The use of molecular imaging of gene expression by radiotracers in gene therapy

INTRODUCTION: Progress with gene-based therapies has been hampered by difficulties in monitoring the biodistribution and kinetics of vector-mediated gene expression. Recent developments in non-invasive imaging have allowed researchers and clinicians to assess the location, magnitude and persistence of gene expression in animals and humans. Such advances should eventually lead to improvement in the efficacy and safety of current clinical protocols for future treatments. AREAS COVERED: The molecular imaging techniques for monitoring gene therapy in the living subject, with a specific highlight on the key reporter gene approaches that have been developed and validated in preclinical models using the latest imaging modalities. The applications of molecular imaging to biotherapy, with a particular emphasis on monitoring of gene and vector biodistribution and on image-guided radiotherapy. EXPERT OPINION: Among the reporter gene/probe combinations that have been described so far, one stands out, in our view, as the most versatile and easy to implement: the Na/I symporter. This strategy, exploiting more than 50 years of experience in the treatment of differentiated thyroid carcinomas, has been validated in different types of experimental cancers and with different types of oncolytic viruses and is likely to become a key tool in the implementation of human gene therapy.     

四环素调控 HSV-tk 基因在 HeLa 细胞内表达及抗肿瘤作用

目的 探讨应用四环素调控单纯疱疹病毒胸苷激酶（HSV-tk）基因在 HeLa 细胞内表达及抗肿瘤作用。方法 将含有四环素阻遏蛋白基因的质粒载体 pcDNA6/TR 与含有四环素反应元件及 HSV-tk 基因的质粒载体 pcD-NA3．1-TetO2-TKI 先后稳定转染 HeLa 细胞,给予不同浓度的四环素类似物强力霉素,应用 RT-PCR 检测细胞内HSV-tk 基因表达,应用 MTT 法测定转染 HSV-tk 基因的 HeLa 细胞对不同浓度无氧鸟苷（GCV）杀伤的敏感性。结果 转染两种质粒载体的 HeLa 细胞经不同浓度的强力霉素作用48 h 后,RT-PCR 结果显示,随着强力霉素浓度的增大,HSV-tk 基因表达量逐渐增加,强力霉素4μg/ml 时,基因表达的水平达到高峰;加入强力霉素后,该细胞对GCV 杀伤细胞的敏感性增加。结论 本研究成功地应用四环素基因表达调控系统调控了 HSV-tk 基因在 HeLa 细胞内表达及杀伤肿瘤功能,为进一步探讨自杀基因杀伤肿瘤的可调控性提供理论基础。     

心血管疾病基因治疗的分子影像学研究进展

近年来,利用基因治疗心血管疾病的实验与临床前期研究取得了较快的发展.分子影像学是在分子或细胞水平上研究活体生命或疾病过程的特定分子事件的新技术,为心血管疾病的基因治疗提供了一种实时、无创地检测方法,促进了这一治疗模式的发展和完善.本文主要综述分子影像学在心血管疾病基因治疗中的应用,包括分子探针、成像技术(包括核医学、MRI、超声、光学成像)的进展.     

A comprehensive review on controls in molecular imaging: lessons from MMP‐2 imaging

Metalloproteinases (MMPs), including MMP‐2, play critical roles in tissue remodeling and are involved in a large array of pathologies, including cancer, arthritis and atherosclerosis. Their prognostic value warranted a large investment or resources in the development of noninvasive detection methods, based on probes for many current clinical and pre‐clinical imaging modalities. However, the potential of imaging techniques is only matched by the complexity of the data they generate. This complexity must be properly assessed and accounted for in the early steps of probe design and testing in order to accurately determine the efficacy and efficiency of an imaging strategy. This review proposes basic rules for the evaluation of novel probes by addressing the specific case of MMP targeted probes. Copyright © 2014 John Wiley & Sons, Ltd.     

分子影像学报告基因显像的研究进展

报告基因显像技术是分子影像学中重要的显像技术,其可对多种不同的生物学和分子遗传学过程进行显像,有望更快地应用于临床.本文综述分子影像学中报告基因显像的一般原则、分类、相关影像学技术及潜在的临床应用.     

Improved Noninvasive In Vivo Tracking of AAV-9 Gene Therapy Using the Perchlorate-Resistant Sodium Iodide Symporter from Minke Whale

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前列腺癌分子影像学诊断与治疗进展

前列腺癌严重威胁着男性健康,近年来我国前列腺癌的发病率也迅速增加,早期发现前列腺癌对于提高生存率具有十分积极的意义。目前各前列腺癌指南中仍需通过超声引导下的前列腺活检确诊,再通过主动监测、根治性切除术、放疗及局部放疗等手段进行治疗。但前列腺活检增加了尿潴留、血尿等不必要的风险,还会遗漏多达1/3的癌组织,且目前的治疗方法因缺乏特异性而对患者进行过度或不足的治疗。近年来,对前列腺癌的诊断和治疗已经迈入分子水平,通过分子影像学进行无创精准的诊断和治疗展现出巨大的发展前景。本文将从核医学分子成像、MR分子成像、光学分子成像、超声分子成像4种常见分子影像学技术在前列腺癌的应用进展进行回顾并作一综述。     

