【作者】 李永金；

【导师】 陈永昌；

【作者基本信息】 江苏大学 ， 临床检验诊断学， 2010， 博士

【摘要】 整合素(integrins)是一类重要细胞粘附分子受体,它介导细胞与细胞之间、细胞与胞外基质(extracellular matrix, ECM)的粘连,也参与细胞内外信息传递。整合素由一个α亚基和一个p亚基构成。目前已经发现18种α亚基和9种p亚基,这些亚基通过不同组合方式构成至少25种整合素。在它们当中,整合素α5β,是纤维连接蛋白的受体,当细胞接触到胞外基质纤维连接蛋白,整合素α5p1被激活。然而,我们身体内大部分纤维连接蛋白是可溶性的,可溶性的纤维连接蛋白主要存在血浆中,随着血液到达我们身体的每一个部分。还没有证据表明可溶性的纤维连接蛋白是否也活化整合素介导的信号通路。RhoA是小G蛋白超家族中Rho家族主要成员,它介导的信号与细胞骨架的重排,迁移,增殖和基因表达等相关。研究发现,多种肿瘤细胞过度表达RhoA,当RhoA的活性被抑制时,癌细胞浸润性生长和转移也被抑制。许多的研究资料证明整合素和RhoA之间有着关联,但确切的机制尚不清楚。PKA是与cAMP信号通路相关的蛋白激酶。PKA是由4个亚基组成的一种别构酶,由两个催化亚基C和两个调节亚基R所构成。当cAMP结合R亚基后,全酶快速解离,C亚基变得有催化活性,可以磷酸化特异性底物。有资料显示PKA能够抑制RhoA的活性和功能。整合素、RhoA和PKA之间可能的相互作用还不清楚。研究目的：探讨可溶性纤维连接蛋白／整合素、cAMP/PKA和RhoA介导的信号通路之间的关系。主要研究可溶性纤维连接蛋白对细胞骨架、RhoA活性及cAMP含量和PKA活性的作用。研究方法：(1)以异硫氰四甲基罗丹明(TRITC)标记的鬼笔环肽,对细胞骨架微丝F-actin进行染色,观察细胞骨架的变化。(2)将培养好的PC-3细胞,药物处理后,通过倒置显微镜动态观察细胞形态的变化。(3)通过pull down assay方法,观察RhoA活性的变化。(4)通过放射免疫的方法,检测cAMP的含量。(5)通过免疫荧光方法,观察PKACα的分布。(6)采用Western Blot方法,观察PKACα及的p-VASP蛋白的含量。(7)将报告基因质粒CRE-luciferase及参照基因质粒P-Galactosidase共同转染入SGC-7901细胞,药物处理后,用荧光虫素酶报告基因方法分析报告基因表达的活性。(8)将野生型Ht31质粒与GFP质粒共同转染SGC-7901细胞,用药物G418进行筛选,并用有限稀释法得到稳定表达Ht31的细胞。(9)将稳定表达AKAP抑制性肽段Ht31的SGC-7901细胞接种到二十四孔板中。药物处理后,细胞用鬼笔环肽进行染色,并在荧光显微镜下观察细胞骨架微丝F-actin的形成变化。研究结果：(1)可溶性纤维连接蛋白可以使PC-3细胞细胞发生明显的形态改变,使用与RhoA相关的激酶Rock的抑制剂Y27632及α5β1抗体后,这种作用被取消。(2)可溶性纤维连接蛋白提高RhoA活性,具有时间和剂量依从性；事先使用α5β1抗体后,可溶性纤维连接蛋白提高RhoA活性作用被抑制；PKA对RhoA活性有抑制作用,纤维连接蛋白可减弱这种作用。(3)可溶性纤维连接蛋白可抑制由Forskolin或百日咳毒素(PTX)导致的cAMP含量的升高。(4)可溶性纤维连接蛋白可抑制由Forskolin或百日咳毒素(PTX)导致的PKACa含量的升高。(5)可溶性纤维连接蛋白可抑制由Forskolin导致的p-VASP含量的增加和CRE依赖的基因转录水平的升高。对cAMP导致的p-VASP含量的增加和CRE依赖的基因转录水平的升高影响不大。(6)筛选得到稳定表达Ht31的细胞克隆用聚丙烯酰胺凝胶电泳鉴定,证实在37KD和27KD左右分别有两条特异蛋白条带。(7)可溶性纤维连接蛋白促进F-actin的形成,且具有剂量依从性。PKA对纤维连接蛋白引起的F-actin形成有抑制作用。PKA在锚定被破坏时,这种抑制作用被削弱。结论(1)可溶性的纤维连接蛋白也通过α5β1活化整合素介导的信号通路。(2)可溶性纤维连接蛋白能促进细胞骨架微丝F-actin的形成,使细胞发生明显的形态改变,这些效应与RhoA、PKA相关。(3)可溶性纤维连接蛋白能够提高RhoA活性,并可拮抗PKA对RhoA活性的抑制作用。(4)可溶性纤维连接蛋白能够对cAMP/PKA介导的信号通路有抑制作用,可能的作用靶点是腺苷酸环化酶。我们的研究结果不仅有助于我们了解纤维连接蛋白／整合素、cAMP/PKA信号和RhoA介导的信号转导通路之间的关系,而且还为癌症治疗策略提供了新靶点。更多还原

