粘着斑激酶和丝裂原活化蛋白激酶对纤粘连蛋白诱导平滑肌细胞迁移和增殖的影响

目的　研究粘着斑激酶和丝裂原活化蛋白激酶在细胞外基质成分诱导血管平滑肌细胞迁移和增殖中的作用。方法　通过纤粘连蛋白 (FN)诱导培养的平滑肌细胞迁移和增殖 ,以免疫沉淀和Western印迹法检测粘着斑激酶 (FAK)和丝裂原活化蛋白激酶 (p4 2 / 4 4MAPK)及其磷酸化的表达量。将FAK反义寡核苷酸 (ODN)经脂质体转染细胞 ,观察其对FAK和p4 2 / 4 4MAPK磷酸化、细胞迁移以及增殖的影响。结果　不同浓度FN(5、10、2 0、4 0、6 0 μg/ml)在有效诱导平滑肌细胞迁移和增殖时FAK和p4 2 / 4 4MAPK也呈明显表达 ,2 0 μg/mlFN可使其磷酸化处于较高的表达量。脂质体可有效地介导ODN转染 ,转染效率为 86 7%± 4 5 %。转染后FAK以及p4 2 / 4 4MAPK磷酸化表达量明显减少 ,10、2 0、4 0和 6 0 μg/mlFN组迁移细胞数也分别显著减少 (2 3 2 6 %、2 1 6 3%、19 31%、17 88% ,P <0 0 5 ) ,5～ 6 0 μg/ml不同浓度的FN组 ,细胞增殖减少 2 7 6 7%～ 4 6 6 7% (P <0 0 5 )。 结论　活化的FAK和p4 2 / 4 4MAPK是细胞外基质诱导平滑肌细胞迁移和增殖的重要信号分子 ,二者之间存在着密切的联系 ,由其介导的信号转导促进了这一过程 ,反义FAKODN可有效地对此进行抑制     

粘着斑激酶活性对气道上皮细胞粘附迁移的影响

目的 研究粘着斑激酶(FAK)磷酸化对细胞外基质成份诱导的气道上皮细胞粘附、迁移的影响，探讨FAK活化对气道上皮细胞损伤后修复的影响机制。方法 通过纤维连接蛋白(FN)诱导培养的气道上皮细胞，以计数法测定细胞粘附率，以损伤实验测定细胞迁移速度，以Western blot和免疫共沉淀检测FAK表达水平及其酪氨酸磷酸化程度；以反义FAK寡核苷酸(ODNs)经脂质体转染细胞，观察其对FAK表达和磷酸化、细胞粘附和迁移的影响。结果 FN诱导的气道上皮细胞粘附率和迁移速度明显增高，同时FAK表达水平及其酪氨酸磷酸化程度显著增高；经脂质体转染了反义FAK寡核苷酸的气道上皮细胞粘附率和迁移速度明显降低，同时FAK表达水平及其酪氨酸磷酸化程度降低，且与气道上皮细胞粘附率和迁移速度的降低成显著正相关。结论 FAK是细胞外基质诱导气道上皮细胞粘附、迁移的重要信号分子，其活化促进气道上皮细胞的粘附和迁移，在气道上皮细胞损伤后修复过程中起重要作用。     

FIP200对呼吸道上皮细胞细胞粘附迁移的影响

目的研究FIP200（FAK—family interacting protein of 200 kDa）对细胞外基质成份诱导的呼吸道上皮细胞粘附和迁移的影响及其作用机制。方法通过纤连接蛋白（finbronectin，FN）诱导培养呼吸道上皮细胞的粘附迁移，以FIP200反义寡核苷酸（ODN）经脂质体转染细胞；以计数法测定细胞粘附率，以损伤实验测定细胞迁移速度，以Western blot检测FIP200和FAK蛋白表达水平：以免疫共沉淀检测FIP200和FAK结合情况和FAK磷酸化程度。结果与40mg／LFN组相比，经脂质体转染了FIP200反义寡核苷酸的气道上皮细胞粘附率和迁移速度明显增高，FIP200表达水平显著降低，FAK表达水平无明显变化，与FIP200结合的FAK显著降低，FAK酪氨酸磷酸化程度明显增高。结论内源性FIP200和FAK的结合抑制FAK的活化，从而抑制呼吸道上皮细胞的粘附和迁移，内源性FIP200作为FAK的抑制剂而存在；FN能够促使FAK和FIP200结合的解离而活化FAK，从而促进细胞粘附和迁移。     

