克唑替尼在ALK阳性中晚期非小细胞肺癌中的疗效观察

目的：探讨克唑替尼治疗晚期间变淋巴瘤激酶(anaplastic lymphoma kinase,ALK)融合基因阳性中晚期非小细胞肺癌(non-small cell lung cancer,NSCLC)的近期疗效及毒副反应。方法：回顾性分析43例ALK阳性的中晚期NSCLC患者,服用克唑替尼治疗,服用至病情进展或出现不可耐受的毒副反应,随访12个月,观察疗效。结果：克唑替尼治疗ALK阳性NSCLC的疾病控制率(disease control rate,DCR)为93%(3/43),客观缓解率(objective response rate,ORR)为62%(26/43),中位无进展生存时间(progression free survival,PFS)为7.0个月(95% CI,6.0~8.0月),不良反应主要为消化道症状,其次是谷丙转氨酶升高,视觉障碍,大部分为1~2级。结论：克唑替尼作为NSCLC患者的多靶点靶向治疗,具有良好的疗效及安全性,不良反应轻微。     

卡博替尼、克唑替尼和奥西替尼 对小鼠髓源抑制性细胞亚型和凋亡的影响

目的探讨卡博替尼、克唑替尼和奥斯替尼(AZD9291)对小鼠骨髓或脾脏源性抑制细胞(MDSCs)亚群比例、凋亡及增殖的影响。方法免疫磁珠法分离BALB/c小鼠(雄性)骨髓G-MDSCs和M-MDSCs,分别加入卡博替尼(0.01、0.1、0.3和0.9μmol/L)、克唑替尼(0.2、2、20和200μg/mL)或AZD9291(0.01、0.1、1和10μmol/L)培养24 h,采用流式细胞术(FCM)检测3种靶向药物对MDSCs亚型的影响,CCK-8法检测MDSCs增殖;流式细胞分选仪分选小鼠骨髓Gr-1~+CD11b~+细胞,检测Gr-1~+CD11b~+细胞凋亡。结果克唑替尼处理组骨髓粒细胞型MDSCs(G-MDSCs)、单核细胞型MDSCs(M-MDSCs)比例均下降(P0.05);卡博替尼处理组脾脏G-MDSCs比例下降(P0.05);卡博替尼、克唑替尼处理骨髓MDSCs后早期凋亡比例增加(P0.05),AZD9291处理的MDSCs凋亡比例无明显变化。结论卡博替尼和克唑替尼可能通过诱导MDSCs凋亡降低MDSCs比例。     

克唑替尼治疗非小细胞肺癌的研究

非小细胞肺癌（NSCLC）是肺癌常见类型,早期诊断难度较大,5年生存率低。克唑替尼作为抑制Met/ALK/ROS的ATP竞争性的多靶点蛋白激酶抑制剂,是治疗NSCLC的分子靶向药物,在控制变性淋巴瘤激酶（ALK）阳性NSCLC患者疾病发展中具有重要意义,可有效延长患者无进展生存期,且较化疗毒副反应更小,患者耐受性高,但克唑替尼的耐药性也是临床关注重点。本文主要对克唑替尼治疗NSCLC、克唑替尼药理作用及其临床效果、耐药后治疗等进行综述,旨在为更好的发挥该药治疗效果,为临床治疗NSCLC提供参考依据。     

克唑替尼对ALK阳性非小细胞肺癌脑转移的疗效分析

目的：非小细胞肺癌(non-small cell lung cancer,NSCLC)脑转移是影响NSCLC患者生存期及生存质量的重要因素,本研究将探讨克唑替尼在间变性淋巴瘤激酶(anaplastic lymphoma kinase, ALK)阳性NSCLC脑转移患者中的疗效。方法：筛选20例ALK阳性NSCLC脑转移患者,给予克唑替尼治疗,观察临床疗效。结果：克唑替尼治疗前基线有脑转移患者,颅内疗效部分缓解(partial response,PR)12例,疾病稳定(stable disease,SD)7例,疾病进展(progressive disease,PD)1例。客观缓解率(objective response rate,ORR)为60%,疾病控制率(disease control rate,DCR)为95%。中位无进展生存期(progression free survival,PFS)为6个月(95%CI：3.92~8.08)。结论：克唑替尼治疗ALK阳性NSCLC脑转移患者有效。     

5-氟尿嘧啶诱导人肺腺癌A549细胞自噬

目的 探讨5-氟尿嘧啶(5-FU)是否诱导人肺腺癌A549细胞发生自噬.方法 吖啶橙(AO)荧光染色和透射电镜观察自噬泡的变化;细胞免疫化学法测定LC3-Ⅱ的表达;流式细胞术及吖啶橙染色结合激光扫描共焦显微镜检测细胞凋亡;DNA电泳检测凋亡梯形条带的产生.结果 吖啶橙荧光染色和透射电镜观察结果显示,5-氟尿嘧啶处理细胞...     

