活体生物发光成像技术及其在病毒感染研究中的应用

生物发光是动物活体光学成像技术之一,因其反应灵敏、操作简单、数据精确,而被广泛地应用于生命科学研究多个领域,观测活体动物体内病毒复制、肿瘤生长等生命过程.生物发光技术采用荧光素酶基因标记细胞或病毒,与外源注射的底物荧光素发生反应,在冷CCD成像系统下显像并进行数据记录、分析.本文简要介绍活体生物发光成像这一新技术的原理...     

生物光学成像技术在干细胞应用中的研究进展

干细胞目前已应用在多种疾病的治疗中,前景十分广阔,但干细胞存活、分布和迁移等具体机制仍未明确,需要通过长期有效、无副作用的干细胞示踪技术进一步研究.生物光学成像(OI)技术与干细胞相结合,操作简单、成像直观,并有高灵敏度和高特异性,可以实时、无创监测干细胞在动物活体内的生物学活动.本文就OI示踪技术的原理及其在干细胞应...     

动物活体成像生物发光稳定细胞株的构建

目的:构建组成型表达萤光素酶基因的载体,建立稳定高表达萤光素酶的MCF-7乳腺癌细胞株,检测其对细胞增殖的影响及在传代后的表达效果.方法:以载体pGIA.20为模板,PCR扩增萤火虫萤光素酶基因,将其克隆到载体pIRESpuro2上,将获得的pIRESpuro2-Luc经酶切和测序验证后,转染293T、MCF-7及ZR...     

活体动物体内光学成像技术的研究进展及其应用

活体动物体内光学成像是利用基因改构进行内源性成像试剂或外源性成像试剂标记细胞、蛋白或DNA，从而非侵入性地报告小动物体内的特定生物学事件的技术。活体成像可以直观灵敏地监测基因的表达模式、标记和示踪细胞、探讨蛋白间的相互作用，因而这一技术被广泛地用于分析基因的表达模式、评价基因治疗效果、评估肿瘤的发生和转移、监测移植器官等。简要综述了现有活体动物体内光学成像技术的基本原理、技术进展和相关应用。     

在体生物发光成像技术在生物医学中的应用

在体生物发光成像技术采用萤光素酶基因标记细胞及DNA,利用灵敏的光学检测仪器,实时直观地监测活体小动物体内感染性疾病的发生发展、肿瘤的生长及转移及引发的免疫反应、特定基因的表达等诸多生物过程。通过对同一组实验对象在不同时间点进行记录,跟踪同一观察目标(标记细胞、病原微生物或基因)的移动及变化,与传统的动物实验方法相比,在体生物发光成像得到的数据具有更高的可信度。近年来因其灵敏度较高、不涉及放射性物质、所得结果直观等优势,该技术已普遍应用于生物医学、药物开发等研究领域。     

活体动物体内光学成像技术的研究进展

生物发光和荧光成像作为近年来新兴的活体动物体内光学成像技术,以其操作简便及直观性成为研究小动物活体成像的一种理想方法,在生命科学研究中得以不断发展。利用这种成像技术,可以直接实时观察标记的基因及细胞在活体动物体内的活动及反应。利用光学标记的转基因动物模型可以研究疾病的发生发展过程,进行药物研究及筛选等。本文综述了现有活体动物体内光学成像技术的原理、应用领域及发展前景,比较了生物发光与几种荧光技术的不同特点和应用。     

小动物活体成像技术的应用

小动物活体成像技术在国内外得到越来越多的普及应用,极大地促进了生命科学特别是肿瘤研究的发展。本文就小动物活体成像技术的原理、标记方法和实际应用做简单介绍。     

鼠伤寒沙门菌动物模型活体成像技术的研究进展

鼠伤寒沙门菌的体内实验有利于开展食物中毒、胃肠炎、伤寒热等肠道传染病的防治。由于在活体内检测鼠伤寒沙门菌的动态变化存在瓶颈,使细菌致病机制的研究、疫苗及药物研发滞后。近年来应用小动物成像技术在活体中追踪转化了荧光素酶基因的鼠伤寒沙门菌越来越受到人们关注,综述该技术的应用现状及缺憾之处。     