Focal adhesion kinase antisense oligodeoxynucleotides inhibit human pulmonary artery smooth muscle cells proliferation and promote human pulmonary artery smooth muscle cells apoptosis

Background Pulmonary artery smooth muscle cell (PASMC) proliferation plays an important role in pulmonary vessel structural remodelling. At present, the mechanisms related to proliferation of PASMCs are not clear. Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein tyrosine kinase. Recent research indicates that FAK is implicated in signalling pathways which regulate cytoskeletal organization, adhesion, migration, survival and proliferation of cells. Furthermore, there are no reports about the role of FAK in human pulmonary artery smooth muscle cells (HPASMCs). We investigated whether FAK takes part in the intracellular signalling pathway involved in HPASMCs proliferation and apoptosis, by using antisense oligodeoxynucleotides (ODNs) to selectively suppress the expression of FAK protein. Methods Cultured HPASMCs stimulated by fibronectin (4μg/ml) were passively transfected with ODNs, sense FAK, mismatch sense and antisense-FAK respectively. Expression of FAK, Jun NH2-temlinal kinase (JNK), cyclin-dependent kinase 2 (CDK 2) and caspase-3 proteins were detected by immunoprecipitation and Western blots. Cell cycle and cell apoptosis were analysed by flow cytometry. In addition, cytoplasmic FAK expression was detected by immunocytochemical staining. Results When compared with mismatch sense group, the protein expressions of FAK, JNK and CDK 2 in HPASMCs decreased in antisense-FAK ODNs group and increased in sense-FAK ODNs group significantly. Caspase-3 expression upregulated in HPASMCs when treated with antisense ODNs and downregulated when treated with sense ODNs. When compared with mismatch sense ODNs group, the proportion of cells at G1 phase decreased significantly in sense ODNs group, while the proportion of cells at S phase increased significantly. In contrast, compared with mismatch sense ODNs group, the proportion of cells at G1 phase was increased significantly in antisense-FAK ODNs group. The level of cell apoptosis in antisense-FAK group was higher than in the mismatch sense group and the latter was higher than sense-FAK group. In addition, the sense-FAK ODNs group was strongly stained by immunocytochemistry, whereas the antisense-FAK ODNs group was weakly stained. Conclusions The results suggest that FAK relates to the proliferation of HPASMCs Antisense-FAK ODNs inhibit HPASMCs proliferation and facilitate their apoptosis. It is possible that FAK via JNK, CDK 2 signalling pathways enhances HPASMCs proliferation and via caspase-3 inhibits HPASMCs apoptosis.     

低氧状态下黏着斑激酶对人肺动脉平滑肌细胞增殖的影响机制

目的 探讨低氧状态下黏着斑激酶(FAK)对人肺动脉平滑肌细胞(HPASMC)增殖的影响机制.方法 采用FAK寡核苷酸转染法对HPASMC进行低氧处理,免疫沉淀法测定胞质蛋白FAK、生长因子受体结合蛋白2(Grb2)及吻蛋白的活性,Western印迹法测定胞质蛋白FAK、Grb2及吻蛋白的蛋白表达,免疫组织化学法测定胞质FAK、Grb2及吻蛋白表达.结果 低氧处理后胞质FAK、Grb2及吻蛋白的活性增加.胞质FAK、Grb2及吻蛋白的蛋白表达值在低氧处理1.5 h时分别为43.4±1.4、69.7±1.9、59.3±1.6,在低氧处理24 h时分别为41.3±1.3、71.3±1.5、59.4±1.8,较常氧处理1.5 h(35.7±1.2、48.7±1.3、33.2±1.8)和24 h时(41.3±1.3、50.2±1.7、38.9±1.9)明显增加,差异均有统计学意义(均P＜0.05).免疫组织化学结果也显示低氧情况下胞质FAK、Grb2及吻蛋白的表达增强.结论 低氧状态下HPASMC增殖与胞质FAK、Grb2及吻蛋白有关.这些胞质蛋白在低氧情况下对细胞生长和分化具有调控作用.

