Focal adhesion kinase-related non-kinase ameliorates liver fibrosis by inhibiting aerobic glycolysis via the FAK/Ras/c-myc/ENO1 pathway

BACKGROUND Hepatic stellate cell(HSC)hyperactivation is a central link in liver fibrosis development.HSCs perform aerobic glycolysis to provide energy for their activation.Focal adhesion kinase(FAK)promotes aerobic glycolysis in cancer cells or fibroblasts,while FAK-related non-kinase(FRNK)inhibits FAK phosphorylation and biological functions.AIM To elucidate the effect of FRNK on liver fibrosis at the level of aerobic glycolytic metabolism in HSCs.METHODS Mouse liver fibrosis models were established by administering CCl4,and the effect of FRNK on the degree of liver fibrosis in the model was evaluated.Transforming growth factor-β1 was used to activate LX-2 cells.Tyrosine phosphorylation at position 397(pY397-FAK)was detected to identify activated FAK,and the expression of the glycolysis-related proteins monocarboxylate transporter 1(MCT-1)and enolase1(ENO1)was assessed.Bioinformatics analysis was performed to predict putative binding sites for c-myc in the ENO1 promoter region,which were validated with chromatin immunoprecipitation(ChIP)and dual luciferase reporter assays.RESULTS The pY397-FAK level was increased in human fibrotic liver tissue.FRNK knockout promoted liver fibrosis in mouse models.It also increased the activation,migration,proliferation and aerobic glycolysis of primary hepatic stellate cells(pHSCs)but inhibited pHSC apoptosis.Nevertheless,opposite trends for these phenomena were observed after exogenous FRNK treatment in LX-2 cells.Mechanistically,the FAK/Ras/c-myc/ENO1 pathway promoted aerobic glycolysis,which was inhibited by exogenous FRNK.CONCLUSION FRNK inhibits aerobic glycolysis in HSCs by inhibiting the FAK/Ras/c-myc/ENO1 pathway,thereby improving liver fibrosis.FRNK might be a potential target for liver fibrosis treatment.

Inhibition of focal adhesion kinase enhances antitumor response of radiation therapy in pancreatic cancer through CD8+ T cells

Objective:Pancreatic ductal adenocarcinoma(PDAC)is a deadly malignancy,due in large part to its resistance to conventional therapies,including radiotherapy(RT).Despite RT exerting a modest antitumor response,it has also been shown to promote an immunosuppressive tumor microenvironment.Previous studies demonstrated that focal adhesion kinase inhibitors(FAKi)in clinical development inhibit the infiltration of suppressive myeloid cells and T regulatory(T regs)cells,and subsequently enhance effector T cell infiltration.FAK inhibitors in clinical development have not been investigated in combination with RT in preclinical murine models or clinical studies.Thus,we investigated the impact of FAK inhibition on RT,its potential as an RT sensitizer and immunomodulator in a murine model of PDAC.Methods:We used a syngeneic orthotopic murine model to study the effect of FAKi on hypofractionated RT.Results:In this study we showed that IN10018,a small molecular FAKi,enhanced antitumor response to RT.Antitumor activity of the combination of FAKi and RT is T cell dependent.FAKi in combination with RT enhanced CD8+T cell infiltration significantly in comparison to the radiation or FAKi treatment alone(P<0.05).FAKi in combination with radiation inhibited the infiltration of granulocytes but enhanced the infiltration of macrophages and T regs in comparison with the radiation or FAKi treatment alone(P<0.01).Conclusions:These results support the clinical development of FAKi as a radiosensitizer for PDAC and combining FAKi with RT to prime the tumor microenvironment of PDAC for immunotherapy.

