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

细胞外基质三维结构对培养肝星状细胞形态和基因表达的调控

• 批准号: 11680689
• 负责人: SATO Mitsuru
• 金额: $ 2.37万
• 依托单位: Akita University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680689/
• 关键词: Hepatic stellate cell; Extracellular matix; Interstitial collagen; Intracellular signaling; Differential display; Microtubule-associated protein; Gene expression; Matrix metalloproteinase; I型コラーゲン; 微小管; 低分子量Gタンパク質; インテグリン

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

作为对细胞外基质（ECM）成分的反应，特别是作为基质的间质胶原，培养的肝星状细胞（hsc）在形态上表现出明显的变化，并延长了许多类似于体内结构的细胞过程。我们的研究结果表明，在培养的hsc中，过程伸长的诱导依赖于细胞表面整合素与间质胶原的结合、细胞内信号传导、微管相关蛋白2 (MAP2)，最后是微管重组。在培养的造血干细胞中，除了细胞形态外，ECM成分还影响了增殖能力、胶原合成和排泄活性。采用原位酶谱法、明胶酶谱法和逆转录聚合酶链反应（RT-PCR）分析了培养的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MMP膜型（MT1-MMP）的表达具有调节作用，并参与了部分MMP的活化过程。因此，造血干细胞周围的ECM成分似乎在转录和/或翻译阶段或翻译后加工过程中控制基底膜成分（包括TV型胶原和层粘胶蛋白）或基质成分（如I型或III型胶原）的重组。由于造血干细胞在I型胶原包膜培养皿中培养时没有发生过程延伸，因此造血干细胞似乎识别出I型胶原的二级或三级结构。然而，细胞如何识别I型胶原蛋白的二级或三级结构尚不清楚。我们分别在聚苯乙烯表面、I型胶原包被表面、I型胶原凝胶表面和I型胶原get表面培养hsc，然后利用RT-PCR和差异展示法检测不同ECM组分mRNA种类的差异表达。利用I型胶原凝胶培养的造血干细胞中表达的特异性mrna的鉴定可能有助于我们阐明造血干细胞识别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

项目成果

Kojima N,Sato M, et al.: "Alteration in distribution of focal adhesion components by signaling inhibitors in hepatic stellate cells and fibroblasts cultured on type I collagen gel"Cells of Hepatic Sinusoid. 7. 24-25 (1999)

Kojima N、Sato M 等人：“在 I 型胶原凝胶上培养的肝星状细胞和成纤维细胞中信号抑制剂改变粘着斑成分的分布”肝窦细胞。

Li Y-L,Sato M, et al.: "Regulatory role of extracellular matrix components in expression of matrix metalloproteinases in cultured hepatic stellate cells"Cell Structure and Function. 24. 255-261 (1999)

Li Y-L，Sato M，等人：“细胞外基质成分在培养的肝星状细胞中基质金属蛋白酶表达中的调节作用”细胞结构和功能。

Sato M et al.: "Nuclear deviation in hepatic parenchymal cells on sinusoidal surfaces in arctic animals"Cell Structure and Function. 26(in press). (2001)

Sato M 等人：“北极动物肝窦表面肝实质细胞的核偏差”细胞结构和功能。

Sato M, et al.: "Intracellular signaling for process elongation in cultured hepatic stellate cells on type I collagen gel"Cells of Hepatic Sinusoid. 7. 32-33 (1999)

Sato M 等人：“I 型胶原凝胶上培养的肝星状细胞中过程伸长的细胞内信号传导”肝正弦细胞。

Sato M et al.: "Intracellular signaling for process elongation in culturted hepatic stellate cells on type I collagen gel"Cells Hepatic Sinusoid. 7. 32-33 (1999)

Sato M 等人：“I 型胶原凝胶上培养的肝星状细胞中过程伸长的细胞内信号传导”细胞肝正弦波。