EphB4反义寡核苷酸对人脑胶质瘤细胞增殖、凋亡及迁移的作用

目的观察EphB4反义寡核苷酸(ASODN)对人脑胶质瘤细胞增殖、凋亡及迁移的影响,探讨EphB4的生物学功能。方法人工合成EphB4 ASODN,脂质体转染U87细胞。分别应用RT-PCR以及免疫组织化学染色方法检测EphB4 mRNA和蛋白质,评估阻抑效应;并应用MTT法和流式细胞术检测细胞的增殖和凋亡;采用Boyden侵袭小室法检测细胞迁移和侵袭力。结果(1)由脂质体转染的EphB4 ASODN对U87细胞EphB4 mRNA和蛋白质表达具有不同程度的抑制作用,且呈现一定的时间-剂量依赖性,600nmol/LASODN作用于U87细胞72h,EphB4 mRNA相对表达量为0.30±0.05,EphB4蛋白表达水平亦明显减弱。(2)各实验组EphB4 ASODN对U87细胞增殖均具有抑制作用,呈明显时间-剂量依赖性;以600nmol/LASODN作用72h,U87细胞生长抑制率达到68.70%,而对照组则无明显抑制作用。(3)EphB4 ASODN诱导U87细胞凋亡呈剂量依赖性。(4)EphB4 ASODN组U87细胞的跨膜数为17.82±2.35,与空白对照组及NSODN组比较差异具有统计学意义(P<0.01)。结论EphB4在人脑胶质瘤细胞增殖、凋亡、迁移和侵袭过程中发挥重要作用,可能成为脑胶质瘤基因治疗的一个新靶点。     

Development of patient derived organoids for cancer drug screening applications

Cancer is a disease characterised by abnormal cell growth that can invade or spread to other regions of the body. Organoids are three-dimensional ex vivo tissue cultures made from embryonic stem cells, induced pluripotent stem cells, progenitor cells or tissue that serve as a physiological model for cancer research. These are designed to recapitulate the in vivo properties of tumours. Importantly, effective recapitulation of the structure of tissues and function is believed to predict patient response, allowing for the creation of personalised therapy in a timely manner that may be used in the clinic. This Review discusses the pre-clinical model and different types of human organoids as models for the development of high throughput drug screening and also aims to highlight how organoids are shaping the future of cancer research.     

Preparation of a novel radiotracer targeting the EphB4 receptor via radiofluorination using spiro azetidinium salts as precursor

The visualization of Eph receptors, which are overexpressed in various tumor entities, using selective small molecule Eph inhibitors by means of positron emission tomography is a promising approach for tumor imaging. N‐(Pyrimidinyl)indazolamines represent a class of compounds, which are known to have high affinity especially for the EphB4 receptor. Radiofluorination of these compounds could provide a highly specific imaging agent and was investigated using a classical nucleophilic introduction of [¹⁸F]fluoride as well as a less common nucleophilic ring‐opening reaction of azetidinium salts. In the past, radiofluorinations using azetidinium precursors were demonstrated to result in high radiochemical yields in short periods. For this purpose, an azetidinium precursor based on the N‐(pyrimidinyl)indazolamine lead compound was developed, and radiofluorination was successfully accomplished. The respective [¹⁸F]radiotracer was quickly prepared with high radiochemical purity >97% and in a radiochemical yield of 34%.     

Presenilin mediates neuroprotective functions of ephrinB and brain-derived neurotrophic factor and regulates ligand-induced internalization and metabolism of EphB2 and TrkB receptors

