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1.
人脑源性神经营养因子cDNA在COS7细胞中的表达及活性…   总被引:1,自引:0,他引:1  
本文从质粒M13mp18-hBDNF中酶切回收入及源性神经营养因子(hBDNF)全长基因,构建真核表达载体pCMV4-hBDNF。利用脂质体的方法转染COS7细胞,对转染后的COS7细胞提取RNA进行狭缝杂交分析和免疫细胞化学反应,分别从转录及翻译水平上检测BDNF基因在COS7细胞中的表达。实验还证实在COS7细胞中表达的hBDF蛋白可分泌至胞外并可促进中脑黑质细胞的发育和生长,具有良好的生物学  相似文献   

2.
HCV NS5B基因片段克隆入BAC-TO-BAC^TM重组杆状病毒表达系统的pFASTHTc载体质粒,转化DH10BAC^TM感受态细菌获得重组的Bacmid质粒,将重组Bacmid质粒转染Sf细胞,获得的重组杆状病毒可表达目的蛋白。免疫印迹和体外活性检测表明,所表达蛋白为HCV NS5B蛋白,具有多聚酶活性。  相似文献   

3.
用酶组织化学和免疫组织化学双标技术,观察了正常SD大鼠基底前脑内侧隔核(MS)、斜角带垂直支(VDB)和水平支(HDB)中NOS阳性神经元的形态和分布及NOS与胆碱能神经元标志物ChAT、NGF受体(NGF-R)和AChE之间的共存关系。结果发现,MS、VDB和HDB的头端NOS阳性神经元较多、胞体较大、突起多,尾端NOS阳性神经元数目较少、胞体较小、突起少而短。NOS+ChAT双标神经元占NOS阳性神经元总数的90%,占ChAT阳性神经元总数的39%;NOS+NGF-R双标神经元占NOS阳性神经元总数的83%,占NGF-R阳性神经元总数的40%;NOS+AChE双标神经元占NOS阳性神经元总数的96%,占AChE阳性神经元总数的39%。这些结果为研究Alzheimer'sdisease病理过程中基底前脑隔区胆碱能神经元退变与NO的关系提供了形态学依据。  相似文献   

4.
hBMP—2cDNA在COS细胞和小鼠肌肉中的表达   总被引:4,自引:1,他引:3  
研究了骨形态发生蛋白BMP-2cDNA在COS细胞和小鼠肌肉中的表达的情况,从pSPS65BMP-2质粒中回收BMP-2cDNA,删除5端的非翻译序列,插入pSVL载体中,构建了含有BMP-2全长编码序列的重组表达质粒pSVLBMP-2将表达质粒导入COS-7细胞中,细胞RNA点杂交结果表明,转染BMP-2基因的细胞内BMP-2的mRNA水平明显升高,细胞培养上清的ELISA显示,转染BMP-2c  相似文献   

5.
Ms—SOD对CHO细胞电离辐射敏感性的影响   总被引:6,自引:1,他引:6  
近年来的研究发现,IL-1和TNF最重要的辐射防护因子,因IL-01和TNF都能造反性诱导Mn-SOD的高度表达,因此认为Mn-SOD可能有辐射防护作用。通过转染有义和反义Mn-SODcNDA小于CHO细胞,进一步说明了Mn-SOD在抗电离辐射损伤中的作用。  相似文献   

6.
低分子量硫酸葡聚糖对小鼠造血干细胞动员作用的研究   总被引:3,自引:0,他引:3  
给小鼠静脉注射低分子量(<10 ̄4u)硫酸葡聚糖(DS)15mg/kg后外周血中白细胞、单个核细胞(mononuclearcek,MNC)、CFU-GM、BFU-E和CFU-Mix产率等指标出现时相性变化。给药后1h开始升高,2h达到高峰、分别为药前值的2.2、2.6、3.8、4.4和3.0倍,7h时趋向正常。给药后2h上述各类细胞在外周血中的含量随着DS的剂量增加而增加。白细胞、MNC计数在DS180mg/kg时达到峰值,均为对照组的4倍。240mg/k8时未见明显增加。不同剂量DS对各系祖细胞均有不同程度的动员作用,DS剂量15-30mg/kg效果最好,每升血中CFU-GM、BFU-E、CFU-Mix的数量分别相当于对照组的5.0、11.9和8.8倍。其峰值出现时间与白细胞、MNC不同,表明DS对不同类型细胞的作用机制也不尽一致。经口给小鼠投以DS240和48omg/kg后,未见外周血中白细胞、MNC计数有显著性升高,提示对造血干细胞没有动员作用。  相似文献   

