【作者】 王静；

【导师】 马勇杰；

【作者基本信息】 天津医科大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 目的:Girdin是由日本学者在2005年首次发现的一种肌动蛋白结合蛋白。研究发现Girdin在促进细胞的迁移及血管生成等方面起到重要的作用。细胞迁移不仅是细胞进行重要生理活动的基础,同时也是肿瘤发生、发展等病理过程中的重要步骤和关键环节,本研究将Girdin与肿瘤运动和侵袭相关联,通过小RNA干扰技术降低Girdin在恶性胶质瘤LN229和U87细胞中的表达,深入研究其对恶性胶质瘤侵袭和凋亡能力的影响,为临床治疗恶性胶质瘤提供新的靶点。方法:第一部分:1.用小RNA干扰技术降低恶性胶质瘤细胞株LN229和U87中Girdin蛋白的表达水平,通过Western blotting技术验证其降表达效率,并筛选出稳定降表达的克隆。2.用划痕实验、趋化运动实验和趋化动力学实验检测Girdin对恶性胶质瘤细胞的运动能力的影响。3.用肌动蛋白聚合实验检测EGF刺激不同时间后Girdin降表达对细胞骨架蛋白actin聚合能力的影响。4.用粘附实验检测Girdin降表达对恶性胶质瘤细胞粘附能力的影响。5.采用Western blotting技术检测Girdin降表达粘附相关蛋白FAK和integrin β1的磷酸化水平的变化;同时用Western blotting技术检测基质金属蛋白酶2(MMP-2)和MMP-9的表达水平,验证Girdin降表达对恶性胶质瘤细胞基质金属蛋白酶的表达水平的影响。6.通过裸鼠皮下成瘤实验验证Girdin的降表达对胶质瘤细胞体内侵袭能力的影响。第二部分:1.用小RNA干扰技术降低恶性胶质瘤细胞株LN229中Girdin蛋白的表达水平,同时采用瞬转的方法降低U87中Girdin的表达,通过Western blotting技术验证其降表达效率。2.应用细胞增殖实验和MTT法检测Girdin对恶性胶质瘤细胞增殖能力的影响。3.DAPI染色、流式细胞术及AnnexinV/PE凋亡试剂盒检测Girdin表达下降对细胞凋亡能力的影响。4.Western blotting技术检测Girdin表达下降后,凋亡相关蛋白分子细胞色素C(cytochromeC,Cyt-C)、Bad 的表达情况,5.将稳定降表达Girdin的细胞及对照组细胞分别接种于裸鼠皮下观察移植瘤在裸鼠体内的生长情况。6.免疫组织化学染色方法检测Girdin表达下降后Cyt-C、Bad在裸鼠移植瘤组织中的表达情况。结果:第一部分:1.胶质瘤细胞株LN229和U87经转染后,Girdin蛋白表达水平明显下降。2.与对照组相比,Girdin降表达后胶质瘤细胞的迁移能力和趋化运动能力较对照组细胞显著下降(P<0.05)。3.与对照组相比,Girdin降表达导致LN229细胞的F-actin聚合能力明显下降(P<0.05)。4.Girdin降表达后,恶性胶质瘤细胞株LN229和U87的粘附能力比对照组显著降低(P<0.05)。同时Western blotting结果显示磷酸化的integrin β1及FAK表达水平较对照组降低。5.体外细胞侵袭实验结果显示,Girdin降表达后细胞的侵袭能力较对照组细胞显著减弱(P<0.05)。同时与细胞侵袭相关的MMP-2和MMP-9的表达也明显降低。6.体内侵袭实验进一步证实,实验组裸鼠发生组织浸润的数量较对照组显著减少(P<0.05)。第二部分:1.Western blotting检测结果显示转染小RNA干扰技术有效降低LN229细胞中Girdin的表达。合成的小RNA片段转染U87细胞后Girdin的表达水平降低。2.与对照组相比,Girdin降表达后LN229细胞的增殖能力明显下降(P<0.05)。3.DAPI染色、流式细胞术及AnnexinV/PE凋亡试剂盒等检测结果发现Girdin表达下降LN229和U87细胞的凋亡能力均明显下降(P<0.05)。4.Western blotting检测显示线粒体释放的Cyt-C比对照组增加;同时实验组细胞内Bad蛋白表达水平也明显升高(P<0.05)。5.体内实验显示,Girdin表达下降后移植瘤的生长能力较对照组细胞显著减弱(P<0.05)。6.免疫组织化学染色结果显示,Girdin表达下降的移植瘤组织中Cyt-C在胞浆中的表达较对照组明显增加;与对照组相比较,siGirdin组细胞中Bad表达水平升高。结论:1.Girdin参与调控恶性胶质瘤细胞的迁移过程。其过程可能是通过调节恶性胶质瘤细胞骨架重塑来实现的。2.Girdin通过调节integrin β1及FAK的磷酸化水平从而调控恶性胶质瘤细胞的粘附能力。3.Girdin通过调节MMP-2和MMP-9的表达影响恶性胶质瘤细胞向细胞外基质侵袭的过程。4.降低Girdin的表达能够抑制恶性胶质瘤细胞的生长,促进细胞凋亡,这些作用可能是通过调节Bad和Cyt-C的表达而实现的。更多还原