抑制自噬增强MCF-7乳腺癌细胞对依托泊苷的敏感性

目的探讨自噬抑制剂(Baf)在乳腺癌MCF-7细胞对依托泊苷(VP-16)的药物敏感性的影响。方法实验分为对照组(NC)、Baf组、VP-16和VP-16+Baf组。用MTT法检测细胞生存活力、GFP-LC3质粒转染后荧光显微镜检测绿色荧光的分布、Western blot检测蛋白表达和流式细胞仪检测细胞凋亡。结果 15μmol/L的VP-16使细胞活力降低,而在收集细胞前12 h时加入10 nmol/L的Baf进一步降低了细胞活力(P0.01);VP-16明显增加MCF-7细胞的LC3Ⅱ的表达和GFP-LC3绿色荧光斑点的聚集,而减少了P62的蛋白表达;与VP-16组比较,VP-16+Baf组细胞P62的蛋白表达增多,凋亡蛋白cleaved-PARP的表达和细胞凋亡比例也明显增多(P0.01)。结论 VP-16抑制乳腺癌细胞增殖的过程中诱导了保护性自噬,抑制自噬促进了凋亡性死亡,可以增加癌细胞对VP-16的敏感性。     

miR-30a抑制Ang2诱导的大鼠心肌细胞系H9c2自噬

目的 探索miR-30a对血管紧张素2(Ang2)诱导的大鼠心肌细胞自噬的影响.方法 1)将H9c2细胞分为对照组(control)、Ang2处理组(Ang22×10-6 mol/L)、去甲肾上腺素阳性对照组(NE 5×10-6 mol/L).处理细胞48 h,RT-qPCR检测miR-30a表达;Western bl...     

miR-30a通过靶向调控Beclin-1、ATG5影响食管癌细胞自噬及增殖过程北大核心CSCD

目的:探究miR-30a通过靶向调控Beclin-1、ATG5表达对食管癌细胞自噬及增殖过程的影响。方法:靶基因预测、双荧光素酶报告实验验证miR-30a对Beclin-1、ATG5的靶向调控作用。GEO、Human Protein Atlas和GEPIA在线分析TCGA数据库和GTEx项目中食管癌组织及癌旁组织miR-30a、Beclin-1、ATG5表达、患者预后生存期。免疫组化检测45例食管癌及癌旁组织标本Beclin-1、ATG5表达,并分析Beclin-1、ATG5表达与患者临床病理参数的关系。采用脂质体LipofectamineTM3000将靶向Beclin-1、ATG5的miR-30a-mimic转染至Eca-109细胞,体外常规培养并分为3组:未转染组(control组)、转染无义RNA的阴性对照组(mimic NC组)、转染miR-30a-mimic的干扰组(miR-30a组)。MTT和克隆形成实验测定细胞增殖活力;qRT-PCR检测miR-30a、Beclin-1、ATG5 mRNA表达;划痕实验检测细胞迁移能力;Transwell小室实验检测细胞迁移和侵袭;Western blot检测Beclin-1、ATG5、LC3Ⅱ、p62蛋白表达。透射电镜观察细胞自噬泡形成情况。结果:生信分析结果显示,食管癌组织中miR-30a表达高于正常组织,Beclin-1、ATG5表达低于正常组织,且预后生存期与miR-30a表达呈负相关,与Beclin-1、ATG5表达呈正相关(P<0.05)。与control组和mimic NC组相比,miR-30a组细胞增殖、迁移和侵袭能力、p62蛋白表达明显升高,Beclin-1、ATG5、LC3Ⅱ表达、细胞自噬活力明显降低(P<0.05)。而control组与mimic NC组上述指标差异无统计学意义(P>0.05)。结论:miR-30a靶向调控Beclin-1、ATG5表达;Beclin-1、ATG5在食管癌组织和细胞中表达均降低,具有肿瘤抑制基因作用,可能通过抑制癌细胞增殖、迁移和侵袭、促进自噬发挥抑癌作用,为通过靶向调控自噬途径治疗食管癌提供了新思路。     