铁蛋白报告基因在磁共振分子影像学中的研究进展

铁蛋白是体内铁储存的主要形式之一,它普遍存在于各种生物中.铁蛋白在细胞中的过度表达能够增加细胞对铁的摄取,促使细胞内、外铁重新分布,导致细胞内铁的净含量增加,磁共振成像时表现为横向弛豫率的增加,T2值的减低.近年来,铁蛋白作为一种新的内源性MRI报告基因逐渐引起人们的重视.现就铁蛋白作MRI报告基因的机制、研究现状及其发展前景做一概述.     

Recent advances in the development of nanoparticles for multimodality imaging and therapy of cancer

This review explores recent work directed toward the development of nanoparticles (NPs) for multimodality cancer imaging and targeted cancer therapy. In the growing era of precision medicine, theranostics, or the combined use of targeted molecular probes in diagnosing and treating diseases is playing a particularly powerful role. There is a growing interest, particularly over the past few decades, in the use of NPs as theranostic tools due to their excellent performance in receptor target specificity and reduction in off-target effects when used as therapeutic agents. This review discusses recent advances, as well as the advantages and challenges of the application of NPs in cancer imaging and therapy.     

Magnetic resonance nanoparticles for cardiovascular molecular imaging and therapy

Molecular vascular imaging represents a novel tool that promises to change the current medical paradigm of ‘see and treat’ to a ‘detect and prevent’ strategy. Nanoparticle agents, such as superparamagnetic nanoparticles and perfluorocarbon nanoparticle emulsions, have been developed for noninvasive imaging, particularly for magnetic resonance imaging. Designed to target specific epitopes in tissues, these agents are beginning to enter clinical trials for cardiovascular applications. The delivery of local therapy with these nanoparticles, using mechanisms such as contact-facilitated drug delivery, is in the advanced stages of preclinical research. Ultimately, combined diagnostic and therapeutic nanoparticle formulations may allow patients to be characterized noninvasively and segmented to receive custom-tailored therapy. This review focuses on recent developments of nanoparticle technologies with an emphasis on cardiovascular applications of magnetic resonance imaging.     

Dual‐isotope 111In/177Lu SPECT imaging as a tool in molecular imaging tracer design

The synthesis, design and subsequent pre‐clinical testing of new molecular imaging tracers are topic of extensive research in healthcare. Quantitative dual‐isotope SPECT imaging is proposed here as a tool in the design and validation of such tracers, as it can be used to quantify and compare the biodistribution of a specific ligand and its nonspecific control ligand, labeled with two different radionuclides, in the same animal. Since the biodistribution results are not blurred by experimental or physiological inter‐animal variations, this approach allows determination of the ligand's net targeting effect. However, dual‐isotope quantification is complicated by crosstalk between the two radionuclides used and the radionuclides should not influence the biodistribution of the tracer. Here, we developed a quantitative dual‐isotope SPECT protocol using combined ¹¹¹Indium and ¹⁷⁷Lutetium and tested this tool for a well‐known angiogenesis‐specific ligand (cRGD peptide) in comparison to a potential nonspecific control (cRAD peptide). Dual‐isotope SPECT imaging of the peptides showed a similar organ and tumor uptake to single‐isotope studies (cRGDfK–DOTA, 1.5 ± 0.8%ID cm^?3; cRADfK–DOTA, 0.2 ± 0.1%ID cm^?3), but with higher statistical relevance (p‐value 0.007, n = 8). This demonstrated that, for the same relevance, seven animals were required in case of a single‐isotope test design as compared with only three animals when a dual‐isotope test was used. Interchanging radionuclides did not influence the biodistribution of the peptides. Dual‐isotope SPECT after simultaneous injection of ¹¹¹In and ¹⁷⁷Lu‐labeled cRGD and cRAD was shown to be a valuable method for paired testing of the in vivo target specificity of ligands in molecular imaging tracer design. Copyright © 2012 John Wiley & Sons, Ltd.     

心肌梗死分子影像学研究进展

以心肌梗死（MI）为主的缺血性心脏病发病率和死亡率逐年上升。MI发生30 min后,部分心肌将发生不可逆性损伤,于MI早期详细评估受损心肌并及时挽救存活心肌可极大改善预后。分子影像学技术不仅能在体了解心肌缺血后分子和细胞生物学机制,也有助于判断MI预后及评估疗效。本文对基于分子影像学的MI相关研究进展进行综述。     