Abstract：

Objective To explore the mechanisms of focal adhesion kinase (FAK) in the proliferation of human pulmonary artery smooth muscle cells (HPASMCs) under hypoxia.Methods Cultured HPASMCs were passively transfected with FAK oligonucleotides (ODNS) and under normoxia or hypoxia condition.They were divided into four groups:normoxia without fibronectin ( FN), normoxia with FN, hypoxia without FN, hypoxia with FN in vitro respectively.Cytoplasmic FAK, Grb2 and paxillin were observed simultaneously by immunoprecipitation and Western blot.In addition, the expressions of cytoplasmic FAK, Grb2 and paxillin were detected by immunocytochemical staining.Results Immunoprecipitation and Western blot demonstrated that cytoplasmic expressions of FAK, Grb2 and paxillin in HPASMCs increased in hypoxia with FN from 43.4 ± 1.4, 69.7 ± 1.9, 59.3 ± 1.6 to 35.7 ± 1.2, 48.7±1.3, 33.2±1.8 at 1.5 h (all P＜0.05), from41.3±1.3, 71.3 ±1.5, 59.4 ±1.8 to41.3±1.3, 50.2 ± 1.7, 38.9 ± 1.9 at 24 h respectively ( P ＜ 0.01, P ＜ 0.05, P ＜ 0.05).Immunocytochemistry staining showed that the cytoplasmic expressions of FAK, Grb2 and paxillin were enhanced in hypoxia with FN versus normoxia with FN.There were significant differences.Conclusion Hypoxia can induce the activation of cytoplasmic FAK, Grb2 and paxillin so as to regulate the migration, survival and proliferation of HPASMCs.     

Role of connective growth factor in plasminogen activator inhibitor-1 and fibronectin expression induced by transforming growth factor β1 in renal tubular cells

Background Connective tissue growth factor (CTGF) contributes greatly to renal tubulointerstitial fibrosis, which is the final event leading to end-stage renal failure. This study was designed to investigate the effects of CTGF antisense oligodeoxynucleotides (ODNs) on the expressions of plasminogen activator inhibitor-1 (PAI-1) and fibronectin in renal tubular cells induced by transforming growth factor β1 (TGF-β) in addition to the role of CTGF in the accumulation and degradation of renal extracellular matrix (ECM). Methods A human proximal tubular epithelial cell line (HKC) was cultured in vitro. Cationic lipidmediated CTGF antisense ODNs were transfected into HKC cells. After HKC cells were stimulated with TGF-β1 (5 μg/L), the mRNA levels of PAI-1 and fibronectin were measured by RT-PCR. Intracellular PAI-1 protein synthesis was assessed by flow cytometry. The secreted PAI-1 and fibronectin in the medium were determined by Western blot and ELISA, respectively. Results TGF-β was found to induce tubular CTGF, PAI-1, and fibronectin mRNA expression. PAI-1 and fibronectin mRNA expression induced by TGF-β was significantly inhibited by CTGF antisenes. ODNs CTGF antisense ODNs also inhibited intracellular PAI-1 protein synthesis and lowered the levels of PAI-1 and fibronectin protein secreted into the medium. Conclusions CTGF may play a crucial role in the accumulation and degradation of excessive ECM during tubulointerstitial fibrosis, and transfecting CTGF antisense ODNs may be an effective way to prevent renal fibrosis.     