敲低Wnt7a抑制卡介苗诱导的小鼠肺泡上皮细胞自噬

目的 探究无翅基因7a(Wnt7a)对牛结核分枝杆菌卡介苗(BCG)感染的肺泡上皮细胞自噬水平的调控作用。方法使用干扰Wnt7a慢病毒及BCG单独作用或共处理TC-1小鼠肺泡上皮细胞,设置小干涉RNA对照(si-NC)组、 si-NC联合BCG组、 Wnt7a小干涉RNA(si-Wnt7a)组和si-Wnt7a联合BCG组。Western blot法检测Wnt7a、微管相关蛋白1轻链3(LC3)、 P62及自噬相关基因5(ATG5)蛋白表达水平,利用免疫荧光细胞化学染色检测LC3的分布以确定细胞自噬情况。结果 与si-NC组相比,BCG感染的TC-1细胞Wnt7a、 LC3和ATG5蛋白表达均升高、 LC3绿色荧光斑点亮度与分布显著增加;与BCG组感染的TC-1细胞相比,si-Wnt7a联合BCG组Wnt7a、 LC3、 P62及ATG5蛋白表达均显著降低,LC3绿色荧光斑点亮度与分布显著降低。结论 敲低Wnt7a抑制BCG诱导的肺泡上皮细胞自噬。     

人脂肪来源间充质干细胞诱导人乳腺癌MCF-7细胞上皮间质转化

目的探讨人脂肪来源间充质干细胞(hAD-MSCs)在乳腺肿瘤微环境中对乳腺癌细胞的影响。方法采用Transwell体系间接共培养人乳腺癌细胞系(MCF-7)和hAD-MSCs,对共培养后的肿瘤细胞进行细胞形态,EMT相关标志以及肿瘤特性的检测。结果与对照组相比,共培养之后的MCF-7细胞发生上皮间质转化。与hAD-MSCs共培养能明显促进乳腺癌细胞的侵袭和迁移,但对细胞周期和增殖能力没有明确的影响。结论人脂肪来源间充质干细胞可诱导乳腺癌细胞上皮间质转化。     

Oestrogen‐induced epithelial–mesenchymal transition of endometrial epithelial cells contributes to the development of adenomyosis

Adenomyosis is an oestrogen‐dependent disease caused by a downward extension of the endometrium into the uterine myometrium. Epithelial–mesenchymal transition (EMT) endows cells with migratory and invasive properties and can be induced by oestrogen. We hypothesized that oestrogen‐induced EMT is critical in the pathogenesis of adenomyosis. We first investigated whether EMT occurred in adenomyotic lesions and whether it correlated with serum 17β‐oestradiol (E2) levels. Immunohistochemistry was performed on adenomyotic lesions and corresponding eutopic endometrium samples from women with adenomyosis. Endometria from women without endometrial disorders were used as a control. In the epithelial component of adenomyotic lesions, vimentin expression was up‐regulated and E‐cadherin expression was down‐regulated compared to the eutopic endometrium, suggesting that EMT occurs in adenomyosis. In adenomyosis, the serum E2 level was negatively correlated with E‐cadherin expression in the epithelial components of the eutopic endometrium and adenomyotic lesions, suggesting the involvement of oestrogen‐induced EMT in endometrial cells. In oestrogen receptor‐positive Ishikawa endometrial epithelial cells, oestrogen induced a morphological change to a fibroblast‐like phenotype, a shift from epithelial marker expression to mesenchymal marker expression, increased migration and invasion, and up‐regulation of the EMT regulator Slug. Raloxifene, a selective oestrogen receptor modulator, abrogated these effects. To determine the role of oestrogen‐induced EMT in the implantation of ectopic endometrium, we xenotransplanted eutopic endometrium or adenomyotic lesions from adenomyosis patients into ovariectomized SCID mice. The implantation of endometrium was oestrogen‐dependent and was suppressed by raloxifene. Collectively, these data highlight the crucial role of oestrogen‐induced EMT in the development of adenomyosis and suggest that raloxifene may be a potential therapeutic agent for adenomyosis patients. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

HAS-2沉默抑制大肠癌细胞上皮间质转化

目的本研究主要探讨HAS-2沉默前后对大肠癌细胞上皮间质转化(EMT)的影响。方法分别用Realtime PCR及Western blot方法检测HAS-2 siRNA转染前后E-cadherin、Vimentin、Slug、Snail mRNA、蛋白表达水平变化,用免疫荧光法观察HAS-2 siRNA转染前后E-Cadherin、Vimentin表达的变化。结果 E-Cadherin蛋白和mRNA在大肠癌细胞的表达水平在HAS-2 siRNA转染组比阴性对照组明显上调(0.05),而Vimentin蛋白和mRNA,Snail mRNA and Slug蛋白在大肠癌细胞的表达水平在HAS-2 siRNA转染组表达水平比阴性对照组明显下调(0.05)。结论 HAS-2沉默对大肠癌细胞EMT有抑制作用。     