Activation of EphB receptors by ephrinB (efnB) ligands on neuronal cell surface regulates important functions, including neurite outgrowth, axonal guidance, and synaptic plasticity. Here, we show that efnB rescues primary cortical neuronal cultures from necrotic cell death induced by glutamate excitotoxicity and that this function depends on EphB receptors. Importantly, the neuroprotective function of the efnB/EphB system depends on presenilin 1 (PS1), a protein that plays crucial roles in Alzheimer's disease (AD) neurodegeneration. Furthermore, absence of one PS1 allele results in significantly decreased neuroprotection, indicating that both PS1 alleles are necessary for full expression of the neuroprotective activity of the efnB/EphB system. We also show that the ability of brain-derived neurotrophic factor (BDNF) to protect neuronal cultures from glutamate-induced cell death depends on PS1. Neuroprotective functions of both efnB and BDNF, however, were independent of γ-secretase activity. Absence of PS1 decreases cell surface expression of neuronal TrkB and EphB2 without affecting total cellular levels of the receptors. Furthermore, PS1-knockout neurons show defective ligand-dependent internalization and decreased ligand-induced degradation of TrkB and Eph receptors. Our data show that PS1 mediates the neuroprotective activities of efnB and BDNF against excitotoxicity and regulates surface expression and ligand-induced metabolism of their cognate receptors. Together, our observations indicate that PS1 promotes neuronal survival by regulating neuroprotective functions of ligand-receptor systems.     

Molecular mechanisms underlying the effects of acupuncture on neuropathic pain

Acupuncture has been used to treat neuropathic pain for a long time, but its mechanisms of action remain unknown. In this study, we observed the effects of electroacupuncture and manual acu-puncture on neuropathic pain and on ephrin-B/EphB signaling in rats models of chronic constriction injury-induced neuropathic pain. The results showed that manual acupuncture and elec-puncture significantly reduced mechanical hypersensitivity following chronic constriction injury, es-pecially electroacupuncture treatment. Real-time PCR results revealed that ephrin-B1/B3 and EphB1/B2 mRNA expression levels were significantly increased in the spinal dorsal horns of chronic constriction injury rats. Electroacupuncture and manual acupuncture suppressed the high sion of ephrin-B1 mRNA, and elevated EphB3/B4 mRNA expression. Electroacupuncture signifi-cantly enhanced the mRNA expression of ephrin-B3 and EphB3/B6 in the dorsal horns of neuro-pathic pain rats. Western blot results revealed that electroacupuncture in particular, and manual acupuncture, significantly up-regulated ephrin-B3 protein levels in rat spinal dorsal horns. The re-sults of this study suggest that acupuncture could activate ephrin-B/EphB signaling in neuropathic pain rats and improve neurological function.     

Decreased expression of receptor tyrosine kinase of EphB1 protein in renal cell carcinomas

Receptors tyrosine kinase of Eph superfamily plays an important role in human cancers. We previously found that EphB1 subtype is down-regulated in gastric cancer, colorectal cancer and ovary serous carcinoma. Fore the more, the decreased expression of EphB1 is related to invasion and metastasis in cancers. Although EphB1 has been revealed as an important receptor in cancers, our understanding of its roles in renal cell carcinoma (RCC) is limited. In the present study, using specific anit-EphB1 polyclonal antibody and immunohistochemistry, we evaluated EphB1 protein expression levels in RCC specimens surgically resected from 82 patients (including 62 conventional clear-cell RCC, 10 papillary, and 10 chromophobic RCC cases). We found EphB1 protein is positively expressed in the epithelium of renal tubules. Decreased expression of EphB1 was found in all RCC carcinomas compared with expression in the normal epithelium of renal tubules. EphB1 protein moderately expressed in chromophobic RCC, weakly expressed in clear-cell RCC and negatively expressed in papillary RCC. Our results indicate that EphB1 may be involved in carcinogenesis of RCC, the molecular mechanisms of down-regulation of EphB1 including genetic and epigenetic alterations and the dedicated roles of EphB1 in occurrence and progress of RCC need to be explicated in next step.     

Complementary and layered expression of Ephs and ephrins in developing mouse inner ear

The distributions of the Eph-class receptors EphA4 and EphB1, and their ligands ephrin-A2, ephrin-B1, and ephrin-B2, were analysed by immunostaining in the mouse inner ear. Complementary patterns of EphA4 and its potential ligand ephrin-A2 were found, with ephrin-A2 in many of the structures lining the cochlear duct and within the cochlear nerve cells, and EphA4 in the deeper structures underlying the cochlear duct and in the cells lining the nerve pathway. EphB1 and its potential ligands ephrin-B1 and ephrin-B2 showed a segregated layered expression in the lateral wall of the cochlear duct (the external sulcus), which together with EphA4 expressed in the area, form a four-layered structure with an alternating pattern of receptors and ligands in the different layers. This arrangement gives the potential for different bidirectional Eph-mediated interactions between each of the layers. The results suggest that the Eph system in the cochlea may have a role in maintaining cell segregation during phases of cochlear development.     