单细胞技术在干细胞研究中的应用

干细胞是具有自我更新和分化潜能的异质性细胞群体。基于细胞群体水平的干细胞研究不能满足深入认识干细胞生物学本质及实际应用的需要。近年来,单细胞相关技术不断发展和成熟,并正在干细胞基础研究及其相关领域中获得迅速应用。该文以造血干细胞为主要例举,就实验研究中常用的单细胞分离、单细胞克隆分析、单细胞移植、单细胞实时定量PCR及单细胞测序等技术原理及其应用进行综述。     

荧光成像技术及其在生命科学中的应用

近年来,荧光成像技术发展迅速,其成像系统通常为目前最先进的分析检测仪器之一的激光共聚焦显微镜,荧光探针是荧光成像技术的核心之一。作为新兴光学成像技术,荧光成像技术在生命科学领域中应用广泛,可用于蛋白质及金属离子检测,肿瘤疾病的诊断,并为药物新剂型的研究提供了新思路。     

利用非侵入性光学成像技术监测在体肿瘤的生长

利用电穿孔技术将gfp表达质粒转染于SP2／0细胞,通过G418筛选获得稳定表达GFP的小鼠骨髓瘤细胞株SP2／0—GFP细胞。将稳定的转化细胞移植于同系BALB／c小鼠后腿、耳部皮下及腹腔成瘤,每隔一天对小鼠肿瘤成像,跟踪肿瘤的生长过程。结果表明,探测到的GFP荧光强度和发光范围与肿瘤的生长或消亡相关,从而证明可以利用GFP作为肿瘤细胞的标记,结合光学成像技术,对肿瘤生长过程实时追踪。     

间充质干细胞的细胞成像技术比较与应用

间充质干细胞是一类具有强大增殖、多向分化潜能和免疫调节能力的多功能细胞,研究显示间充质干细胞移植可能治疗多种难治性疾病,例如帕金森病、脊髓损伤以及肿瘤等。但是,人们对移植后的细胞在宿主内的存活、分布、增殖、分化、免疫排斥反应以及成瘤特性等问题尚不清楚,所以许多疾病经过细胞移植治疗后的进展及转归情况仍难以获得确切的科学证据。而细胞成像技术(包括放射性核素成像、超声成像、磁共振成像以及光学成像)可以在体外或者体内实现对间充质干细胞实时、无创的示踪,在以间充质干细胞为研究基础的细胞移植治疗和细胞组织再生的医学领域里有着巨大的应用潜力。该文综述近十年来细胞成像技术应用于示踪间充质干细胞移植疗法的研究进展,旨在比较当下多种热门细胞成像技术的优劣,进而找寻更合适的干细胞示踪策略,为干细胞移植治疗的基础和临床研究提供进一步的理论证据支持和研究思路。     

The cellular form of the prion protein guides the differentiation of human embryonic stem cells into neuron-, oligodendrocyte-, and astrocyte-committed lineages

Prion protein, PrP^C, is a glycoprotein that is expressed on the cell surface beginning with the early stages of embryonic stem cell differentiation. Previously, we showed that ectopic expression of PrP^C in human embryonic stem cells (hESCs) triggered differentiation toward endodermal, mesodermal, and ectodermal lineages, whereas silencing of PrP^C suppressed differentiation toward ectodermal but not endodermal or mesodermal lineages. Considering that PrP^C might be involved in controlling the balance between cells of different lineages, the current study was designed to test whether PrP^C controls differentiation of hESCs into cells of neuron-, oligodendrocyte-, and astrocyte-committed lineages. PrP^C was silenced in hESCs cultured under three sets of conditions that were previously shown to induce hESCs differentiation into predominantly neuron-, oligodendrocyte-, and astrocyte-committed lineages. We found that silencing of PrP^C suppressed differentiation toward all three lineages. Similar results were observed in all three protocols, arguing that the effect of PrP^C was independent of differentiation conditions employed. Moreover, switching PrP^C expression during a differentiation time course revealed that silencing PrP^C expression during the very initial stage that corresponds to embryonic bodies has a more significant impact than silencing at later stages of differentiation. The current work illustrates that PrP^C controls differentiation of hESCs toward neuron-, oligodendrocyte-, and astrocyte-committed lineages and is likely involved at the stage of uncommitted neural progenitor cells rather than lineage-committed neural progenitors.     