【Abstract】 Integrin is an important receptor of cellular adhesion molecules and mediates the adhesion between cell and cell or cell and extra-cellular matrix. It is also involved in intracellular and extracellular information transmission. Integrin consists of one a and oneβsubunit. So far,8 (3 subunits and 18 a subunits have been found and the subunits may assemble into 25 instinct integrins. Among them, integrinα5β1 is the receptor of fibronectin. When cells attach to extracellular matrix containing fibronectin, integrinα5β1 is activated. However, a large portion of fibronectin is soluble within the body. Soluble fibronectin exists mainly in the blood plasma and reaches each part of the body through the blood stream. There is no research data addressing whether soluble fibronectin activates integrin-mediated signal transduction.RhoA is a key member of Rho family of small GTP-binding proteins and mediates signaling relating to cytoskeleton arrangement, migration, proliferation and gene expression. Study found that there was a variety of cancer cells over-expressing RhoA and invasive growth and metastasis of cancer cell were inhibited when RhoA activity was inhibited. A lot of research data have addressed the significance of the association between integrin and RhoA. However, the exact mechanism is still unclear. PKA is a protein kinase related to the cAMP signaling pathway. PKA is an isozyme existing as a tetramer of two R-and two C-subunits. Occupancy of the R-subunits by cAMP subsequently results in the rapid dissociation of the holoenzyme, allowing the catalytically active c-subunit to phosphorylate substrates.Research data suggested that PKA could inhibit the activity and the function of RhoA. The possible interaction among integrin, RhoA and PKA is unknown.ObjectivesThis study was designed to investigate the relationship among soluble fibronectin/integrin-, cAMP/PKA-, and RhoA-mediated intracellular signal transduction pathways. This thesis was focused on investigating the effect of soluble fibronectin on cytoskeleton, RhoA activity, cAMP content and PKA activity.Methods(1) To examine cytoskeletal changes, we stained F-actin with rhodamine-conjugated phalloidin.(2) The morphological dynamic changes of the PC-3 cells treated with drug were visualized by a reverse microscope.(3) Rho activity was detected by pull down assay.(4) cAMP concentration was measured by Radioimmunoassay (RIA).(5) The distribution of PKA catalytic subunit was detected by fluorescent microscopy.(6) The amount of the phosphorylation of VASP and PKA catalytic subunit were detected by Western blotting. (7) The reporter gene plasmid pcDNACRE-Luc and plasmid of referrence geneβ-galactosidase were co-transfected into SGC-7901 cells. The reporter gene expression activity of the cells treated with drug was analyzed by luciferase reporter gene assay.(8) The SGC-7901 cells transfected with wild type pcDNA HT31 and pGFP were selected with G418. The cells stably expressing the peptide Ht31 were obtained by Limited-Dilution Method.(9) The SGC-7901/HT31wt cells which stably expressed the peptide Ht31 were grown on 24-well plate. The cells treated with drug were stained with phalloidin and the F-actin was visualized under fluorescent microscope.Results(1) Soluble fibronectin caused obviously morphological changes of PC-3 cells. Y27632, an inhibitor of RhoA related protein kinase Rock, and antibody against integrinα5β1 partially blocked the effect of soluble fibronectin.(2) Soluble fibronectin dose and time-dependently increased RhoA activity. Pre-incubating the cells with the antibody against integrinα5β1 partially inhibited the RhoA activation induced by soluble fibronectin. PKA could inhibit RhoA activity and fibronectin could reduce this effect.(3) The elevation of cAMP level induced by forskolin or PTX could be inhibited by soluble fibronectin.(4) The increase of PKA catalytic subunit indeuced by forskolin or PTX could be inhibited by soluble fibronectin.(5) The increases of VASP phosphorylation and CRE-dependent transcription activity induced by forskolin could be inhibited by soluble fibronectin. However, soluble fibronectin could not inhibite these increase induced by cAMP.(6) The cell clone which stably expressed the peptide Ht31 selected out was identified with SDS-PAGE (SDS-polyacrylamide gel electrophoresis), and two specific protein bands, respectively at 37KD and 27KD, were proved.(7) Soluble fibronectin dose-dependently increased the formation of F-actin. PKA was able to antagonize the soluble fibronectin-induced F-actin formation.This effect was partly impaired upon the disruption of PKA anchorage.Conclusions(1) Soluble fibronectin also binds integrinα5β1 and activates consequent signaling events.(2) Soluble fibronectin increased the formation of F-actin and caused obviously morphological changes. These effects were related to RhoA and PKA.(3) Soluble fibronectin could increase RhoA activity and antagonize the inhibition of PKA on RhoA activity.(4) Soluble fibronectin could inhibit cAMP/PKA mediated signal transduction pathway. The possible acting poin was adenylate cyclase.Our results not only help us to understand the relationship among fibronectin/integrin-, cAMP/PKA-and RhoA-mediated signal transduction pathways, but also provides novel target for strategy of cancer therapy.更多还原