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。     

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     

VEGF-C在宫颈癌细胞的表达及其与细胞黏附和侵袭的关系

目的 VEGF-C在宫颈癌细胞中的表达及其与细胞黏附和侵袭等转移相关生物学行为的关系。方法 采用脂质体转染技术将VEGF-C反义寡核苷酸转染人宫颈癌HeLa细胞,通过黏附实验、Transwell小室,检测转染后细胞黏附和侵袭能力的变化。结果 脂质体介导VEGF-C反义寡核苷酸,转染了VEGF-C反义寡核苷酸的HeLa细胞,能抑制其对细胞外基质的黏附能力;转染后48 h对细胞外基质FN、Matrigel的黏附率较正义组和未转染组明显降低,统计分析差异具有显著性(P＜0.05);Transwell小室实验,计数转染VEGF-C反义寡核苷酸的细胞穿过滤膜的数量,在24 h和48 h较正义组和对照组都减少(P＜0.05)。结论 VEGF-C可影响人宫颈癌HeLa细胞的黏附、侵袭能力等生物学行为。这可能是VEGF-C促进肿瘤细胞淋巴转移的机制之一。     

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。     

CTNNAL1反义寡核苷酸对人支气管上皮细胞增殖和损伤修复的影响

目的 该实验旨在观察CTNNAL1反义寡核苷酸对人支气管上皮细胞增殖和损伤修复的影响.方法 引进人永生化支气管上皮细胞株进行细胞培养,设计合成CTNNAL1反义寡核苷酸和CTNNAL1错义寡核苷酸,分4组转染入人BEC:空白对照组;脂质体-CTNNAL1-ASODN转染组(Lip-CTNNAL1-A-SODN);脂质体-错义CTNNAL1-MSODN转染组(Lip-CTNNAL1-MSODN);脂质体转染组(Lip).荧光定量RT-PCR检测CTNNAL1 mRNA的表达,MTT法检测人BEC的增殖,测定损伤修复指数衡量各组细胞损伤修复速度.结果 CTNNAL1 ASODN能明显抑制人BEC的CTNNAL1 mRNA的表达,CTNNAL1ASODN能抑制人BEC的增殖,延长损伤后修复的速度,且呈剂量依赖性,在1.0μmol/L的CTNNAL1 A-SODN时,抑制作用最明显(P＜0.01).结论 CTNNAL1 ASODN通过沉默人BEC的CTNNAL1 mRNA的表达,抑制人BEC的增殖和损伤修复的速度.     

端粒酶反义寡核苷酸抑制端粒酶活性和诱导肺腺癌A549细胞凋亡的研究

目的 研究端粒酶反义寡核苷酸在肺腺癌A549细胞中抑制端粒酶活性和诱导细胞凋亡的能力.方法 用脂质体将荧光标记的端粒酶反义寡核苷酸转染入肺腺癌A549细胞后,TRAP-PCR-ELISA检测端粒酶活性变化,凋亡细胞用透射电镜(TEM)观察,TUNEL法检测各组细胞凋亡率.结果 转染72h后,端粒酶反义寡核苷酸进入A549细胞内,端粒酶活性显著降低.透射电镜下,端粒酶反义寡核苷酸转染组细胞凋亡增加,细胞凋亡率比其他组明显增高,差异有统计学意义(P＜0.01).结论 端粒酶反义寡核苷酸能靶向端粒酶,抑制其活性,并诱导肺腺癌A549细胞凋亡.     

Par-4反义寡核苷酸拮抗谷氨酸对PC12细胞中ERK1/2的下调作用及其抗凋亡作用

目的研究par-4反义寡核苷酸拮抗谷氨酸对PC12细胞中ERK1/2活性的下调作用及其抗凋亡作用.方法利用脂质体将par-4反义寡核苷酸转染入PC12细胞.谷氨酸诱导PC12细胞凋亡.利用吖啶橙/溴化乙锭荧光染色观察PC12细胞形态,流式细胞分析评价凋亡百分率.Western印迹测定par-4的蛋白质表达量和磷酸化的ERK1/2的蛋白表达量. 结果 (1)谷氨酸诱导PC12细胞中par-4蛋白表达上调,Par-4反义寡核苷酸呈剂量依赖性地拮抗其上调(组间比较,P<0.01).(2)谷氨酸诱导PC12细胞中磷酸化(Thr202/Tyr204)的ERK1/2蛋白水平下调,par-4反义寡核苷酸拮抗其上调(组间比较,P<0.01).(3)Par-4反义寡核苷酸拮抗谷氨酸诱导的PC12细胞凋亡,如予ERK1/2阻断PD98059预处理,则其拮抗作用被下调(组间比较,P<0.05).结论 Par-4反义寡核苷酸拮抗谷氨酸诱导的PC12细胞凋亡,其机制可能与ERK1/2的活化有关.     