Progress in researches about focal adhesion kinase in gastrointestinal tract

Focal adhesion kinase(FAK)is a 125-kDa non-receptor protein tyrosine.Growth factors or the clustering of integrins facilitate the rapid phosphorylation of FAK at Tyr-397 and this in turn recruits Src-family protein tyrosine kinases,resulting in the phosphorylation of Tyr-576 and Tyr-577 in the FAK activation loop and full catalytic FAK activation.FAK plays a critical role in the biological processes of normal and cancer cells including the gastrointestinal tract.FAK also plays an important role in the restitution,cell survival and apoptosis and carcinogenesis of the gastrointestinal tract.FAK is over-expressed in cancer cells and its over-expression and elevated activities are associated with motility and invasion of cancer cells.FAK has been proposed as a potential target in cancer therapy.Small molecule inhibitors effectively inhibit the kinase activity of FAK and show a potent inhibitory effect for the proliferation and migration of tumor cells,indicating a high potential for application in cancer therapy.

Inhibiting focal adhesion kinase:A potential target for enhancing therapeutic efficacy in colorectal cancer therapy

Focal adhesion kinase(FAK) is a major integrin- dep-endent tyrosine phosphorylated protein, recently, FAK association with colorectal cancer(CRC) has gained at-tention. The various cancer-promoting mechanisms that associated with FAK can be implicated in the progression of CRC. The interactions between structural features of FAK and various kinases could be closely related to growth, survival, and metastasis in CRC cells. These interactions include human epithelial growth factor re-ceptor, c-Met, platelet-derived growth factor receptor, vascular endothelial growth factor receptor, and Src. Such interactions can trigger the survival signaling of CRC cells and are also involved signaling downstream of phosphatidylinositol 3-kinase, AKT, and the extracellular regulated kinase. Based on this scientific background, many pharmaceutical companies are taking efforts to develop FAK inhibitors to treat solid cancer including CRC. Although the anti-cancer efficacies have been noted in many studies, the commercial drugs have not been deve-loped yet. Therefore, the FAK research on CRC is expec-ted to gain momentum and be highly appreciated as a potential field for developing the new drugs. Therefore, the studies on FAK that effect on the progression of human CRC s would be possible to suggest various app-roaches to CRC treatment, and FAK could be a potential target as an anticancer candidate for CRC therapies.

Effect of focal adhesion kinase on cytoskeletal arrangement of HepG2 cells induced by hypoxia

对在 hypoxic HepG2 房间和 HepG2 房间的细胞骨架安排上的 FAK siRNA 的效果的焦点的粘附 kinase (FAK ) 表示由组织缺氧导致了的学习客观。方法 HepG2 房间在 21% O 2 和 1% O 2 是有教养的。词法变化在组织缺氧处理以后被观察。西方的污点被用来测量 FAK 表示。指向 FAK 和向量 pGensil-2 的 mRNA 的 siRNA 表示向量 pshRNA-FAK (作为控制) 被构造，然后 transfected 进 HepG2 房间。西方的污点被用来检测 FAK。有组织缺氧导致的 pshRNA-FAK 的 HepG2 房间 transfected 的细胞骨架安排被 phalloidin 分析。有组织缺氧导致的 pshRNA-FAK 的 HepG2 房间 transfected 的迁移的能力被房间迁居试金分析。结果对待组织缺氧的房间与房间分开的大度显示了更多的伸长的形状。FAK 表示在 hypoxic HepG2 房间增加了。FAK 蛋白质水平被 75.64% ±减少 3.12%(P 【 0.01 ) 在 pshRNA-FAK transfection 以后。组织缺氧导致了 HepG2 房间的细胞骨架安排。然而，有组织缺氧导致的 pshRNA-FAK 的 HepG2 房间 transfected 的细胞骨架安排在 1% O 2 被禁止。当房间移植试金出现了，有 pshRNA-FAK 的 HepG 房间 transfected 的移居的数字比控制的显著地低(P 【 0.05 ) 。在 hypoxic HCC 的 FAK 的表示可能有一种靠近的关系到 HepG2 房间的细胞骨架安排的结论由组织缺氧导致了。FAK 表示的起来规定可以是细胞骨架安排的机制和组织缺氧导致的 hepatocellular 癌的侵略之一。

