神经节苷脂GD1a对MMP-9在不同细胞类型中的调控作用

论文摘要

已证明在FBJ细胞中,外源性的神经节苷脂GD1a负调节基质金属蛋白酶-9的表达,而对MMP-2的表达无影响。为了进一步的阐明GD1a和MMP-9的关系,本实验检测了其它细胞系,首先在沉默了GM3合成酶St3ga15和GD1a合成酶St3gal2的Lewis细胞中,发现基质金属蛋白酶的表达显著的增加,这说明了GM3或GD1a能够抑制基质金属蛋白酶的表达。因此,在不同的细胞系中,加入外源GD1a,实验结果发现,在小鼠肺癌Lewis细胞、人子宫颈癌HeLa细胞、人单核THP-1细胞中,外源性GD1a能够抑制MMP-9的表达,但对MMP-2无影响,这和FBJ细胞中的结果类似。然而,在小鼠黑色素瘤细胞B16中,外源性GD1a能够促进MMP-9的表达。这说明在不同的细胞中,神经节苷脂GD1a对MMP-9具有不同的调控作用。为了阐明GD1a对MMP-9的调控途径,比较了小鼠Lewis细胞和小鼠B16细胞。通过HPTLC分析,在小鼠黑色素瘤B16细胞中,不含有内源性的GD1a,但在Lewis细胞中,含有GD1a。通过酶谱法分析,发现和B16细胞比较,在Lewis细胞中能分泌更多的基质金属蛋白酶。因此,假想在这两株细胞中,神经节苷脂通过不同的途径来调节MMP-9的表达。实验结果表明在B16细胞中,GD1a通过caveolae介导信号传递,并且主要通过ERK促进MMP-9的表达;而在Lewis细胞中,GD1a不通过caveolae介导信号传递,但部分通过p38,ERK和JNK抑制MMP-9表达。同时,检测到在B16细胞中,TNF-α主要通过ERK促进MMP-9的表达,在Lewis细胞中,TNF-α部分通过p38,ERK和JNK抑制MMP-9表达。这样得到的结论是在不同的细胞类型中,神经节苷脂GD1a能够激活不同的信号转导途径。

论文目录

• Abstract
• 中文摘要
• Chapter 1.Introduction
• 1.1.Tumor Metastasis and MMPs
• 1.2.Gangliosides
• 1.3.Phosphoinositide 3-kinase/AKT pathway
• 1.4.MAPKs
• 1.5.Caveolae
• 1.6.Summary
• Chapter 2.Meterials and methods
• 2.1.Cell lines and cell culture
• 2.2.Chemicals
• 2.3.RNA extraction and semi-quantitatively RT-PCR
• 2.4.Gelatin zymography
• 2.5.Ganglioside extraction and HPTLC
• 2.6.Western blotting analysis
• 2.7.Stable transfection and monoclone
• Chapter 3.Augment MMPs expression in st3gal5 siRNA and st3gal2 siRNA transfected Lewis cells
• 3.1.Increased MMPs expression in st3gal5 siRNA stable transfected Lewis cells
• 3.2.Augment MMPs expression in st3gal2 siRNA by stable transfection
• Chapter 4.Diverse effect of ganglioside GD1a on MMP-9 regulation in Different Cell lines
• 4.1.Different effect of ganglioside GD1a on MMP-9 expression in different cells on transcriptional level
• 4.2.Similar effect of ganglioside GD1a on MMP-2 expression in different type of cells
• 4.3.GD1a in culture medium decreased MMP-9 but not MMP-2 expression by gelatin zymography
• Chapter 5.The differences between B16 cells and Lewis cells
• 5.1.Compared with Lewis cells,B16 cells did not contain GD1a and MMPs secreation was less.
• 5.2.Some GD1a related singnals expression were examined in B16 cells and Lewis cells
• 5.3.GD1a regulated MMP-9 expression through different MAPK pathways between Lewis cells and B16 cells
• 5.4.TNF-α regulate MMP-9 expression through different MAPK pathways between Lewis cells and B16 cells
• 5.5.MβCD and Nystatin for involvement of caveolae in GD1a signals in B16 cells but not in Lewis cells
• 5.6.Akt is used as GD1a signal pathway both in B16 cells and Lewis cells
• Chapter 6.Conclusions and discussion
• References
• Published paper
• Acknowledgements