反义局部粘着斑激酶抑制肝癌侵袭生长的研究

目的　观察反义局部粘着斑激酶 (FAK)脱氧寡核苷酸 (ODN)转染对Bel740 2肝癌移植瘤侵袭性生长的影响 ,并探讨其作用机制。方法　以LipofectAMINE介导的反义FAKODN转染Bel 740 2肝癌细胞株后 ,于 6只裸鼠双侧颈背部、髋部共 4处皮下接种 ,观察移植瘤生长情况 ,并行肿瘤微血管密度 (MVD)的免疫组织化学检测及MMP2与TIMP2表达的逆转录 聚合酶链反应(RT PCR)检测。结果　反义FAKODN转染使裸鼠皮下移植瘤的生长受到显著抑制 ,抑瘤率为3 5 .7% ;MVD在反义转染组 ( 9.5 99± 1.2 84)显著低于对照组 [( 13 .915± 2 .618) ,P <0 .0 5 ] ;MMP2表达在反义转染组 ( 0 .199± 0 .0 61)显著低于对照组 ( 0 .42 1± 0 .118) ,TIMP2表达在反义转染组( 0 .461± 0 .15 3 )则显著高于对照组 [( 0 .2 98± 0 .10 4) ,P <0 .0 5 ]。结论　信号转导分子FAK表达阻断可显著抑制Bel 740 2肝癌细胞株在裸鼠体内的侵袭性生长 ,病理性肿瘤血管生成减少及MMP2、TIMP2表达改变与其抗肿瘤作用密切相关。     

粘着斑激酶在SACC-LM及SACC-83细胞中的活性及表达变化

目的:探讨粘着斑激酶(FAK)在涎腺腺样囊性癌细胞中的活性及表达变化。方法:同时采用逆转录-聚合酶链反应(RT-PCR)和Western blot法检测肺高转移性涎腺腺样囊性癌(SACC-LM)及涎腺腺样囊性癌(SACC)83细胞系中FAK mRNA和蛋白表达;同时测定FAK酶活力。所得实验数据采用SPSS12.0统计软件进行,t检验进行组间比较。结果:SACC-LM细胞中的FAK活性为(18.320±2.050)pmol·min~(-1)·μg~(-1),SACC-83细胞中的FAK活性为(0.873±0.091)pmol·min~(-1)·μg~(-1)。与SACC-83细胞相比,SACC-LM细胞中FAK具有较高的活性(t=13.865,P<0.01)。FAKmRNA在SACC-LM细胞中的表达量为0.883±0.052,明显高于其在SACC-83细胞中的表达量(0.637±0.081,t=4.952,P<0.05)。在SACC-LM细胞中FAK蛋白的表达高于在SACC-83细胞中的表达。结论:FAK蛋白活性变化及蛋白表达与SACC的侵袭有关。     

粘着斑激酶与肾脏疾病

粘着斑激酶是一种非受体型酪氨酸激酶 ,是细胞内多条信号传导通路的交汇点 ,与细胞粘附、铺展、迁移、增殖和凋亡有关 ,在介导肾脏疾病的发生过程中起重要作用     

粘着斑激酶与肾脏疾病

粘着斑激酶是一种非受体型酪氨酸激酶，是细胞内多条信号传导通路的交汇点，与细胞粘附、铺展、迁移、增殖和凋亡有关，在介导肾脏疾病的发生过程中起重要作用。     

粘着斑激酶家族与肝癌关系的研究进展

粘着斑激酶（focal adhesion kinase,FAK）是蛋白质酪氨酸激酶（protein tyrosine kinase,PTK）的一个家族,它属于非受体型酪氨酸蛋白激酶,这个家族的成员包括FAK和新近发现的富含脯氨酸的蛋白酪氨酸激酶2／细胞粘附激酶β     