Lithium stabilizes the polarized lens epithelial phenotype and inhibits proliferation, migration, and epithelial mesenchymal transition

Posterior capsule opacification (PCO) is a common complication of cataract surgery caused by epithelial mesenchymal transition (EMT) and aberrant lens cell growth. One path to prevention depends on maintaining the quiescent lens epithelial phenotype. Here we report that lithium chloride (LiCl) is a potent stabilizer of the lens epithelial phenotype. In lens epithelial explants (controls), at low cell density, cells readily depolarized, spread out, and proliferated. By contrast, in the presence of LiCl, cells did not spread out or exhibit migratory behaviour. Using concentrations of 1-30 mM LiCl we also showed that cell proliferation is inhibited in a dose-dependent manner. Confocal microscopy and immunohistochemistry for ZO-1 and E-cadherin showed that LiCl treatment maintained tight junctions at the apical margins of cells. Taken together with measurements of cell heights, this showed that the cells in LiCl-treated explants maintained the apical baso-lateral polarity and cobblestone-like packing that is characteristic of lens epithelial cells in vivo. Significantly, the effects of LiCl also extended to blocking the potent EMT/cataract-promoting effects of transforming growth factor beta (TGFbeta) on lens epithelial cells. In TGFbeta-treated explants, cells progressively dissociated from one another, taking on various elongated spindle shapes and strongly expressing alpha-smooth muscle actin (alpha-SMA). These features are characteristic of PCO. In both rat and human capsulorhexis explants, LiCl treatment effectively blocked the accumulation of alpha-SMA and maintained the cells in a polarized, adherent, cobblestone-packed monolayer. These findings highlight the feasibility of applying molecular strategies to stabilize lens epithelial cells and prevent aberrant differentiation and growth that leads to cataract.     

A biomaterial model of tumor stromal microenvironment promotes mesenchymal morphology but not epithelial to mesenchymal transition in epithelial cells

The stromal tissue surrounding most carcinomas is comprised of an extracellular matrix densely packed with collagen-I fibers, which are often highly aligned in metastatic disease. Here we developed an in vitro model to test the effect of an aligned fibrous environment on cancer cell morphology and behavior, independent of collagen ligand presentation. We grew cells on a biomimetic surface of aligned electrospun poly-l-lactic acid (PLLA) fibers and then examined the effect of this environment on growth rate, morphology, cytoskeletal organization, biochemical and genetic markers of epithelial to mesenchymal transition (EMT), cell surface adhesion, and cell migration. We grew a phenotypically normal breast epithelial cell line (MCF10A) and an invasive breast cancer cell line (MDA-MB-231) on three different substrates: typical flat culture surface (glass or plastic), flat PLLA (glass coated with PLLA) or electrospun PLLA fibers. Cells of both types adopted a more mesenchymal morphology when grown on PLLA fibers, and this effect was exaggerated in the more metastatic-like MDA-MB-231 cells. However, neither cell type underwent the changes in gene expression indicative of EMT despite the changes in cell shape, nor did they exhibit the decreased adhesive strength or increased migration typical of metastatic cells. These results suggest that changes in cell morphology alone do not promote a more mesenchymal phenotype and consequently that the aligned fibrous environment surrounding epithelial cancers may not promote EMT solely through topographical cues.     

阻断核心岩藻糖基化修饰抑制肾小管上皮细胞的间充质转化

目的: 探讨阻抑核心岩藻糖基化修饰对肾小管上皮细胞间充质转化(EMT)过程的影响。方法: 利用转化生长因子β₁(TGF-β₁)建立肾小管上皮HK-2细胞EMT的模型,应用RNAi技术沉默HK-2细胞的α-1,6-岩藻糖基转移酶(FUT8)基因表达,光镜下观察FUT8 基因沉默后细胞形态变化,免疫印迹及免疫细胞化学方法测定细胞表型标记物蛋白E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、α-平滑肌肌动蛋白(α-SMA)和成纤维细胞特异性蛋白-1(FSP-1)的表达变化,流式细胞仪测定细胞凋亡。 结果: TGF-β₁孵育48 h后,HK-2细胞失去原有的上皮细胞形态,呈现纤维细胞形态,纤维细胞表型标记蛋白α-SMA、FSP-1及N-cadherin表达明显升高,而上皮细胞表型标记蛋白E-cadherin表达明显下降,同时伴有 FUT8 基因表达上调,细胞凋亡增加,而提前转染FUT8 siRNA能明显减弱上述这些反应。结论: FUT8催化的核心岩藻糖基化修饰参与HK-2细胞的EMT过程;阻断核心岩藻糖基化修饰,能有效阻断肾小管上皮细胞的EMT过程。