Focal adhesion kinase and Src phosphorylations in HGF-induced proliferation and invasion of human cholangiocarcinoma cell line, HuCCA-1

AIM: To study the role of focal adhesion kinase (FAK) and its association with Src in hepatocyte growth factor (HGF)-induced cell signaling in cholangiocarcinoma progression.METHODS: Previously isolated HuCCA-1 cells were re-characterized by immunofluorescent staining and reverse transcriptase-polymerase chain reaction assay for the expression of cytokeratin 19, HGF and c-Met mRNA. Cultured HuCCA-1 cells were treated with HGF and determined for cell proliferation and invasion effects by MTT and invasion assays. Western blotting, immunoprecipitation, and co-immunoprecipitation were also performed to study the phosphorylation and interaction of FAK and Src. A novel Src inhibitor (AZM555130) was applied in cultures to investigate the effects on FAK phosphorylation inhibition and on cell proliferation and invasion.RESULTS: HGF enhanced HuCCA-1 cell proliferation and invasion by mediating FAK and Src phosphorylations.FAK-Src interaction occurred in a time-dependent manner that Src was proved to be an upstream signaling molecule to FAK. The inhibitor to Src decreased FAK phosphorylation level in correlation with the reduction of cell proliferation and invasion.CONCLUSION: FAK plays a significant role in signaling pathway of HGF-responsive cell line derived from cholangiocarcinoma. Autophosphorylated Src, induced by HGF, mediates Src kinase activation, which subsequently phosphorylates its substrate, FAK, and signals to cell proliferation and invasion.

Degradation of FAK-targeting by proteolytic targeting chimera technology to inhibit the metastasis of hepatocellular carcinoma

Liver cancer is a prevalent malignant cancer,ranking third in terms of mortality rate.Metastasis and recurrence primarily contribute to the high mortality rate of liver cancer.Hepatocellular carcinoma(HCC)has low expression of focal adhesion kinase(FAK),which increases the risk of metastasis and recurrence.Nevertheless,the efficacy of FAK phosphorylation inhibitors is currently limited.Thus,investigating the mechanisms by which FAK affects HCC metastasis to develop targeted therapies for FAK may present a novel strategy to inhibit HCC metastasis.This study examined the correlation between FAK expression and the prognosis of HCC.Additionally,we explored the impact of FAK degradation on HCC metastasis through wound healing experiments,transwell invasion experiments,and a xenograft tumor model.The expression of proteins related to epithelial-mesenchymal transition(EMT)was measured to elucidate the underlying mechanisms.The results showed that FAK PROTAC can degrade FAK,inhibit the migration and invasion of HCC cells in vitro,and notably decrease the lung metastasis of HCC in vivo.Increased expression of E-cadherin and decreased expression of vimentin indicated that EMT was inhibited.Consequently,degradation of FAK through FAK PROTAC effectively suppressed liver cancer metastasis,holding significant clinical implications for treating liver cancer and developing innovative anti-neoplastic drugs.