粘着斑激酶在增生性瘢痕成纤维细胞中的表达和意义

目的探讨粘着斑激酶（FAK）在增生性瘢痕成纤维细胞（HSFB）中的表达和意义。方法运用细胞免疫组化技术，测定HSFB和正常皮肤成纤维细胞（NSFB）中的FAK、整合素α1、转化生长因子受体1（TGF-βR1）及α肌动蛋白（α—SMA）的表达；同时将HSFB分别用抗FAK、整合素α1、TGF-βR1及α—SMA抗体进行阻断培养，并测定培养前后整合素α1、TGF—βR1、α—SMA及FAK的表达。结果与NSFB相比，HSFB中的FAK、整合素α1、TGF—βR1、α—SMA的表达增高，差异具有显著意义（P〈0．05）。分别用抗整合素α1、TGF-βR1、α—SMA抗体进行阻断培养后，HSFB中的FAK表达下降（P〈0．01）；阻断FAK表达也可分别导致了整合素α1、TGF-βR1、α—SMA的表达降低（P〈0．01）。结论FAK对整合素α1、TGF-βR1及α—SMA的蛋白合成具有重要的调控作用，与增生性瘢痕的发生、发展关系密切。减少FAK在成纤维细胞中的过度表达，可能是抑制瘢痕增生、软化瘢痕的新途径。     

粘着斑激酶在增生性瘢痕成纤维细胞中的表达和意义

目的探讨粘着斑激酶(FAK)在增生性瘢痕成纤维细胞(HSFB)中的表达和意义。方法运用细胞免疫组化技术,测定HSFB和正常皮肤成纤维细胞(NSFB)中的FAK、整合素α1、转化生长因子受体1(TGF-βR1)及α肌动蛋白(α-SMA)的表达;同时将HSFB分别用抗FAK、整合素α1、TGF-βR1及α-SMA抗体进行阻断培养,并测定培养前后整合素α1、TGF-βR1、α-SMA及FAK的表达。结果与NSFB相比,HSFB中的FAK、整合素α1、TGF-βR1、α-SMA的表达增高,差异具有显著意义(P<0.05)。分别用抗整合素α1、TGF-βR1、α-SMA抗体进行阻断培养后,HSFB中的FAK表达下降(P<0.01);阻断FAK表达也可分别导致了整合素α1、TGF-βR1、α-SMA的表达降低(P<0.01)。结论FAK对整合素α1、TGF-βR1及α-SMA的蛋白合成具有重要的调控作用,与增生性瘢痕的发生、发展关系密切。减少FAK在成纤维细胞中的过度表达,可能是抑制瘢痕增生、软化瘢痕的新途径。     

黏着斑激酶在转化生长因子β1诱导的人肾小管上皮细胞转分化中的作用

目的 观察转化生长因子（TGF）β1诱导的正常人近端肾小管上皮细胞（HK-2）转分化（EMT）过程中黏着斑激酶（FAK）的表达及下调FAK的表达后对TGF-β1诱导的HK-2细胞转分化进程的影响。 方法 应用TGF-β1（10 μg/L）刺激HK-2细胞,采用RT-PCR、Western印迹和免疫荧光方法分别检测E钙黏蛋白（E-cadherin）、α平滑肌肌动蛋白（α-SMA）、FAK mRNA和蛋白的表达及磷酸化（p）-FAK（Tyr397）的蛋白表达。应用Lipofectmine2000将FAK siRNA转染HK-2细胞,采用Western印迹观察下调表达FAK对上述指标的影响。 结果 TGF-β1刺激后,HK-2细胞α-SMA蛋白和mRNA水平上调,E-cadherin蛋白和mRNA表达下调。FAK蛋白和mRNA随时间的延长表达逐渐增多,48 h达到高峰。p-FAK（Tyr397）蛋白表达趋势与FAK相同。脂质体转染siRNA后FAK的mRNA和蛋白分别下调了50%和41%,下调表达FAK后可以显著抑制TGF-β1诱导的HK-2细胞α-SMA蛋白的上调表达,逆转 E-cadherin蛋白的下调表达。 结论 在TGF-β1诱导的HK-2细胞转分化进程FAK蛋白表达上调,敲低FAK蛋白表达后可以部分减轻EMT的程度,提示FAK在TGF-β1诱导的肾小管上皮细胞转分化和肾脏纤维化中发挥一定的作用。     

大肠癌患者外周血淋巴细胞粘着斑激酶表达的临床意义

目的：探讨大肠癌患者外周血淋巴细胞粘着斑激酶(FAK)的表达及其临床意义。方法 结果 结论 方法：应用流式细胞仪(flow cytometry, FCM)检测40例大肠癌患者及20例正常人外周血淋巴细胞中FAK的表达。结果：大肠癌患者外周血淋巴细胞FAK阳性表达率为60.0%，与正常人(25.0%)比较，差异有显著性意义(P＜0.05)。并且外周血淋巴细胞FAK表达与大肠癌临床分期、组织学分化程度、淋巴转移有关。结论：大肠癌患者外周血淋巴细胞FAK异常表达与荷瘤状况有关，对大肠癌的早期诊断、临床分期、组织学分型、淋巴转移的预测等可能有一定帮助。     

