Cpf1 和 Cas9核酸酶靶向EGFR-L858R突变的研究

表皮生长因子受体(EGFR)单核苷酸突变(2573TG,L858R)占所有EGFR突变的90%.使突变的EGFR失活对有此突变的病人非常有利.这里,应用双荧光报告分析的方法分析规律成簇间隔短回文重复(CRISPR)系统中Cpf1和Cas9在靶向EGFR-L858R突变的编辑效率.在EGFR-L858R突变位点的附近,有两个Cpf1前间区序列邻近基序(PAMs)——TTTN.并且,2573TG突变形成了一个Cas9的PAM——NGG.因此本文通过构建两条AsCpf1的gRNAs(gRNA1和gRNA2)和一条SpCas9的gRNA(gRNA3)在体外通过双荧光蛋白分析系统去评估SpCas9和AsCpf1特异性靶向等位基因的能力.结果证实了AsCpf1和SpCas9都能够特异性的编辑突变的EGFR(2573TG).     

壳寡糖纳米载体细胞内递送EGFR脱氧核酶的研究

设计靶向EGFR mRNA的脱氧核酶(EGFR DRz),以壳寡糖(COS)为材料,建立了一种有效的纳米基因细胞内传递体系,并研究其介导的靶向EGFR的脱氧核酶在Hela细胞内的生物学效应.流式结果表明COS-EGFR DRz复合体转染效率为88.7%,与脂质体转染试剂的89.7%相比无显著差异.半定量RT-PCR结果显示,经壳寡糖纳米载体递送的EGFR DRz能有效地靶向切割Hela细胞内的EGFR mRNA,使其表达下降.进一步的流式分析显示细胞被阻滞在G0～G1期,并且出现凋亡现象,其中COS组的凋亡率为19.3%,大于对照组脂质体的凋亡率13.0%.研究表明,COS较脂质体有相似的转染效率和更低的毒性,是一种潜在的、有效的脱氧核酶递送载体.     

The epidermal growth factor receptor family: Biology driving targeted therapeutics

The epidermal growth factor family of receptor tyrosine kinases (ErbBs) plays essential roles in regulating cell proliferation, survival, differentiation and migration. The ErbB receptors carry out both redundant and restricted functions in mammalian development and in the maintenance of tissues in the adult mammal. Loss of regulation of the ErbB receptors underlies many human diseases, most notably cancer. Our understanding of the function and complex regulation of these receptors has fueled the development of targeted therapeutic agents for human malignancies in the last 15 years. Here we review the biology of ErbB receptors, including their structure, signaling, regulation, and roles in development and disease, then briefly touch on their increasing roles as targets for cancer therapy.     

The role of P-glycoprotein/cellular prion protein interaction in multidrug-resistant breast cancer cells treated with paclitaxel

We previously reported that treatment with P-glycoprotein (P-gp) substrates promotes in vitro invasion in multidrug-resistant (MDR) breast cancer cells. This effect is initiated by the P-gp pump function and mediated by interaction of P-gp with some unknown component(s). However, the underlying mechanism(s) remains poorly understood. Here we confirm a novel physical interaction between P-gp and cellular prion protein (PrP^c). Blocking P-gp activity or depletion of PrP^c inhibited paclitaxel (P-gp substrate)- induced invasion. Paclitaxel further facilitated the formation of P-gp/PrP^c clusters residing in caveolar domains and promoted the association of P-gp with caveolin-1. Both caveolin-1 and the integrity of caveolae were required for the drug-induced invasion. In addition, the P-gp/PrP^c complex also played an important role in anti-apoptotic activity of MCF7/Adr cells.These data provide new insights into the mode by which MDR breast cancers evade cytotoxic attacks from P-gp substrates and also suggest a role for P-gp/ PrP^c interaction in this process. Received 4 September 2008; received after revision 16 November 2008; accepted 18 November 2008     

Effect of kinase-negativeEGFR gene on differentiation of embryonic germ cell line EG4

EG4 cells derived from primordial germ cells (PGCs) of 10.5 d post coitum 129/svJ mouse embryos can be used as a model system for in vitro differentiation study due to their pluripotential development ability. EG4 cell lines with stable expression of kinase-negative EGFR cDNA, designated EG4-EGFR^d, were generated by gene transfection. We found that: (ⅰ) EG4-EGFR^d cells share the similar morphology and growing character with wildtype cells that can maintain undifferentiated state in long term culture. (ⅱ) Treatment of EG4 cells with RA resulted in differentiation of adipocyte, while in mutant clones of EG4-EGFR^d, adipocytes were sparse or absent under the same condition, indicating the role of EGFR expressed during adipocyte development. (ⅲ) Histological analysis showed that predominant tissues in teratocarcinomas derived from EG4-EGFR^d cells and wildtype cells are different. A large amount of undifferentiated cells was present in those coming from mutant cell clones. In addition some cardiac and skeletal muscles are prominently differentiated cell types. EG4 wildtype cells produced multiple differentiated cell types of three primary germ layers such as cartilage, epithelia and neural tube. These studies suggested that EGFR-dependent differentiation was inhibited in kinase-negative EG4 cells.     