survivin反义寡核苷酸激活caspase-3诱导SGC7901胃癌细胞凋亡的实验研究

目的研究转染生存素（survivin）反义寡核苷酸诱导SGC7901胃癌细胞凋亡及其可能的作用机制。方法实验分为空白对照组（Control组）、脂质体组（Lip组）、正义寡核苷酸组（Sodn组）及反义寡核苷酸组（Asodn组）。人工合成正、反义寡核苷酸,经脂质体将survivin正、反义寡核苷酸转染人胃癌细胞48h后,MTT检测细胞增殖抑制率,Western blot检测caspase-3蛋白表达改变,Hoechst染色观察细胞凋亡形态改变及流式细胞仪检测凋亡率。结果Hoechst染色荧光显微镜下Control组、Lip组及Sodn组细胞核呈正常的蓝色,而Asodn组细胞核染色增强、核质浓缩、核碎裂,呈凋亡改变;Asodn组细胞增殖抑制率、细胞凋亡率、caspase-3蛋白表达均增高,与Control组、Lip组及Sodn组相比差异均有统计学意义（P〈0．05）。结论survivin反义寡核苷酸可诱导胃癌细胞凋亡,其作用机制可能是通过激活caspase-3表达,启动凋亡信号传导。     

survivin反义寡核苷酸对肝癌耐药细胞的作用及与化疗的关系

目的研究survivin反义寡核苷酸对人肝癌耐药细胞株的增殖和凋亡情况,以及对阿霉素化疗敏感性的影响。方法将人肝癌耐药细胞系SMMC-7721/ADM分为脂质体转染组、阿霉素组、正义寡核苷酸转染组、正义寡核苷酸转染 阿霉素组、反义寡核苷酸转染组、反义寡核苷酸转染 阿霉素组共6组。MTT法检测细胞相对存活率,流式细胞仪分析细胞凋亡率变化,RT-PCR和Westernblot印迹法检测细胞survivinmRNA和蛋白表达。结果survivin-ASODN作用后的人肝癌耐药细胞SMMC-7721/ADM的细胞凋亡率明显增高(P<0.05)。survivinmRNA和survivin蛋白表达:脂质体转染对照组、阿霉素组、正义寡核苷酸转染对照组、正义寡核苷酸转染对照 阿霉素组之间survivin蛋白表达无明显差异(P>0.05)。400ng/mlsurvivingASODN组和400ng/mlASODN 阿霉素组survivinmRNA较其他4组显著降低(P<0.05),但其两组之间表达无明显差异(P>0.05)。结论survivin反义寡核苷酸能降低肝癌耐药细胞survivin表达,增强人肝癌耐药细胞对阿霉素的化疗敏感性。     

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。     

Activated focal adhesion kinase involved in adhesion and migration of vascular smooth muscle cells stimulated by fibronectin

Objective To study the effects of focal adhesion kinase (FAK) phosphorylation on smooth mu scle cells (SMCs) adhesion and migration stimulated by fibronectin.Methods Adhesion and migration of cultured SMCs were stimulated by different concentrati ons of fibronectin (FN), FAK and its phosphorylation were detected by immunoprec ipitation and Western blot. FAK antisense oligodeoxynucleotides (ODNs) were tra nsfected into SMCs by cationic lipid to investigate its modulatory effects on ty rosine phosphorylation. SMCs adhesion and migration were also measured by morph ological enumeration and modified Boyden Chambers, respectively.Results FAK were expressed when SMCs adhesion and migration were successfully simulated by different concentrations of FN. FAK phosphorylation were detected only at 20 μg/ml FN or more. FAK antisense ODNs were transfected efficiently by cationi c lipid and FAK phosphorylation was inhibited substantially. The SMCs migration rate in the 5-60μg/ml FN groups was reduced by 17.89%-27.67%. Cell migrat ion stimulated by FN at 10, 20, 40 and 60μg/ml were reduced by 23.26%, 21.6 3%, 19.31% and 17.88%, respectively (P&lt;0.05).Conclusions FAK phosphorylation and FAK-mediated signal transduction play important roles i n SMCs adhesion and migration stimulated by ECM. The process can be inhibited e ffectively by FAK antisense ODNs.     