7.
大鼠卵巢绒毛膜促性腺激素受体在CHO中的表达和扩增   总被引:1,自引:0,他引:1  
本文报道了利用二氢叶酸还原酶放大系统将大鼠LH/hCG受体(记为F-hCGR)及其胞外肽段(记为T-hCGR)在中国苍鼠卵巢细胞(CHO)中的表达。SDS-PAGE分析表明,F-hCGR为一条蛋白质带,其表观分子量为92kd,而T-hCGR为35kd和37kd两条带。表达受体对其配基hCG表现出高的亲合力,F-hCGR的解离常数为7×10-9mol/L,T-hCGR为6.4×10-9mol/L。表达F-hCGR的转染CHO细胞可结合125I-hCG,而表达T-hCGB者不结合125I-hCG。这提示F-hCGR主要存在于细胞质膜表面上。表达F-hCGR的转染CHO细胞能刺激。cAMP的形成,而表达T-hCGR者不能刺激cAMP形成。免疫荧光定位结果表明,T-hCGR主要分布于质膜的细胞质侧以及胞内其他一些细胞器膜上。用免疫亲和层析可以得到纯化的T-hCGR。  相似文献   

8.
为了构建含人凝血因子Ⅷ基因的重组腺病毒载体系统,首先构建了含FⅧ(B结构域缺失型)的载体质粒pAd-hFⅧ和插入内含子结构的pAdI-hFⅧ,经脂质体介导,证明两质粒的目的基因均能在COS-7细胞中表达有凝血活性的FⅧ因子,其中经纯化的载体质粒pAd-hFⅧ与pBHG10经脂质体介导,共转染至293包装细胞,经同源重组和E1蛋白反式互补,形成E1/E3缺失型重组腺病毒载体颗粒Ad-hFⅧ.PCR检则到病理293培养液上清的病毒DNA中具有目的基因扩增片段,证明病毒DNA含有FⅧ基因.经扩增、纯化、滴度测定,用于感染离体细胞COS-7、A549、L929、293.证实该病毒能在上述细胞中高效转移和表达目的基因,其表达量具有一定范围内的剂量依赖性.COS-7细胞中,MOI>1000时,有细胞毒效应,表达量反而下降.经耳缘静脉注射Ad-hFⅧ至家兔后,1~4周能测到一定水平的FⅧ蛋白,1周内升至最高峰.免疫组化研究表明感染的离体细胞和家兔肝等组织均有FⅧ表达,其中肝脏表达最高.为甲型血友病的基因治疗开辟了新途径  相似文献   

9.
研究了骨形态发生蛋白BMP-2cDNA在COS细胞和小鼠肌肉中的表达的情况,从pSPS65BMP-2质粒中回收BMP-2cDNA,删除5'端的非翻译序列,插入pSVL载体中,构建了含有BMP-2全长编码序列的重组表达质粒pSVLBMP-2。将表达质粒导入COS-7细胞中,细胞RNA点杂交结果表明,转染BMP-2基因的细胞内BMP-2的mRNA水平明显升高;细胞培养上清的ELISA显示,转染BMP-2cDNA后,细胞分泌产生的BMP-2显著增加。小鼠实验发现,在肌肉内用注射法导入BMP-2重组质粒后,局部组织内BMP-2的mRNA转录水平也明显提高。  相似文献   

10.
福尔马林固定云南鲴的DNA提取及其细胞色素b基因序列分析   总被引:12,自引:0,他引:12  
消武汉  吴春花 《动物学研究》1997,18(3):24-24,252,258,8
福尔马林固定云南鲴的DNA提取及其细胞色素b基因序列分析DNAEXTRACTEDFROMFORMALIN-FIXEDXenocyprisyunnanensisANDSEQUENCEANALYSISOFITSCYTOCHROMEBGENE关键词福尔马林...  相似文献   

11.
A super lens system is proposed to achieve subdiffraction limit demagnification imaging. The super lens system consists of a hyperlens with planar input and output surfaces, a metal superlens, and a plasmonic reflector. By employing the hyperlens to transform evanescent waves into propagating waves and employing the metal superlens and the plasmonic reflector to amplify evanescent waves, the super lens system can produce a subdiffraction limit image with relatively high electric field intensity. The reduction factor of the super lens system depends on the geometric parameters of the hyperlens. Simulation results show that an image with a half-pitch resolution of about one tenth the operating wavelength and a reduction factor of about 2.2 can be produced by the super lens system. The proposed super lens system has potential applications in nanolithography.  相似文献   