Transgenic and knock-out mice for deciphering the roles of EGFR ligands

Generation of genetically engineered mice with either gain-of-function or loss-of-function mutations is the most popular technique for determining gene functions and the interrelationship between molecules in vivo. These models have provided a wealth of information about the developmental and physiological roles of oncogenes and growth factors. To date, transgenic techniques have been used extensively to study the functions of the epidermal growth factor (EGF) family. This review highlights some of the major recent findings pertinent to the EGF receptor (EGFR) and its ligands with special reference to elucidating how EGF and its related growth factors work together to regulate reproduction, growth and development. Finally, future investigations on ligand-ligand communications, EGFR and its ligands in neural stem cell research, and the mechanisms of EGFR signaling and trafficking in cells are also suggested. Received 24 May 2002; received after revision 15 July 2002; accepted 16 July 2002     

Boron-based Drug Design for Cancer Therapy

1 Results Selective inhibition of protein tyrosine kinases is gaining importance as an effective therapeutic approach for the treatment of a wide range of human cancers.The epidermal growth factor receptor (EGFR) protein tyrosine kinase is one of the important kinases that play a fundamental role in cell growth signal pathways.We focused on the 4-anilinoquinazoline framework,which is observed in both compounds as a common structure.A boron atom has a vacant orbital and interconverts with ease between th...     

EGFR信号转导机制及靶向治疗

综述了EGFR基本结构特征及其介导细胞信号转导的机制,论述了基于EGFR靶向治疗的机理及研究现状。指出,EGFR是最早被发现并研究的RTK家族成员,其蛋白结构分成胞外域、跨膜区与胞内域三部分。EGFR介导细胞信号转导的核心机制是配体EGF与EGFR胞外域结合,通过变构作用与二聚化作用,使胞内域通过反式激活完成对受体末端酪氨酸残基的磷酸化,进而招募下游信号因子完成细胞信号转导过程。质膜结构与组成、EGFR跨膜区的组成及胞外域的有无、EGFR的内吞及泛素依赖性降解过程都调控EGFR细胞信号转导过程。阻断EGFR信号通路可抑制表皮肿瘤细胞成长。EGFR已经成为表皮肿瘤治疗的重要靶点。     

无菌大鼠深二度烫伤感染金黄色葡萄球菌机体炎性反应的研究

目的 为探究无菌大鼠深二度烫伤创面感染金黄色葡萄球菌后机体的炎性反应,试验应用无菌大鼠及SPF级大鼠分别建立深二度烫伤模型和烫伤后金黄色葡萄球菌感染模型,分析愈合过程、炎性反应及病理变化。方法20只6周龄雌性Wistar无菌大鼠,在超净工作台内采用恒温恒压烫伤仪94℃作用8 s造成大鼠深二度烫伤,随机分为两组,一组伤口感染浓度为1×1~08CFU/m L金黄色葡萄球菌0. 5 m L,一组不做处理,创面保持无菌状态; 20只6周龄雌性SPF级Wistar大鼠,在相同条件下烫伤,随机分为两组,一组伤口感染浓度为1×10~8CFU/m L金黄色葡萄球菌0. 5 m L,一组不做处理。分别观察4组大鼠的伤口结痂时间、脱痂时间和愈合时间,并在烫伤前(0 h),烫伤后24 h、48 h、3d、5 d、7d、10 d、14 d时取血清,检测肿瘤坏死因子-α(TNF-α)、白细胞介素-1α(IL-1α)和表皮生长因子受体(EGFR)的变化;烫伤后24 h、3d、7 d、10 d伤口处皮肤HE染色,观察病理变化。结果 感染金黄色葡萄球菌无菌大鼠伤口脱痂及愈合时间显著短于其他3组(P<0. 05);感染金黄色葡萄球菌SPF级大鼠,结痂、脱痂及愈合时间显著长于其他3组(P<0. 05);感染金黄色葡萄球菌SPF级烫伤大鼠组由于双重损伤使血清炎性因子TNF-α、IL-1α水平升高,且在烫伤后72 h显著高于其他3组(P<0. 05);无菌大鼠由于对烫伤及金黄色葡萄球菌刺激反应激烈,烫伤后血清TNF-α、IL-1α和EGFR水平迅速升高,且在烫伤24 h时显著高于2组SPF大鼠(P<0. 05),在烫伤48 h时血清EGFR水平显高于两组SPF大鼠(P<0. 05)。病理显示,感染金黄色葡萄球菌SPF烫伤大鼠伤口处新生血管及肉芽组织生成时间均长于其他3组;感染金黄色葡萄球菌无菌烫伤大鼠伤口处新生血管及肉芽组织生成时间均短于其他3组。结论 无菌大鼠由于其免疫系统处于休眠状态,受到烫伤和金黄色葡萄球菌的双重刺激,迅速激活其全身免疫系统,加快了伤口愈合; SPF烫伤大鼠感染金黄色葡萄球菌后产生的多种免疫逃避分子加重了机体的炎性反应,延缓了伤口愈合。     

治疗NSCLC的EGFR靶向性药物设计与筛选

计算了现有的治疗非小细胞肺癌(NSCLC)常见药物的相关参数,总结了以表皮生长因子受体(EGFR)酪氨酸激酶为靶标的药物分子的药效团特征.在Gefitinib的基础上进行了药物分子的组合化学设计,通过虚拟筛选得到了分子对接得分较高的结构分子,并对其进行了预测和模拟.结果表明:新设计筛选出的2种分子在药代动力学和毒性方面...