黏着斑激酶表达下调对肝癌细胞黏附迁移侵袭行为的影响

目的研究下调黏着斑激酶（FAK）表达对人肝癌细胞HCC-LM3黏附迁移侵袭行为的影响及可能涉及的机制。 方法根据处理方法不同,将人肝癌细胞HCC-LM3分为未处理组（肿瘤细胞未经处理）、对照组（肿瘤细胞转染空载体,FAK表达未改变）和FAK-shRNA组（肿瘤细胞稳定低表达FAK）。分别采用细胞黏附实验、划痕实验、Transwell实验检测3组肝癌细胞的黏附、迁移、侵袭能力,Western blotting检测细胞黏附侵袭相关蛋白paxillin、p130Cas以及基质金属蛋白酶MMP-2与MMP-9蛋白的表达及活化情况。 结果FAK表达下调后,细胞黏附能力显著受到抑制,细胞迁移能力和侵袭能力均明显下降;细胞黏附分子p130Cas、paxillin的蛋白总量表达无明显改变,而磷酸化水平明显降低,其活化形式p-paxillin和p-p130Cas表达则明显受到抑制;MMP-2、MMP-9蛋白表达水平则在下调FAK表达后明显降低（P<0.01）。 结论在人肝癌细胞HCC-LM3中,下调FAK表达可以影响肝癌细胞黏附迁移侵袭能力,其机制可能是通过调节相关细胞黏附分子的表达或活化来实现。     

内源性大麻素对人肝癌细胞生长及黏附和侵袭性的影响

目的 观察内源性大麻素(AEA)对人肝癌高转移细胞MHCC97H细胞生长、黏附、侵袭性等的抑制作用.方法 不同浓度AEA(100、50、0μmol/L)作用细胞后,应用体外细胞-基质黏附实验方法,检测细胞对Fibronectin黏附能力的改变;应用体外细胞趋化实验和侵袭实验,检测细胞的运动功能及对人工基底膜的侵袭能力;应用噻唑蓝(MTT)比色法检测细胞生长抑制率.结果 (1)不同浓度的AEA(100、50、0 μmol/L)作用细胞3、6、12 h后,细胞黏附抑制率分别为:3 h(24.4%、20.6%和20.8%)、6 h(23.6%、21.3%和19.6%)及12 h(25.2%、24.1%和22.8%),组间及组内比较差异无统计学意义(P＞0.05).(2)100μmol/L的AEA作用细胞48 h后,平均每个视野的透膜细胞数为(13.00±4.30)个,与对照组(15.70±3.30)个比较,差异无统计学意义(P＞0.05);平均每个视野的侵袭细胞数为(4.10±1.65)个,与对照组(3.40±1.24)个比较,差异无统计学意义(P＞0.05).(3)不同浓度的AEA(100、50、0μmol/L)作用细胞24 h后,细胞生长抑制率分别为(39.30 ±9.17)%、(35.60±8.25)%和(28.70±0.04)%,组间比较差异无统计学意义(P＞0.05).结论 AEA对体外生长的人肝癌细胞MHCC97H的生长、黏附和侵袭性等方面无明显抑制作用.

Abstract：

Objective To evaluate the inhibitory effects of anandamide (AEA) on proliferation and adhesion and invasion in human hepatocellular carcinoma cell line MHCC97H. Methods Mmethyl thiazol tetrazolium (MTT) method, cell-matrix adhesion assay, and Transwell invasion chamber and matriger were used to analyze the effect of AEA on proliferation ability, adherence ability, and migration,chemotaxis and invasion of MHCC97H cells, respectively. Results After MHCC97H cells were treated with AEA at the different concentrations (100, 50, 0 μmol/L) for 3, 6 and 12 h, the inhibition ratio of cell adhesion was 24.4%, 20.6% and 20.8% for 3 h, 23.6%, 21.3% and 19.6% for 6 h, and 25.2%, 24.1% and 22. 8% for 12 h, respectively (P＞0.05). After MHCC97H cells were treated with AEA (100 μmol/L) for 24 h, the migration, adhesion and invasion was not markedly inhibited (P＞0. 05 ). Conclusion AEA did not show inhibition effects on adhesion, invasiveness and proliferation of MHCC97H cells.     