12.
Correlative light and electron microscopy (CLEM) has become a powerful tool in life sciences. Particularly cryo‐CLEM, the combination of fluorescence cryo‐microscopy (cryo‐FM) permitting for non‐invasive specific multi‐colour labelling, with electron cryo‐microscopy (cryo‐EM) providing the undisturbed structural context at a resolution down to the Ångstrom range, has enabled a broad range of new biological applications. Imaging rare structures or events in crowded environments, such as inside a cell, requires specific fluorescence‐based information for guiding cryo‐EM data acquisition and/or to verify the identity of the structure of interest. Furthermore, cryo‐CLEM can provide information about the arrangement of specific proteins in the wider structural context of their native nano‐environment. However, a major obstacle of cryo‐CLEM currently hindering many biological applications is the large resolution gap between cryo‐FM (typically in the range of ~400 nm) and cryo‐EM (single nanometre to the Ångstrom range). Very recently, first proof of concept experiments demonstrated the feasibility of super‐resolution cryo‐FM imaging and the correlation with cryo‐EM. This opened the door towards super‐resolution cryo‐CLEM, and thus towards direct correlation of structural details from both imaging modalities.  相似文献   

13.
We propose a near-infrared super resolution near field imaging system with an array of metallic nanoshell particle chain. The imaging array can plasmonically transfer the near field components of dipole sources and the super resolution images can be reconstructed in the output plane. By decreasing the metallic nanoshell’s thickness of the fixed size nanoparticle, the plasmon resonance wavelength of the isolate nanoshell particle is red-shifted to the near-infrared region. The operation wavelength of the imaging array is correspondingly red-shifted to the near-infrared region. In this paper, we study the incoherent and coherent super resolution imaging. The field intensity distributions at the different planes of imaging process are calculated using the finite element method. The simulation results demonstrate that the array has super resolution imaging capability at near-infrared wavelengths in the incoherent and coherent manners. The results also show that the image formation highly depends on the source coherence. In the same structural parameters, the reconstructed images under the illumination of incoherent light source reach to the higher image quality and spatial resolution than the images under the illumination of coherent light source of in phase. By reasonably designing parameters of the imaging array, the approximate spatial resolutions of λ/13 in incoherent case and λ/10 in coherent case are obtained at the near-infrared wavelength of 764 nm. Furthermore, the image–array distance and the chains’ spacing also affect the image reconstruction.  相似文献   

14.
Either modulated illumination or temporal fluctuation analysis can assist super‐resolution techniques in overcoming the diffraction limit of conventional optical microscopy. As they are not contradictory to each other, an effective combination of spatial and temporal super‐resolution mechanisms would further improve the resolution of fluorescent images. Here, a super‐resolution imaging method called fluctuation‐enhanced Airyscan technology (FEAST) is proposed, which achieves ~40 nm lateral imaging resolution and is useful for a range of fluorescent proteins and organic dyes. It was demonstrated not only to obtain different subcellular super‐resolution images, but also to improve the accuracy of counting the average human epidermal growth factor receptor 2 (HER2) copy number for diagnosis in breast cancer. Furthermore, the combination of FEAST and sample expansion microscopy (Ex‐FEAST) improves the lateral resolution to ~26 nm.  相似文献   

15.
Localization‐based super‐resolution microscopy relies on the detection of individual molecules cycling between fluorescent and non‐fluorescent states. These transitions are commonly regulated by high‐intensity illumination, imposing constrains to imaging hardware and producing sample photodamage. Here, we propose single‐molecule self‐quenching as a mechanism to generate spontaneous photoswitching. To demonstrate this principle, we developed a new class of DNA‐based open‐source super‐resolution probes named super‐beacons, with photoswitching kinetics that can be tuned structurally, thermally and chemically. The potential of these probes for live‐cell compatible super‐resolution microscopy without high‐illumination or toxic imaging buffers is revealed by imaging interferon inducible transmembrane proteins (IFITMs) at sub‐100 nm resolutions.  相似文献   