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.     

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.     

Effect of Cigarette Smoke Extract on the Proliferation of Human Airway Epithelial Cells and Expression and Activation of FAK

The effect of cigarette smoke extract (CSE) on the proliferation of human airway epithelial cells and the possible mechanism was studied. After airway epithelial cells were treated with different concentrations of CSE for 24 h, the cell proliferation was measured by MTT and the distribution of different cell cycles by flow cytometry. The FAK expression level was detected by Western blot and the degree of tyrosine phosphorylation by immunoprecipitation. The results showed that CSE could inhibit the proliferation of human airway epithelial cells, arrest the epithelial cells in G1 phase of cell cycle, dramatically decrease the number of epithelial cells in S and G2 phases； Meanwhile CSE could decrease the expression level of FAK and the degree of its tyrosine phosphorylation. The above effects of CSE were concentration-dependent. The expression of FAK and the degree of its phosphorylation was positively correlated to the increased number of epithelial cells in G1 phase, and negatively to the number of epithelial cells in S and G2 phases. It was concluded that the mechanism by which CSE could inhibit the proliferation of human epithelial cells was contributed to the increased expression and activation of FAK.     

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。     

双氢青蒿素对卵巢上皮癌细胞黏附、迁移和侵袭能力的影响

目的 观察双氢青蒿素对体外培养的卵巢上皮癌细胞黏附、迁移和侵袭能力的影响,并探讨相关的分子作用机制.方法 以不用药物处理的SKOV3和OVCAR3卵巢上皮癌细胞为对照组,利用基质胶贴壁黏附法检测双氢青蒿素(研究组)对癌细胞体外黏附能力的影响,用跨膜小室模型检测双氢青蒿素对癌细胞迁移和侵袭能力的影响.用Western印迹和RT-PCR法分别检测双氢青蒿素对癌细胞聚焦黏附激酶(FAK)磷酸化的影响以及对基质金属蛋白酶(MMP)及其组织抑制因子(TIMP)表达的影响.结果 (1)与对照组相比,双氢青蒿素使SKOV3和OVCAR3细胞的体外黏附能力分别降低76.1%和57.9%(P<0.05),迁移能力降低59.3%和69.7%(P<0.05).(2)SKOV3和OVCAR3细胞的侵袭能力弱,双氢青蒿素对其侵袭能力无明确抑制作用.(3)与对照组比较,双氢青蒿素分别使SKOV3和OVCAR3细胞FAK蛋白磷酸化水平降低42.9%和44.8%(P<0.05).(4)SKOV3和OVCAR3细胞中未检测到MMP9的表达,双氢青蒿素能诱导TIMP1的表达,对MMP2和TIMP2的表达无影响.结论 双氢青蒿素对卵巢上皮癌细胞侵袭能力的影响不明显,但能抑制其黏附和迁移能力,后两者可能与药物抑制癌细胞FAK的磷酸化水平有关.     

香烟烟雾提取物对气道上皮细胞增殖和FIP200表达的影响

目的: 研究香烟烟雾提取物(CSE)对气道上皮细胞增殖和细胞周期的影响及其作用机制. 方法: 利用MTT法研究CSE对气道上皮细胞增殖的影响,采用流式细胞术分析气道上皮细胞细胞周期的影响,以RT-PCR和Western blot检测气道上皮细胞FIP200 mRNA和蛋白表达,以免疫共沉淀检测FIP200与局灶粘着斑激酶(FAK)连接状况. 结果: CSE干预的气道上皮细胞MTT吸光度比对照组明显降低,G1期细胞增多,S和G2期细胞明显减少(P<0.01),FIP200 mRNA, FIP200的表达水平以及FIP200与FAK的连接均显著增高(P<0.01),且随CSE干预时间和CSE浓度而增大;气道上皮细胞FIP200表达水平和FIP200与FAK连接的增高均与G1期细胞增多呈正相关(P<0.01),与S和G2期细胞减少呈负相关(P<0.01). 结论: CSE显著抑制气道上皮细胞的增殖,FIP200的高表达可能是其作用机制之一.