Extracellular pressure stimulates adhesion of sarcoma cells via activation of focal adhesion kinase and Akt

Background

The effect of extracellular pressure on adhesion and adhesiogenic focal adhesion kinase (FAK) and Akt signaling in sarcomas was investigated.

Methods

Human sarcoma cells (HT-1080 fibrosarcoma, KHOS-240S osteosarcoma, and A-673 rhabdomyosarcoma) were subjected to increased pressure followed by adhesion assay. Two cell lines were pretreated with the FAK inhibitor 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15) or Akt IV inhibitor, followed by Western analysis for activated FAK and Akt. Parallel studies were conducted in cells from a resected human fibrous histiosarcoma.

Results

Pressure increased adhesion in all 3 sarcoma lines and primary histosarcoma cells by 7% to 18% (n = 6; P < .01 each). Pressure activated FAK and Akt (n = 5; P < .01). Inhibiting FAK or Akt inhibited FAK or Akt phosphorylation and the stimulation of adhesion by increased pressure (n = 5 each; P < .01 each).

Conclusions

Pressure increases sarcoma cell adhesiveness via Akt and FAK. Perioperative manipulation or forces in lymphatic or circulatory systems may potentiate local recurrence or distant metastasis.     

E-钙黏蛋白及黏着斑激酶在直肠癌组织中的表达及意义

目的 探讨E-钙黏蛋白(E-CD)及黏着斑激酶(FAK)和磷酸化黏着斑激酶(FAK py397)在直肠癌组织中的表达水平及其临床意义.方法 收集2001年至2002年武汉大学人民医院手术切除的直肠癌及对应癌旁组织石蜡标本30例和2006年至2007年手术切除的直肠癌新鲜标本及对应癌旁组织35例,应用免疫组织化学法、Western blot检测E-CD、FAK和FAK py397的表达,采用X2检验分析相关数据.结果 正常直肠黏膜中E-CD呈细胞膜阳性表达,直肠癌组织中E-CD细胞膜表达缺失,直肠癌组织中E-CD、FAK及FAK py397的表达与肿瘤分化程度、浸润深度和淋巴结转移有关(χ~2=7.099、18.358、25.612,12.316、28.823、23.168,8.927、18.122、22.620,P＜0.05);与年龄、性别无关(χ~2=0.439、1.899、3.676,0.541、4.051、1.135,P＞0.05);癌组织和癌旁组织中FAK及FAK py397表达率分别为83%(54/65)、68%(44/65)和31%(20/65)、26%(17/65),两者比较差异有统计学意义(χ~2=33.707,34.163,20.897,P＜0.05).结论 直肠癌组织中E-CD细胞膜表达缺失、FAK和FAK py397的表达水平明显升高,与肿瘤分化程度、浸润深度和淋巴结转移有关,可预测肿瘤的生物学行为.     

黏着斑激酶在机械通气相关性肺损伤中的作用

背景 机械通气相关性肺损伤(ventilator-induced lung injury,VILI)是指应用呼吸机过程中由于机械通气的各种因素与肺部原发病共同作用导致的肺组织损伤.肺损伤过程中肺微血管的完整性破坏及通透性改变是引起VILI的根本原因.紧密连接(tight junction,TJ)及黏附连接(adherent junction,AJ)是细胞连接的主要方式,对维持肺的正常功能起着重要作用.黏着斑激酶(focal adhesion kinase,FAK)主要作用于细胞膜黏附连接的黏着斑部位,能维持黏着斑蛋白在细胞膜的表达,对AJ及血管通透性具有重要作用. 目的 通过对FAK在VILI中的作用的研究指导临床实践. 内容 探讨机械通气对FAK的影响及FAK对AJ、TJ的影响和作用. 趋向 进一步明确FAK在VILI发生过程中的作用,为临床预防及治疗VILI提供新思路及理论依据.     

