【作者】 王璞；

【导师】 张景海； 山形达也；

【作者基本信息】 沈阳药科大学 ， 微生物与生化药学， 2007， 博士

【摘要】 神经节苷脂是脊椎动物细胞膜的主要组成成分，由神经鞘氨醇和脂肪酸组成的神经酰胺组成了神经节苷脂的疏水残基。神经节苷脂已经被确认为肿瘤抗原、微生物及其产生毒素的受体，并且参与了细胞生长、分化。神经节苷脂通过调节细胞与细胞之间的相互作用来进一步调节肿瘤细胞的侵蚀与转移。最近，发现GM3参与小鼠黑色素瘤B16细胞形态、细胞运动性、生长和转移的调节。在半乳糖转移酶-6(B4Gal-T6)顺反cDNA转染的细胞中，初步验证了GM3的生物学功能(SM-1和CSSH-1分别是质粒和B4Gal-T6 cDNA正义链转染的单克隆细胞；CM-1和CAH-3是质粒和B4Gal-T6 cDNA反义链转染的单克隆细胞)。另外，通过siRNA沉默GM3也表现出与CAH-3相似的调节细胞形态、细胞运动性和生长的特性。细胞变圆并且按照与对照细胞不同的方式生长。GM3合成酶沉默的单克隆细胞B11通过调节MMP-9的表达来降低细胞的运动性。然而，与对照细胞相比，细胞内的GM3的量降低，细胞的恶性程度加剧。实验表明，GM3沉默的细胞更易于在不含血清的培养基或软琼脂的培养基中生长。接下来的实验证明了GM3通过其下游分子Arhgdib来调节细胞的生物学功能。与此同时，经过基因筛选发现，Arhgdib是GM3的效应因子，GM3通过PI3K／Akt／mTOR途径来调节Arhgdib的表达，尤其是PDK1在Arhgdib的调节过程中起到了非常重要的作用。另外，进一步的实验证明了Arhgdib沉默的细胞中细胞的形态、细胞的运动性和生长发生了明显的改变，而这种变化恰恰与GM3沉默的B11细胞相似。这些结果有力的证明了GM3是通过Arhgdib调节细胞生物学功能的。最后发现TNFα是Arhgdib的下游分子。更多还原

【Abstract】 Glycosphingolipids (GSLs) are characteristic components of vertebrate plasma membranes and have the same hydrophobic residue, ceramide, which consists of a sphingosine and a fatty acid. GSLs have been defined as tumor antigens, receptors for microbes and their toxins, and possible modulators of cell proliferation, differentiation, and cell-cell interactions through which GSLs exert its function on cell invasiveness and metastasis. Recently, we found that GM3 regulated cell behaviors including cell shape, cell motility, proliferation and metastasis in B16 cells by transfering cells with B4Gal-T6 sense or antisense cDNA (SM-1 and CSSH-1 are mock and B4Gal-T6 sense cDNA transfectant monoclonal cell lines respectively; CM-1 and CAH-3 are mock and B4Gal-T6 antisense cDNA transfectant monoclonal cell lines respectively).Furthermore, GM3 down-regulation by siRNA targeting St3gal5 (GM3 synthase) resulted in change the cell behaviors including cell shape, cell motility and proliferation. The St3gal5 siRNA transfected cells (one of monoclonal transfectants was B11 cells) were round and refractile and tended to grow distinctly from parental B16 cells. The motility of B11 cells was decreased and cell motlility was possibly mediated by MMP-9. B11 cells proliferated easily in serum free condition or soft agar, possibly because GM3 down regulation enhances its malignancy compared with control cells. Arhgdib was found as a downstream molecule through which GM3 exerts its function on cell behaviors. We further verified that GM3 regulated Arhgdib expression via PI3K/Akt/mTOR pathway. PDK1 in particular was shown to play a critical role in GM3 mediating Arhgdib expression. Moreover, knocking down Arhgdib obviously changed cell shape, modulated cell motility and proliferation just as GM3 expression was suppressed in B16 cells. These results suggested concretely that GM3 modulated cell behaviors via Arhgdib. Finally, TNFαwas screened as a target of Arhgdib.更多还原