根皮素通过抑制FAK改善肾小管间质纤维化

目的探究根皮素能否通过抑制黏着斑激酶(FAK)改善肾小管间质纤维化。方法肾小管上皮细胞NRK-52E经TGF-β1(10 ng/mL)诱导建立肾纤维化细胞模型,并构建单侧输尿管梗阻(UUO)肾间质纤维化小鼠模型。通过免疫荧光及Western blot法检测细胞及小鼠肾脏组织中α-SMA、FN的蛋白表达水平,明确根皮素体内外抗肾纤维化作用;检测实验小鼠血清中肌酐(Cr)和尿素氮(BUN)水平,分析根皮素的肾保护作用;以苏木精-伊红(HE)染色和Masson染色观察根皮素对UUO小鼠肾脏病理学改变和胶原纤维沉积的影响;Western blot检测TGF-β1诱导的NRK-52E细胞和UUO小鼠肾组织中FAK、p-FAK的蛋白表达水平;通过分子对接预测根皮素与FAK蛋白的潜在结合模式和结合能力;使用FAK抑制剂PND-1186,探究根皮素改善肾间质纤维化的作用机制。结果根皮素可逆转TGF-β1所致的肾小管上皮细胞及UUO小鼠肾组织中α-SMA、FN的蛋白表达水平升高,且显著降低UUO小鼠血清中Cr、BUN水平,改善UUO小鼠肾小管扩张、炎性细胞浸润及间质胶原沉积;根皮素可以显著抑制UUO小鼠肾组织中FAK、p-FAK蛋白的表达水平,分子对接提示根皮素与FAK蛋白之间具有良好相互作用。根皮素与PND-1186均可改善TGF-β1诱导的NRK-52E细胞纤维化。结论根皮素通过抑制FAK信号通路在体内外发挥抗肾间质纤维化作用。

人黏着斑激酶(FAK)原核表达及多克隆抗体制备与鉴定

目的克隆、表达与纯化黏着斑激酶(FAK)基因C端黏着斑定位序列(第798~1041位氨基酸),制备兔抗FAK多克隆抗体并鉴定。方法PCR法体外扩增FAK基因C端序列(2671~3402 bp),克隆至pCZN1,构建pCZN1-FAK重组表达载体,将重组表达载体转化到大肠杆菌表达菌株BL21(DE3)感受态细胞,用异丙基β-D-硫代吡喃半乳糖苷(IPTG)诱导表达;利用镍-次氮基三乙酸(Ni-NTA)金属鳌合亲和层析树脂进行蛋白纯化,并免疫新西兰大白兔制备多克隆抗体;通过间接ELISA和Western blot法进行效价和特异性鉴定。结果成功构建pCZN1-FAK重组表达载体,FAK蛋白主要以包涵体形式表达。目的蛋白纯化后,制备的兔抗FAK多克隆抗体效价达1∶512000,且与外源和内源FAK蛋白发生特异性反应。结论成功克隆、表达与纯化FAK蛋白,制备兔抗FAK多克隆抗体,可用于内源FAK蛋白特异性检测。

金雀异黄酮抑制OPN-FAK信号通路对非小细胞肺癌迁移和侵袭的影响

目的:探讨金雀异黄酮(GS)抑制骨桥蛋白(OPN)/黏着斑激酶(FAK)信号通路对非小细胞肺癌(NSCLC)迁移和侵袭的影响。方法:qRT-PCR检测四种NSCLC细胞系(H596、H520、A549、H1703)及正常人肺上皮细胞(BEAS-2B)中OPN、FAK mRNA表达水平;以A549细胞为研究目标,分别设置对照组、GS低浓度(GS-L)组(10μmoL/L)、GS中浓度(GS-M)组(20μmoL/L)、GS高浓度(GS-H)组(40μmoL/L)、重组骨桥蛋白(rOPN)组(15ng/mL)、GS-H+rOPN组(40μmoL/L GS+15ng/mL rOPN)。分别利用MTT法、流式细胞仪检测上述各组细胞增殖率、凋亡率;划痕实验及Transwell实验检测细胞迁移、侵袭变化;Western blot及qRT-PCR检测OPN/FAK信号通路相关基因和蛋白的表达。结果:与BEAS-2B细胞相比,A549细胞中OPN、FAK mRNA表达水平变化最为显著(P<0.05)。与对照组相比,GS-L组、GS-M组、GS-H组A549细胞增殖率、迁移率、侵袭及迁移数、OPN/FAK信号通路相关基因和蛋白表达显著降低,凋亡率显著增加(P<0.05);rOPN组A549细胞增殖率、迁移率、侵袭及迁移数、OPN/FAK信号通路相关基因和蛋白表达显著增加,凋亡率显著降低(P<0.05)。与GS-H组相比,GS-H+rOPN组A549细胞增殖率、迁移率、侵袭及迁移数、OPN/FAK信号通路相关基因和蛋白表达显著增加,凋亡率显著降低(P<0.05)。与rOPN组相比,GS-H+rOPN组A549细胞增殖率、迁移率、侵袭及迁移数、OPN/FAK信号通路相关基因和蛋白表达显著降低,凋亡率显著增加(P<0.05)。结论:GS可以抑制NSCLC-A549细胞的迁移、侵袭及增殖,促进其凋亡,可能与抑制OPN/FAK信号通路有关。