mRNA-Seq and MicroRNA-Seq Whole-Transcriptome Analyses of Rhesus Monkey Embryonic Stem Cell Neural Differentiation Revealed the Potential Regulators of Rosette Neural Stem Cells

Rosette neural stem cells (R-NSCs) represent early stage of neural development and possess full neural differentiation and regionalization capacities. R-NSCs are considered as stem cells of neural lineage and have important implications in the study of neurogenesis and cell replacement therapy. However, the molecules regulating their functional properties remain largely unknown. Rhesus monkey is an ideal model to study human neural degenerative diseases and plays intermediate translational roles as therapeutic strategies evolved from rodent systems to human clinical applications. In this study, we derived R-NSCs from rhesus monkey embryonic stem cells (ESCs) and systematically investigated the unique expressions of mRNAs, microRNAs (miRNAs), and signalling pathways by genome-wide comparison of the mRNA and miRNA profilings of ESCs, R-NSCs at early (R-NSCP1) and late (R-NSCP6) passages, and neural progenitor cells. Apart from the R-NSCP1-specific protein-coding genes and miRNAs, we identified several pathways including Hedgehog and Wnt highly activated in R-NSCP1. The possible regulatory interactions among the miRNAs, protein-coding genes, and signalling pathways were proposed. Besides, many genes with alternative splicing switch were identified at R-NSCP1. These data provided valuable resource to understand the regulation of early neurogenesis and to better manipulate the R-NSCs for cell replacement therapy.     

Neural Stem and Progenitor Cells of Human Embryos and Fetuses as a Basis of New Biomedical Technologies

Isolation and cultivation of stem and progenitor cells of human embryos and fetuses at the age of 7–12 weeks of gestation have been described. The embryonic cells of human brain formed neurospheres with heterogenous composition. Cell differentiation took place not only in the presence of serum or as a result of attachment of neurosphere to a sublayer, but also in floating neurospheres in the presence of mitogens. In most neurospheres, the nestin-immunopositive cells were located near the surface while the cells stained for -tubulin III and glial fibrillar acid protein, as compact groups inside the neurospheres.     

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (<10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t_1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.     

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage.An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues.     

基于双荧光系统的HepG2肝细胞癌原位移植裸鼠模型建立及活体荧光成像动态定量分析

目的:建立一种可以实时、定量、动态监测的肝细胞癌原位移植模型,并利用活体荧光成像系统对裸鼠体内原位肝细胞癌生长进行分析。方法:利用慢病毒包装系统包装pCDH-GFP-Luc质粒,将绿色荧光蛋白(GFP)和萤光素酶(Luc)基因通过病毒感染的方式整合到HepG2肝癌细胞染色体中,利用流式细胞术分选GFP+细胞,扩增培养后,将该细胞注射到裸鼠皮下进行成瘤,成瘤后分离肿瘤组织接种裸鼠肝脏,将造模成功的裸鼠分为对照组和治疗组,分别灌胃给与0.5%羧甲基纤维素钠(CMC-Na)和50 mg/kg索拉非尼,2/d,连续28 d,每7 d利用活体荧光成像系统观察肝癌细胞在对照组和治疗组裸鼠肝脏内的生长情况。实验结束后,分离裸鼠肝脏肿瘤,拍照称重。结果:建立了稳定表达双荧光的人肝癌细胞系HepG2-GFP-Luc,体外发光强度与表达萤光素酶的细胞数量呈正相关(R2=0.9945);建立了肝细胞癌原位移植活体荧光成像模型,对照组和治疗组肝脏内肿瘤细胞荧光强度随时间的延长逐渐增加,治疗组荧光强度明显低于对照组。定量分析结果显示,在第24、31和38 d,治疗组荧光总光子数值显著低于对照组;治疗组平均瘤重显著低于对照组。结论:建立了一种肝细胞癌原位移植荧光成像模型,可通过活体成像系统对肿瘤大小进行动态定量分析,为抗肝癌药物的药效学评价提供了实时定量分析动物模型。