【Abstract】 ObjectivesGirdin was first discovered by Japanese researchers in 2005.The phosphorylated Girdin plays an essential role in both the remodeling of the actin cytoskeleton and tumor progression including migration,invasion,metastasis and angiogenesis.In our study,we used the siRNA technology to knock down the expression of Girdin,and then to investigate the function of Girdin down-regulation in migration,invasion,apoptosis,and the related molecular mechanisms.MethodsPart one:1.Glioma cells LN229 and U87 were respectively transfected with Girdin small RNA interference plasmids and transient transfection to disrupt Girdin expression,which was identified by Western Blotting analysis.2.Using scratch assay,chemotaxis assay and chemokinesis assay to detect the influence of Girdin down-regulation on migration of glioma cells.3.F-actin polymerization assay to explain the role of Girdin down-regulation in actin polymerization stimulated by EGF in different time.4.Adhesion assay detected the influence of down-regulated Girdin on the adhesion of glioma cells.5.Using western blotting assay to detected the expression of phospho-integrin β1,phospho-FAK,MMP-2 and MMP-9 in glioma cells and control glioma cells.6.We used the invasion in vitro assay and subcutaneous mouse xenograft model in vivo to validate the role of Girdin in glioblastoma invasion.Part two:1.We used small interfering RNA technology to knock down Girdin expression level in glioma cells LN229.U87 cells were transfected with small interfering RNA duplex oligoribonucleotide to disrupt Girdin expression.2.Proliferation assay and MTT assay were applied to detect the effect of Girdin-reduction on LN229 cells proliferation.3.DAPI staining,flow cytometry and AnnexinV/PE staining assay were used to analyze the function of Girdin-reduction in cells apoptosis.4.Western blotting was applied to detect the expression of apoptosis related proteins cytochrome C and Bad.5.We used Nu/Nu nude mice to set up xenograft animal model to explore the role of Girdin in glioblastoma tumors growth in vivo.6.Immunohistochemistry staining was used to analyze the expression of Cyt-C and Bad.ResultsPart one:1.After transfected LN229 cells and U87 cells by small interference RNA,the expression of Girdin significantly decreased.2.Girdin-reduced glioma cells showed decreased chemotaxis ability compared with control cells(P<0.05).3.In the Girdin down regulated glioma cells,the actin polymerization in response was significantly reduced.(P<0.05).4.The adherent Girdin-reduced cells was significantly decreased and the expression of phospho-integrin β1 and phospho-FAK were down regulated compared with control cells(P<0.05).5.The invasion assay in vitro demonstrated that the invasion ability of Girdin-reduced cells was significantly reduced,the expression of MMP-2 and MMP-9 were also down regulated compared with control cells(P<0.05).6.The in vivo further demonstrated that the invasion frequency of Girdin down regulated mice group was significant lower than the control group(P<0.05).Part two:1.Compared with the control cells,Girdin-reduction impaired the proliferation in LN229 cells(P<0.05).2.The results of DAPI staining assay,flow cytometry and AnnexinV/PE staining assay showed that the experssion of Girdin-reduction induced the apoptosis ability in LN229 and U87 cells.3.Compared with the control cells,the expression of Cyt-C and Bad were increased in siGirdin/LN229 cells by Western blotting analysis.4.Animal experiments indicated that reduction of Girdin inhibited tumor growth in vivo(P<0.05).5.Immunohistochemistry staining showed that the content of Cyt-C and Bad in cytoplasm were up-regulated in siGirdin/LN229 group,compared with the control cells(P<0.05).Conclusions1.Girdin participated in EGF-induced glioma cells migration by regulating the cytoskeletal rearrangement.2.Girdin accommodated the activation of integrin β1 and FAK to influent the adhesion ability in glioblastoma cells.3.Girdin mediated the glioma cells invasion by regulating the expression of MMP-2 and MMP-9.4.Reduction of Girdin in glioma cells inhibited cell growth and induced cell apoptosis through regulating the activation of Bad and Cyt-C.更多还原