基于FAK/ERK途径探讨NRP-1对结直肠癌细胞的影响

目的探究神经纤毛蛋白-1(neuropilin-1,NRP-1)对结直肠癌(colorectal cancer,CRC)细胞行为的作用,及其对黏着斑激酶(focal adhesion kinase,FAK)/胞外信号调节激酶(extracellular signal-regulated kinases,ERK)信号途径的影响。方法将CRC细胞系DLD-1随机分为对照组、CT-707组、sh-NRP-1组、sh-NRP-1+CT-707组,按分组使用sh-NRP-1慢病毒液感染细胞与FAK抑制剂CT-707处理细胞,通过荧光显微镜观察感染慢病毒后细胞内增强型绿色荧光蛋白表达情况,实时荧光定量PCR和Western blot法检测细胞内NRP-1mRNA与蛋白的表达变化。CCK-8法和EdU染色检测细胞增殖能力,细胞划痕实验观察细胞划痕愈合率,Transwell实验检测细胞迁移与侵袭,Western blot法测定细胞内FAK/ERK相关蛋白表达情况。结果感染sh-NRP-1重组慢病毒的DLD-1细胞中可见明显绿色荧光,细胞内NRP-1mRNA相对表达量和蛋白相对表达量均显著下调(P<0.05),表明转染成功。与对照组比较,CT-707组和sh-NRP-1组的细胞增殖活性显著下降,EdU阳性百分比显著降低,细胞划痕愈合率显著降低,细胞迁移数目与侵袭数目均显著减少,FAK蛋白磷酸化水平和ERK蛋白磷酸化水平均显著下调(P<0.05);与sh-NRP-1组比较,sh-NRP-1+CT-707组细胞增殖活性显著下降,EdU阳性百分比和细胞划痕愈合率均显著降低,细胞迁移数目与侵袭数目也均显著减少,FAK蛋白磷酸化水平和ERK蛋白磷酸化水平均显著下调(P<0.05)。结论下调NRP-1表达能够发挥抑制CRC细胞增殖、迁移及侵袭的作用,该机制可能与调控FAK/ERK信号转导途径有关。

吉马酮调节FAK/YAP信号通路对非小细胞肺癌细胞顺铂耐药性的影响

目的 探讨吉马酮(GMC)调节局部黏着斑激酶(FAK)/Yes相关蛋白(YAP)通路对非小细胞肺癌(NSCLC)细胞顺铂(DDP)耐药性的影响。方法 将A549/DDP细胞分为CK组、低剂量GMC组(GMC-L组,5μmol/L)、中剂量GMC组(GMC-M组,10μmol/L)、高剂量GMC组(GMC-H组,20μmol/L)、GMC-H+pcDNA组(20μmol/L+转染pcDNA)、GMC-H+pcDNA-FAK组(20μmol/L+转染pcDNA-FAK)。检测各组细胞增殖、凋亡、迁移、侵袭情况;检测多药耐药基因1(MDR1)、增殖细胞核抗原(PCNA)、B淋巴细胞瘤-2(Bcl-2)、基质金属蛋白酶(MMP)-2、MMP-9、磷酸化的FAK(p-FAK)、YAP蛋白表达情况。结果 与CK组比较,GMC-L组、GMCM组、GMC-H组A549/DDP细胞OD450值、克隆形成率、划痕愈合率、侵袭细胞数、MDR1、PCNA、Bcl-2、MMP-2、MMP-9、p-FAK蛋白及核YAP蛋白表达降低,细胞凋亡率升高,且呈浓度依赖性(P<0.05);与GMC-H组、GMC-H+pcDNA组比较,GMC-H+pcDNA-FAK组A549/DDP细胞OD_(450)值、克隆形成率、划痕愈合率、侵袭细胞数、MDR1、PCNA、Bcl-2、MMP-2、MMP-9、p-FAK蛋白及核YAP蛋白表达升高,细胞凋亡率降低(P<0.05)。结论 GMC可能通过抑制FAK/YAP信号通路减弱NSCLC细胞DDP耐药性。