EphB2在先天性肛门直肠畸形直肠末端表达的研究

目的 研究酪氨酸蛋白激酶受体--EphB2在先天性肛门直肠畸形直肠末端组织中的mRNA表达和蛋白表达的水平,探讨EphB2与先天性肛门直肠畸形发生的关系.方法 RT-PCR方法和Western蛋白印迹方法,检测31例不同类型先天性肛门直肠畸形直肠后壁末端、5例后天瘘及8例正常直肠后壁末端EphB2在蛋白水平和mRNA水平的表达情况,应用单因素方差分析比较正常组和畸形组、不同类型的畸形组之间EphB2表达水平的差异.结果 ①先天性肛门直肠畸形末端肠壁EphB2基因mRNA相对表达量明显低于正常对照组(0.99±0.11,P=0.00).不同类型的畸形组之间,高位组(0.43±0.07)和中位组(0.47±0.10)之间差异无统计学意义(P=0.33),但二者明显低于低位组(0.64±0.06,P=0.00).后天瘘组(0.97±0.09)与正常对照组比较差异没有统计学意义(P=0.63);②Western蛋白印迹结果显示,先天性肛门直肠畸形高位组(0.21±0.05)、中位组(0.24±0.04)和低位组蛋白表达水平均明显低于后天瘘组(0.51±0.05)和正常对照组(0.52±0.03,P=0.00).不同类型的畸形之间,高位组和中位组之间无明显差异(P=0.11),但二者明显低于低位组(P＜0.01).后天瘘组与正常对照组比较差异没有统计学意义(P=0.67).结论 EphB2在先天性肛门直肠畸形直肠末端低表达.EphB2的表达和肛门直肠畸形的发生有关,EphB2在先天性肛门直肠畸形的发生中可能具有重要作用.     

EphB2基因多态性与先天性肛门直肠畸形的相关性研究

目的 探讨EphB2受体基因第6外显子的-1395A/G多态性在辽宁地区汉族人群中的分布及其与先天性肛门直肠畸形(CAM)的关系.方法 采用PCR-RFLP方法,对65例CAM患儿和115名健康儿童EphB2基因-1395A/G多态位点进行基因型检测,用SHEsis在线统计软件分析等位基因频率、基因型频率及其组间差异.结果 EphB2受体基因第6外显子的-1395A/G多态A等位基因频率及AA基因型频率在CAM组(85.4%、75.4%)与正常对照组(17.0%、4.3%)间差异有显著统计学意义(P＜0.01).结论 EphB2受体基因第6外显子的-1395A/G多态与CAM存在相关性.     

EphB4受体及其配体EphrinB2 mRNA在子宫内膜癌组织中的表达及意义

目的:探讨红细胞生成素诱导的肝细胞受体EphB4及其配体EphrinB2mRNA在子宫内膜癌组织中的表达及意义。方法:采用逆转录PCR(RT-PCR)法检测33例子宫内膜癌和10例正常子宫内膜组织中EphB4、EphrinB2mRNA的表达率及相对含量,并分析二者与子宫内膜癌临床病理特征的关系。结果:①EphB4mRNA在子宫内膜癌组的表达率和相对含量均高于正常子宫内膜组(P<0.05,P<0.05)。EphrinB2mRNA在子宫内膜癌组的表达率和相对含量也均高于正常子宫内膜组(P<0.05,P<0.01)。②EphB4、EphrinB2mRNA的表达与子宫内膜癌手术病理分期、组织学分级有密切关系(P<0.05)。二者表达与子宫内膜癌不同组织学类型、肌层浸润程度以及有无淋巴结转移无关(P>0.05)。结论:EphB4和EphrinB2的异常表达与子宫内膜癌发生、进展以及恶性程度有关,二者表达趋势呈现一致性,检测EphB4/EphrinB2有助于判断子宫内膜癌预后。