Focal adhesion kinase as a marker of invasive potential in differentiated human thyroid cancer

Background: The FAK gene encodes a 125-kDa tyrosine kinase (p125^FAK) involved in signal transduction pathways used in cell adhesion, motility, and anchorage-independent growth. Because thyroid carcinomas have a wide variability in their propensity for invasion and metastasis, we studied the expression of FAK in a variety of thyroid tissues. Methods: We synthesized a recombinant N-terminal fragment of the human FAK protein and developed a specific polyclonal antisera. Using Western blot analysis, we assessed the levels of p125^FAK expression in 30 human thyroid tissue samples from 27 patients that included paired normal and malignant specimens. Levels of FAK protein in individual tumors were quantitated by densitometric scanning of the immunoblots, and the results were correlated with tumor histology and biologic behavior. Results: The levels of FAK expression were directly correlated with thyroid carcinomas demonstrating the most aggressive phenotypes. The highest levels of p125^FAK were seen in follicular carcinomas and tumors associated with distant metastatic foci. In contrast, neoplastic thyroid tissues with limited invasive potential, such as papillary carcinomas, follicular adenomas, and other nonmalignant thyroid lesions, showed minimal p125^FAK expression. Conclusions: Overexpression of FAK may be part of a mechanism for invasion and metastasis of thyroid cancer. Furthermore, the levels of p125^FAK may serve as a marker of biologic behavior in this disease. Presented at the 48th Annual Cancer Symposium of The Society of Surgical Oncology, Boston, Massachusetts, March 23–26, 1995.     

Heparin-binding EGF-like growth factor (HB-EGF) promotes cell migration and adhesion via focal adhesion kinase

Background

Cell migration and adhesion are essential in intestinal epithelial wound healing and recovery from injury. Focal adhesion kinase (FAK) plays an important role in cell–extracellular matrix signal transduction. We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) promotes intestinal epithelial cell (IEC) migration and adhesion in vitro. The present study was designed to determine whether FAK is involved in HB-EGF–induced IEC migration and adhesion.

Materials and methods

A scrape wound healing model of rat IECs was used to examine the effect of HB-EGF on FAK-dependent cell migration in vitro. Immunofluorescence and Western blot analyses were performed to evaluate the effect of HB-EGF on the expression of phosphorylated FAK (p-FAK). Cell adhesion assays were performed to determine the role of FAK in HB-EGF–induced cell adhesion on fibronectin (FN).

Results

HB-EGF significantly increased healing after scrape wounding, an effect that was reversed in the presence of an FAK inhibitor 14 (both with P < 0.05). HB-EGF increased p-FAK expression and induced p-FAK redistribution and actin reorganization in migrating rat IECs. Cell adhesion and spreading on FN were significantly increased by HB-EGF (P < 0.05). FAK inhibitor 14 significantly inhibited both intrinsic and HB-EGF–induced cell adhesion and spreading on FN (both with P < 0.05).

Conclusions

FAK phosphorylation and FAK-mediated signal transduction play essential roles in HB-EGF–mediated IEC migration and adhesion.     

Expression of growth factor receptors,the focal adhesion kinase,and other tyrosine kinases in human soft tissue tumors

Background: The tyrosine kinases are a family of genes that includes many growth factor receptors and protooncogenes. They appear to have a role in many cancers, but have not been systematically studied in the pathogenesis and progression of human sarcomas. Methods: To characterize the protein tyrosine kinases that are expressed in human sarcomas, we used a polymerase chain reaction (PCR)-based method to construct kinase-specific cDNA libraries from low-grade and high-grade primary tumors. Thereafter, individual tyrosine kinase gene expression was assessed in a panel of sarcoma cell lines and primary tumors using Northern blotting and PCR. Results: We identified 19 species of tyrosine kinase genes, including many growth factor receptors, the human homolog of the focal adhesion kinase (FAK) gene, and a noveltrk-related kinase designated HGK2. Messenger RNA expression analyses showed relative overexpression of the two forms of the platelet-derived growth factor receptors (PDGFRs) with expression of the form restricted to a subgroup of high-grade and metastatic sarcomas. We were unable to demonstrate coexpression of the PDGF isoforms in primary tumors that overexpressed the receptors, suggesting that a PDGF/PDGFR autocrine pathway may not be a central mechanism in the malignant transformation of sarcomas in vivo. FAK expression was observed in a variety of sarcomas, with increased levels in several high-grade and metastatic leiomyosarcomas. Conclusions: When grouped together by histologic cell type and grade, the expression data of the 19 kinases in primary tumors described a greater degree of heterogeneity than is generally appreciated by clinicopathologic classification schemes. This diversity suggests that sarcomas, even those that appear to be clinically similar, arise through a variety of molecular pathways involving tyrosine kinases.Presented at the 46th Annual Cancer Symposium of the Society of Surgical Oncology, Los Angeles, March 18–21, 1993.