超声心肌灌注评分联合血清FAK对急性心肌梗死患者介入术后预后的预测价值

目的分析超声心肌灌注评分联合血清黏着斑激酶(FAK)对急性心肌梗死患者介入术后预后的预测价值。方法选取2019年8月至2022年8月收治的76例行经皮冠状动脉介入治疗(PCI)的急性心肌梗死患者作为研究对象,根据不同预后将其分为预后不良组(20例)和预后良好组(56例),均接受超声心肌灌注评分和血清FAK检测。比较不同预后患者超声心肌灌注评分、血清FAK,并分析其预测价值。结果预后不良组的血小板计数、中性粒细胞计数、肌酸激酶同工酶(CK-MB)、心肌肌钙蛋白I(cTnI)、FAK水平及超声心肌灌注评分高于预后良好组,淋巴细胞计数低于预后良好组,差异具有统计学意义(P<0.05)。多因素Logistic回归分析结果显示,血小板计数、中性粒细胞计数、CK-MB、cTnI、FAK、超声心肌灌注评分是急性心肌梗死患者介入术后预后不良的危险因素,淋巴细胞计数为保护因素,差异具有统计学意义(P<0.05)。超声心肌灌注评分联合血清FAK预测急性心肌梗死患者介入术后预后的效能较高,灵敏度为90.00%,特异度为92.86%,明显高于超声心肌灌注评分、血清FAK单项预测,差异具有统计学意义(P<0.05)。结论超声心肌灌注评分、血清FAK水平与急性心肌梗死患者介入术后预后密切相关,超声心肌灌注评分联合血清FAK具有较高的预后预测价值。

结直肠癌中FAK、FAK pY397、Akt、NF-κB表达及相关性研究

目的:检测黏着斑激酶(FAK)、磷酸化FAK(FAK pY397)、Akt和细胞核因子(NF-κB)在结直肠癌组织中的表达,并探讨其相关性。方法:采用免疫组织化学SP法,检测74例结直肠癌组织与10例正常结肠组织中FAK、FAK pY397、Akt和NF-κB的表达。结果:结直肠癌组织标本中FAK、FAK pY397、Akt和NF-κB的阳性率分别为82.43%、45.95%、52.70%和59.46%,除FAK pY397外,FAK、Akt、NF-κB均与正常结肠壁组织表达的差异显著(P<0.05)。FAK表达与分化程度、淋巴结转移有关(P<0.05),但不受患者性别、年龄、肿瘤的大小及位置、Dukes分期的影响。Akt阳性表达与分化程度显著相关(P<0.05),NF-κB表达与淋巴结转移显著有关(P<0.05),并且Akt、NF-κB表达与FAK表达呈显著正相关(P<0.05)。结论:FAK、Akt、NF-κB在结直肠癌组织中均高表达,且Akt、NF-κB表达与FAK有显著正相关性,提示生存信号传导通路FAK/Akt/NF-κB在结直肠癌的发生、演进中起重要作用。