体外扩增对脐血CD34+细胞粘附特性和趋化功能的影响

目的 探讨在体外扩增过程中,脐血(UCB)干／祖细胞(HSPC)的粘附特性和趋化功能的变化。方法 从新鲜UCB标本中纯化的CD34^ 细胞接种于无血清无基质悬浮扩增体系,分别于培养7、10和14d从扩增细胞中再次纯化CD34^ 细胞,比较扩增前后CD34^ 细胞的几种与归巢密切相关的功能特性,包括归巢相关粘附分子(CAM)的表达,粘附功能和趋化功能。结果 (1)表达各种粘附分子的CD34^ 细胞亚群的数量在扩增过程中明显增加(15～72倍),而且扩增后CD34^ 细胞表面粘附分子CD44、CDlla、CD49e和CD49d的表达与原代CD34^ 细胞持平或升高,CD62L、CD54和CD31的表达则有不同程度的下调;(2)在前10d的扩增中,CD34^ 细胞与FN间的自发粘附率和SDF-1诱导粘附率均呈上升趋势,0、7和10d分别为28％和63％、60%,和70％、63％,和90%,;(3)扩增1周后,CD34^ 细胞的体外迁移能力无明显变化。结论 所建立的短期培养体系可以支持扩增1周的UCB HSPC保持原有粘附和趋化功能,而继续扩增则可能在某种程度上不利于细胞这些能力的保持。     

层粘蛋白、纤维连接蛋白对兔晶体上皮细胞粘附和移行的影响

目的　探讨层粘蛋白 (laninin ,LN)、纤维连接蛋白 (fibronectin ,FN)对兔晶体上皮细胞 (RLECs)粘附性和移行性的影响。方法　观察外源性FN、LN对兔晶体上皮细胞粘附、移行及其细胞骨架的影响。结果　随着FN、LN浓度升高 ,RLECs在相应的细胞外基质上粘附和移行能力增强 ,低浓度时增加幅度较大 ;加入相应抗FN、LN抗体 ,RLECs在相应基质上的移行产生不同程度的抑制作用 ,细胞骨架也不能充分铺展 ,并且这一变化与细胞粘附性和移行性呈正相关。结论　FN、LN可诱导RLECs移行、粘附 ,因而可能在后囊膜混浊的形成中起重要的作用     

Syntenin上调FAK磷酸化水平促进人脑胶质瘤细胞迁移的分子机制

目的探讨Syntenin促进人脑胶质瘤细胞迁移的分子机制。方法采用划痕实验和Western blot检测CHG-5、稳定表达人源性Syntenin的CHG-hS细胞在纤维连接蛋白(fibronectin,FN)和多聚赖氨酸(Poly-l-lysine,PL)两种基质表面迁移能力、FAK磷酸化位点及相关信号分子的变化情况;分别在P38MAPK特异性抑制剂SB239063和PI3K特异性抑制剂LY294002处理后,FN基质上CHG-5、CHG-hS细胞迁移能力和下游信号分子JNK和AKT磷酸化水平的改变。结果细胞划痕实验结果显示,CHG-hS细胞在FN表面的运动能力显著高于CHG-5细胞(P<0.05);而在多聚赖氨酸包被的培养板上,CHG-hS细胞的运动能力与CHG-5组细胞表面无显著性差异(P>0.05)。Western blot检测结果显示,FN作用下CHG-hS细胞中FAK Tyr397、FAK Tyr576、FAK Tyr925位点的磷酸化水平随时间延长而逐渐升高(P<0.05),而FAK FAK Tyr861位点的磷酸化水平没有变化(P>0.05);相关信号Src、JNK、AKT的磷酸化水平也随时间延长而升高(P<0.05)。分别采用SB239063和LY294002处理后,伴随着p-JNK和p-AKT的磷酸化水平减弱,CHG-hS迁移能力下降。结论 Syntenin通过与p-Src结合,随后触发FAK Tyr397、FAK Tyr925、FAK Tyr576位点的磷酸化作用,最大程度的激活FAK并上调JNK、AKT等下游信号分子的磷酸化水平,最终通过Syntenin-Src/FAK/MAPK和Syntenin-Src/FAK/PI3K两条通路增强FN相关胶质瘤细胞的迁移能力。     

