCs，然后用RT-PCR和差异显示方法检测不同ECM成分下HSCs基因的差异表达。鉴定在I型胶原凝胶培养的HSCs中表达的特定mRNAs可能有助于阐明HSCs识别I型胶原的二级或三级结构，并被诱导表达特定mRNAs并改变其形态和功能的机制。较少

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