16.
Determining the organisation of key molecules on the surface of live cells in two dimensions and how this changes during biological processes, such as signaling, is a major challenge in cell biology and requires methods with nanoscale resolution. Recent advances in fluorescence imaging both at the diffraction limit tracking single molecules and exploiting super resolution imaging have now reached a stage where they can provide fundamentally new insights. Complementary developments in scanning ion conductance microscopy also allow the cell surface to be imaged with nanoscale resolution. The challenge now is to combine the information obtained using these different methods and on different cells to obtain a coherent view of the cell surface. In the future this needs to be driven by interdisciplinary research between physical scientists and biologists.  相似文献   

17.
Y. Li  B. Sixou  F. Peyrin 《IRBM》2021,42(2):120-133
Super resolution problems are widely discussed in medical imaging. Spatial resolution of medical images are not sufficient due to the constraints such as image acquisition time, low irradiation dose or hardware limits. To address these problems, different super resolution methods have been proposed, such as optimization or learning-based approaches. Recently, deep learning methods become a thriving technology and are developing at an exponential speed. We think it is necessary to write a review to present the current situation of deep learning in medical imaging super resolution. In this paper, we first briefly introduce deep learning methods, then present a number of important deep learning approaches to solve super resolution problems, different architectures as well as up-sampling operations will be introduced. Afterwards, we focus on the applications of deep learning methods in medical imaging super resolution problems, the challenges to overcome will be presented as well.  相似文献   

18.
Expansion microscopy is a recently introduced imaging technique that achieves super‐resolution through physically expanding the specimen by ~4×, after embedding into a swellable gel. The resolution attained is, correspondingly, approximately fourfold better than the diffraction limit, or ~70 nm. This is a major improvement over conventional microscopy, but still lags behind modern STED or STORM setups, whose resolution can reach 20–30 nm. We addressed this issue here by introducing an improved gel recipe that enables an expansion factor of ~10× in each dimension, which corresponds to an expansion of the sample volume by more than 1,000‐fold. Our protocol, which we termed X10 microscopy, achieves a resolution of 25–30 nm on conventional epifluorescence microscopes. X10 provides multi‐color images similar or even superior to those produced with more challenging methods, such as STED, STORM, and iterative expansion microscopy (iExM). X10 is therefore the cheapest and easiest option for high‐quality super‐resolution imaging currently available. X10 should be usable in any laboratory, irrespective of the machinery owned or of the technical knowledge.  相似文献   

19.
膨胀显微成像技术(expansion microscopy,ExM)是一种新型超分辨成像技术。该技术借助可膨胀水凝胶均匀地物理放大生物样本,在常规光学成像条件下实现超分辨成像。ExM适用于细胞、组织切片等多种类型生物样本。蛋白质、核酸、脂质等生物大分子均可借助ExM进行超分辨成像。ExM可与共聚焦显微镜、光片显微镜、超高分辨显微镜联合使用,进一步提高成像分辨率。近年来,多种从基础ExM拓展而来的衍生技术进一步促进了该技术的实际应用。本文综述了ExM及其衍生技术的基本原理、ExM与不同成像技术联用的研究进展及ExM在不同类型生物样本中的应用进展,并对ExM技术的发展前景做出展望。  相似文献   

20.
Various computational super‐resolution methods are available based on the analysis of fluorescence fluctuation behind acquired frames. However, dilemmas often exist in the balance of fluorophore characteristics, computation cost, and achievable resolution. Here we present an approach that uses a super‐resolution radial fluctuations (SRRF) image to guide the Bayesian analysis of fluorophore blinking and bleaching (3B) events, allowing greatly accelerated localization of overlapping fluorophores with high accuracy. This radial fluctuation Bayesian analysis (RFBA) approach is also extended to three dimensions for the first time and combined with light‐sheet fluorescence microscopy, to achieve super‐resolution volumetric imaging of thick samples densely labeled with common fluorophores. For example, a 700‐nm thin Bessel plane illumination is developed to optically section the Drosophila brain, providing a high‐contrast 3D image of rhythmic neurons. RFBA analyzes 30 serial volumes to reconstruct a super‐resolved 3D image at 4‐times higher resolutions (~70 and 170 nm), and precisely resolve the axon terminals. The computation is over 2‐orders faster than conventional 3B analysis microscopy. The capability of RFBA is also verified through dual‐color imaging of cell nucleus in live Drosophila brain. The spatial co‐localization patterns of the nuclear envelope and DNA in a neuron deep inside the brain can be precisely extracted by our approach.  相似文献   

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