BIO-1211对大鼠支气管收缩和白细胞黏附反应的抑制作用

目的：研究迟缓抗原-4（very late antigen-4，VLA-4）拮抗剂BIO-1211对大鼠支气管收缩和白细胞黏附反应的影响。方法：卵白蛋白致敏大鼠，抗原攻击诱发大鼠支气管收缩反应，采用整体肺功能测定法测定肺阻力（Rt，）和动态肺顺应性（Cdyn）。分离大鼠中性粒细胞，用纤维黏连蛋白（Fn）或血清诱导中性粒细胞产生黏附反应。用组胺和乙酰胆碱诱导豚鼠哮喘反应。结果：给大鼠气雾吸入BIO-1211（1、3、10mg／ml）呈剂量依赖抑制致敏大鼠抗原攻击引起RL的升高和Cdyn降低；BIO-1211（25、50、100、200μg／ml）明显抑制纤维黏连蛋白（Fn）和血清诱导的中性粒细胞黏附反应，其抑制率分别为23．5％、24．6%、61．4％、58．1％和29．99／6、35．9％、35．3％、15．49／6；但BIO-1211（10mg／ml）给豚鼠气雾吸入不能抑制乙酰胆碱和组胺诱导的药物性哮喘。结论：BIO-1211预防致敏大鼠抗原攻击引起的支气管收缩反应，可能与其抑制炎症细胞进入肺组织有关，而对炎症介质无拮抗作用。     

脂质体介导的CD44反义寡核苷酸对人眼小梁细胞粘附功能的影响

目的：观察脂质体介导的CD44反义寡核苷酸对人眼小梁细胞粘附功能的影响，探讨CD44分子在原发性开角型青光眼(POAG)发病过程中可能的作用。方法：采用硫代修饰的CD44反义寡核苷酸，通过脂质体导人体外培养的人眼小梁细胞，RT—PCR、Western印迹及MTT法观察CD44反义寡核苷酸对人眼小梁细胞CD44基因表达及粘附功能的影响。结果：RT—PCR及Western印迹结果提示：CD44反义寡核苷酸抑制人眼小梁细胞CD44表达。MTT法检测显示：CD44反义寡核苷酸对人眼小梁细胞与细胞外基质透明质酸的粘附起抑制作用且呈浓度依赖性。结论：CD44反义寡核苷酸抑制人眼小梁细胞与细胞外基质透明质酸的粘附，粘附分子CD44可能参与了POAG发病过程。     

MicroRNA-7 regulates glioblastoma cell invasion via targeting focal adhesion kinase expression

Background  Invasion growth is the most characteristic biological phenotype of glioblastoma, but the molecular mechanism in glioma cell invasion is poorly understood. Recent data have showed that microRNA plays an essential role in tumor invasion. Our study aimed to explore the mechanism of miR-7 involved in the control of glioblastoma cell invasion.

Methods  Glioma cell invasion was evaluated by transwell and scratch assays after up-regulation of miR-7 using miR-7 mimics in U87 and U251 cells. Luciferase reporter assay was used to determine focal adhesion kinase (FAK) as a target of miR-7. The levels of miR-7, matrix metalloproteinases (MMP)-2 and MMP-9 mRNA were detected by PCR assay, and the levels of FAK, MMP-2, MMP-9, total and phosphorylation serine/threonine kinase (AKT), and extracellular signal-regulated kinase (ERK) 1/2 were measured by Western blotting analysis.

Results  Over-expression of miR-7 inhibited the invasion and migration activity of U87 and U251 cells. And up-regulation of miR-7 reduced FAK protein expression, Further, luciferase reporter assay showed that miR-7 modulated FAK expression directly by binding 3′UTR of FAK mRNA. In addition, miR-7 repressed p-ERK1/2 and p-AKT level, MMP-2 and MMP-9 expression. Finally, the inverse relationship between FAK and miR-7 expression was certificated in human glioma tissues.