FAK在肝细胞癌中的表达及临床病理意义

目的研究FAK在肝细胞癌组织中的表达情况以及其与临床病理特征的联系,并探讨其表达与肝细胞癌患者预后之间的关系。方法利用RT-PCR法检测了52例肝癌组织及对应的癌旁组织中FAK mRNA的表达水平。分析了FAK mRNA的表达水平与肝癌临床病理特征之间的关系。利用免疫组化的方法检测了FAK蛋白在30例肝癌组织及其癌旁组织和10例正常肝脏组织中的表达。Kaplan-Meier生存分析和Cox回归分析了FAK表达与肝癌手术治疗预后的关系。结果肝癌组织中FAK mRNA的表达水平显著高于其癌旁组织(0.48±0.12 vs.0.17±0.07;P<0.05)。FAK mRNA表达水平与肿瘤直径、组织病理分化程度、TNM分期、血管侵犯等临床病理特征显著相关(P<0.05)。Kaplan-Meier生存分析和COX回归模型分析发现FAK表达是影响肝细胞癌术后生存率的因素。30例肝癌组织细胞质中广泛表达FAK蛋白,主要定位于肝癌细胞质内,阳性率为60%(18/30),癌旁组织阳性率为26.3%(8/30),10例正常肝组织阳性表达率为20%(2/10)。结论 FAK在肝癌组织中高表达,这与肝癌的恶性临床病理特征相关,也是判断肝细胞癌手术治疗预后的指标,提示FAK可能成为肝癌潜在的分子标志物或治疗靶点。

FAK和Src在甲状腺乳头状癌中的表达及其在肿瘤侵袭转移中的临床意义及机制研究

目的:探讨黏着斑激酶(FAK)和Src在甲状腺乳头状癌侵袭转移中的临床意义。方法:利用免疫组化检测FAK和Src在100例甲状腺乳头状癌组织中的表达,并分析其与患者临床病理参数的关系。利用Western blot以及qRT-PCR检测FAK和Src在甲状腺癌细胞株中与上皮间质转化(EMT)的相关性。结果:FAK与甲状腺乳头状癌的年龄(P=0.000)、T分期(P=0.176)、N分期(P=0.000)、M分期(P=0.000)、临床分期(P=0.038)、局部复发(P=0.000)、淋巴结侵袭(P=0.014)以及与患者较低生存期(P<0.000 1)密切相关;Src与甲状腺乳头状癌的N分期(P=0.000)、M分期(P=0.002)、淋巴结侵袭(P=0.000)、包膜侵袭(P=0.029)以及患者的较低生存期(P<0.000 1)密切相关。并且高表达FAK与Src的细胞其N-cadherin、Vimentin的表达增高,而E-cadherin的表达降低。结论:FAK以及Src与甲状腺乳头状癌的侵袭转移密切相关,并可能通过EMT的方式促进其侵袭和转移。

低强度脉冲超声波对兔膝骨性关节炎软骨整合素-FAK-MAPKs信号通路蛋白表达的影响

目的:观察低强度脉冲超声波(low-intensity pulsed ultrasound,LIPUS)对兔膝骨性关节炎(osteoarthritis,OA)软骨中整合素-FAK-MAPKs力化学细胞信号转导通路相关蛋白表达的影响。方法:18只成年新西兰大白兔随机平均分成3组,分为正常对照组(normal control,NC),OA模型组(OA group,OA),OA模型照射组(O+L)。OA组接受右侧后肢前交叉韧带切断(ACLT)处理术后4周接受LIPUS假辐射,O+L组同样手术处理,术后4周接受LIPUS辐射,NC组仅切开关节囊。LIPUS作用6周后,处死实验动物,取右后肢,采用HE染色行改良Mankin评分比较各组胫骨平台关节表面病理学改变。同时,采用Western blot技术检测Ⅱ型胶原,MMP-13,整合素β1的蛋白表达水平以及FAK、MAPKs家族蛋白(p38、ERK1/2、JNK)磷酸化水平。结果:1组织学观察及Mankin评分:LIPUS干预后,OA软骨表层轻微不平整,染色轻度缺失,可见软骨细胞增殖;Mankin评分,NC组:4.67±0.57;OA组:10.57±2.55;O+L组:7.66±1.74。与NC组相比,OA组、O+L组Mankin评分明显增高,但OA组增高更为明显(P<0.05);与OA组相比,O+L组Mankin评分明显降低(P<0.05)。2Ⅱ型胶原、MMP-13含量:LIPUS干预后,II型胶原含量较NC组升高,MMP-13含量有明显下降。3整合素β1-FAK-MAPKs信号通路相关蛋白表达:LIPUS干预使整合素β1、磷酸化FAK表达增高;同时MAPKs信号通路相关蛋白ERK1/2、p38磷酸化水平明显下降,而JNK磷酸化水平无明显变化。结论:LIPUS可以减轻骨性关节炎软骨ECM损伤程度,与LIPUS产生机械应力使关节软骨中细胞表面应力受体之一整合素β1及其下游分子黏着斑激酶FAK磷酸化表达增高,进一步下游的磷酸化p38,ERK1/2表达下调有关。LIPUS的机械效应可经力化学转导途径作用于OA关节软骨,为治疗骨性关节炎提供一种新的思路。