Conclusion  To our knowledge, these data indicate for the first time that miR-7 directly regulates cell invasion by targeting FAK in glioblastoma and that miR-7 could be a potential therapeutic target for glioblastoma intervention.

     

Bcl-2反义寡核苷酸对肾癌细胞株的生长抑制作用

目的：研究bcl-2反义寡核苷酸对肾细胞癌细胞的生长抑制作用及剂量一效应关系。方法：设计、合成与已知人类基因无同源性的bcl-2反义寡核苷酸(AS1翻译起始端，AS2编码区)，以阳离子脂质体Lipofectin转染不同的肾癌细胞株，MTS比色实验测定细胞活率。结果：单纯Lipofectin、反义寡核苷酸及正义寡核苷酸处理，对肾癌细胞ACHN生长无抑制作用，而转染入细胞内的反义寡核苷酸可抑制多种肾癌细胞的生长(ACHN、RCW和OSRC-2)，且抑制作用与剂量呈正相关。结论：bcl-2反义寡核苷酸可剂量依赖性抑制多种肾癌细胞的生长。     

PLGA经多巴胺改性后负载纤连蛋白对MC3T3-E1细胞黏附的影响

目的:建立以聚乳酸-羟基乙酸共聚物[poly(lactic-co-glycolic acid),PLGA]为基底,经多巴胺(dopamine,DP)负载纤连蛋白(fibronectin,FN)的材料模型,研究FN对MC3T3-E1细胞黏附的影响。方法:使用红外光谱分析仪,接触角仪和石英晶体损耗检验微量天平测量样品表面基团、膜的亲水性和FN负载量;在磷酸盐缓冲液(PBS)环境中,分别接种MC3T3-E1细胞于各样品,使用延时照相系统每隔3 min观察细胞黏附情况,CCK-8法测量接种细胞后0.5、1.0、4.0、8.0 h黏附细胞的吸光度。结果:红外光谱分析显示DP成功结合于PLGA膜表面,接触角测量表明PLGA膜经DP处理后亲水性改善,石英晶体损耗检验微量天平测得FN的负载量为22.5 ng/cm2,延时照相系统和CCK-8检测结果显示负载FN的PLGA膜上细胞黏附比例和数量显著高于PLGA膜和PLGA的DP涂层膜(P<0.05)。结论:在PBS环境中,MC3T3-E1细胞可在负载FN的PLGA膜上正常黏附。     

猪血浆FN的分离及其性质鉴定

本文用明胶和肝素亲和层析柱等方法,从猪血浆中分离得到大量(0.5mg/ml)的纤维粘连蛋白(FN)。此FN在琼脂糖电泳和聚丙烯酰胺凝胶电泳中呈单一条带,分子量约450000;巯基乙醇还原后呈现分子量约230000的两条带;可以与抗人和抗牛血浆FN抗体发生免疫沉淀反应;并具有强烈促CHO细胞贴壁活性。提示猪血浆是一种分离提取FN的良好资源。     

粘着斑激酶在乳腺癌的表达及其与血管生成的关系

目的 研究乳腺癌组织中粘着斑激酶(FAK)的表达及其与微血管密度(MVD)的关系,探讨FAK介导的细胞信号转导系统对乳腺癌血管生成的作用和意义.方法 应用SP法对88例乳腺癌组织和30例乳腺良性病变组织进行FAK和CD34免疫组化染色,分析FAK蛋白表达和CD34标记的MVD值之间,及其与乳腺癌临床病理参数之间的关系.结果 在88例乳腺癌中FAK的阳性表达率为68.2%(60/88),MVD为(34.52±13.11)/HPE,与良性对照组比较均有显著差异(P＜01);FAK表达和MVD与乳腺癌肿瘤大小、腋窝淋巴结转移、临床分期呈正相关(P＜0.05),与患者年龄、组织病理学分级未见显著相关(P＞0.05);乳腺癌FAK表达与MVD呈正相关(P＜0.01).结论 FAK蛋白表达和微血管形成与乳腺癌的侵袭转移密切相关,FAK表达对乳腺癌血管生成具有促进作用.     

FAK和MMP-9蛋白在胃癌组织中的表达及相关性研究

目的 检测基质金属蛋白酶9(MMP-9)和黏着斑激酶(FAK)在胃癌中的表达,并探讨其相关性.方法 采用免疫组织化学方法,观察60例胃癌及肿瘤与正常组织交界处FAK和MMP-9的表达情况.结果 60例胃癌组织中FAK阳性及强阳性表达率为73.3%(44/60)、56.7%(34/60),MMP-9的阳性表达率为85%(51/60);60例相应交界组织中,FAK阳性及强阳性表达率为56.7%(34/60)、26.7%(16/60),MMP-9阳性表达率为56.7%(34/60).MMP-9和FAK阳性表达率和阳性强度均以胃癌中为高.在癌组织中,MMP-9和FAK在低分化组中的表达高于高、中分化组,在淋巴结转移组中较无淋巴转移组表达高,浸润程度越深表达越高.MMP-9阳性表达强度与FAK的阳性表达强度之间呈显著正相关(P＜0.05).结论 胃癌中FAK与MMP-9的表达密切相关,可作为胃癌生物学行为预测的辅助指标.     

FAK通过和EGFR的相互作用调节MAPK和Akt之间的相对平衡

目的研究上皮生长因子受体和FAK的相互作用以及对下游信号的影响。方法建立聚集粘连激酶(FAK)缺失突变和绿色荧光蛋白(GFP)融合基因del1-693FAK-GFP、del1-100FAK-GFP和FAK-GFP稳定表达细胞系。结果同野生型FAK-GFP相比,N-端1-100氨基酸残基的缺失突变体,缺失1-693氨基酸残基的突变体结合在黏附点的能力被完全抑制。应用等电聚焦和SDS-PAGE双向电泳证明,EGF和纤维连接蛋白诱导FAK磷酸化的位点不同,进一步证实del1-693FAK-GFP、del1-100FAK-GFP,抑制MAPK的磷酸化,增强Akt的磷酸化;而FAK-GFP增强MAPK磷酸化,抑制Akt磷酸化。结论FAK通过和EGFR的相互作用调节MAPK和Akt之间的相对平衡。     

血管内皮下层纤维连接素在血小板粘附中的作用

本文利用扫描电镜和荧光分光光度计技术,研究了血管内皮下层Fn在血小板与血管壁粘附中的作用.当内皮细胞剥脱后血管壁上可见大量血小板粘附:当裸露的内皮下层失与抗Fn抗体反应后,血小板的相对粘附率降低了70.1％(P<0.01),而正常血清对照组无明显变化,说明Fn在血小板粘附中起重要作用.     

Twist shRNA对人宫颈癌细胞体外黏附、铺展及迁移能力的影响

目的通过RNA干扰抑制Twist基因在人宫颈癌HeLa细胞中的表达,观察Twist基因沉默对HeLa细胞体外黏附、铺展及迁移能力的影响。方法根据shRNA设计原则,构建两种靶向Twist基因的短发夹RNA（shRNA）干扰质粒,稳定转染HeLa细胞。通过荧光定量PCR及Western印迹法检测HeLa细胞中Twist基因mRNA和蛋白的表达水平,利用黏附实验、铺展实验和划痕实验检测其对细胞黏附力和迁移力的影响。结果成功构建的Twist shRNA真核表达载体稳定转染HeLa细胞后可显著降低Twist基因的表达。与对照组相比,Twist基因沉默组细胞的黏附、铺展及迁移能力明显下降（P〈0.05）。结论成功构建的Twist shRNA真核表达载体,能有效抑制HeLa细胞黏附、铺展及迁移能力。