反义FAK寡核苷酸抑制FAK-ERK1/2介导的平滑肌细胞迁移和粘附

目的 研究FAK ERK1 2信号通路在平滑肌细胞迁移和粘附中的作用及FAK反义寡核苷酸对其的调控作用。方法 通过纤粘连蛋白 (fibronectin ,FN)诱导平滑肌细胞迁移和粘附 ,以免疫沉淀和Westernblot方法检测粘着斑激酶 (focaladhesionkinase,FAK)和细胞外调控激酶 1 2 (extracellularregulationkinase ,ERK1 2 )及其磷酸化的表达量。将FAK反义寡核苷酸 (antisenseoligodeoxynucleotides,ODNs)经脂质体转染细胞 ,观察转染后反义ODN对FAK和ERK磷酸化、细胞粘附和迁移的影响。结果 FN在有效诱导平滑肌细胞迁移和粘附时FAK和ERK1 2也呈明显表达 ,FN2 0 μg·mL- 1 可使磷酸化处于较高表达量。脂质体可有效地介导ODNs转染。转染后FAK及ERK1 2磷酸化表达量明显减少。结论 由活化的FAK和ERK1 2介导的信号转导促进了细胞外基质诱导的SMCs迁移和增殖 ,反义FAKODNs可有效地对此进行抑制。

PTEN下调FAK磷酸化来抑制EGFR受体突变体引起的胶质瘤细胞侵袭

背景与目的:抑癌基因PTEN在胶质瘤中突变率达20%～40%,且PTEN缺失和表皮生长因子受体突变体EGFRvⅢ同时存在EGFR表达的胶质瘤组织中。PTEN通过直接与FAK作用从而降低其酪氨酸磷酸化来抑制肿瘤细胞侵袭。EGFRvⅢ表达,PTEN缺失都是肿瘤细胞侵袭性增加的原因之一。PTEN功能的丧失可能是EGFRvⅢ表达的肿瘤进一步发展成恶性侵袭性肿瘤的原因之一。本文将探讨在EGFRvⅢ表达和PTEN缺失的肿瘤细胞中转入PTEN以观察其是否能抑制EGFRvⅢ所引起的肿瘤细胞的侵袭。方法:将野生型PTEN及其突变体分别导入人胶质瘤细胞U87ΔEGFR中,利用Transwell-细胞侵袭实验观察细胞侵袭运动变化;免疫印迹实验检查FAK磷酸化变化;FAK表达载体来转染FAK磷酸化程度低的U87ΔEGFR-wtPTEN细胞,观察细胞侵袭运动变化。结果:PTEN和PTEN(G129E)能够抑制U87ΔEGFR细胞侵袭,并且下调FAK397位点磷酸化水平。U87ΔEGFR-wtPTEN细胞中FAK397位点磷酸化水平增加伴随着细胞侵袭能力的提高。结论:PTEN可能通过下调FAK397位点磷酸化水平来抑制EGFRvⅢ引起的胶质瘤细胞侵袭。