SV2A基因真核表达质粒构建及其在HEK293T细胞中的表达

目的构建鼠突触囊泡蛋白2A(SV2A)基因的真核表达质粒,并瞬时转染至人胚肾细胞(HEK293T)中,对其表达进行鉴定。方法以APP/PS1双转基因小鼠海马组织的cDNA为模板,扩增得到长2239 bp的SV2A基因编码序列,将此序列插入到真核表达载体p3×Flag-CMV-10多克隆位点区域中,得到真核表达质粒p3×Flag-CMV-10-SV2A,转化后挑取单克隆菌落经双酶切鉴定后送公司测序,将构建成功的重组质粒转染至HEK293T细胞中,利用蛋白质印迹法(Western blot)检测SV2A基因的表达情况。结果成功构建p3×Flag-CMV-10-SV2A重组质粒,并在转染至HEK293T细胞后,验证了相应蛋白表达。结论利用分子克隆技术成功构建了p3×Flag-CMV-10-SV2A真核表达质粒并在HEK293T细胞中正确表达,为后续实验奠定了基础。     

超声分子影像学研究进展

随着超声分子探针技术的兴起,超声分子成像成为当前医学影像学研究的热点之一.分子探针的设计是超声分子成像研究的重点和先决条件.靶向超声微泡(球)造影剂在分子影像中的研究、应用,愈来愈受到关注,而多学科的融合使其具有更大的发展空间.     

SPECT imaging of fibrin using fibrin‐binding peptides

Noninvasive detection of fibrin in vivo using diagnostic imaging modalities may improve clinical decision‐making on possible therapeutic options in atherosclerosis, cancer and thrombus‐related pathologies such as pulmonary embolism and deep venous thrombosis. The aim of this study was to assess the potential of a novel ¹¹¹In‐labeled fibrin‐binding peptide (FibPep) to visualize thrombi in mice noninvasively using single‐photon emission computed tomography (SPECT). FibPep and a negative control peptide (NCFibPep) were synthesized and their fibrin‐binding properties were assessed in vitro. FibPep showed enhanced binding compared with NCFibPep to both fibrin and blood clots. FibPep bound to fibrin with a dissociation constant (K_d) of 0.8 μ m , whereas NCFibPep displayed at least a 100‐fold lower affinity towards fibrin. A FeCl₃‐injury carotid artery thrombosis mouse model was used to evaluate the peptides in vivo. FibPep and NCFibPep displayed rapid blood clearance and were eliminated via the renal pathway. In vivo SPECT imaging using FibPep allowed clear visualization of thrombi. Ex vivo biodistribution showed significantly increased uptake of FibPep in the thrombus‐containing carotid in comparison to the noninjured carotid (5.7 ± 0.7 and 0.6 ± 0.4% injected dose per gram (%ID g^?1), respectively; p < 0.01; n = 4), whereas nonspecific NCFibPep did not (0.4 ± 0.2 and 0.3 ± 0.0%ID g^?1, respectively; n = 4). In conclusion, FibPep displayed high affinity towards fibrin in vitro and rapid blood clearance in vivo, and allowed sensitive detection of thrombi using SPECT imaging. Therefore, this particular imaging approach may provide a new tool to diagnose and monitor diseases such as atherosclerosis and cancer. Copyright © 2013 John Wiley & Sons, Ltd.     

表皮生长因子受体靶向治疗分子影像研究进展

表皮生长因子受体（EGFR）靶向治疗癌症需要准确评估肿瘤EGFR表达。分子影像技术以分子探针与细胞特定靶分子结合，通过PET/CT检测技术获取图像，可无创、准确、重复地实时评价靶分子表达。本文围绕PET各类EGFR分子探针研究进展进行综述。     

分子影像探针在肺癌诊疗中的应用进展

肺癌作为全球第二高发的癌症类型,占所有新发病例的11.4%,致死率高达所有癌症相关死亡的18.0%。早期发现、诊断与及时治疗对于降低肺癌死亡率至关重要。得益于分子影像学的持续进步,多种分子影像探针在肺癌的早期筛查、诊疗和预后评估中得到了广泛运用,且取得了显著成效。本文重点探讨了当前应用最广泛的 ¹⁸F-FDG探针以及新型非FDG分子影像探针的作用机理、靶点及其在临床实践中的应用,旨在为分子探针在肺癌诊疗领域的未来发展与应用提供参考。     

Solid silica nanoparticles: applications in molecular imaging

Silica and silica‐based nanoparticles have been widely used for therapeutic and diagnostic applications in cancer mainly through delivery of drugs, genes and contrast agents. Development of synthesis methods has provided the possibility of fabricating silica nanoparticles with different sizes in nanometer ranges as well as silica‐based multimodal nanoparticles with many innovative properties and intriguing applications in biomedicine. The surface of silica particles facilitates different methods of surface modifications and allows conjugation of various biomolecules such as proteins and nucleic acids. In this review, different methods of fabrication of silica and silica‐based nanoparticles, their surface modification and the application of these nanoparticles in molecular imaging are discussed. Overall, the aim of this review is to address the development of silica and silica‐based multifunctional nanoparticles that are introduced mainly for molecular imaging applications using optical, magnetic (MRI), X‐ray (computed tomography) and multimodal imaging techniques. Copyright © 2014 John Wiley & Sons, Ltd.     

The cell death and DNA damages caused by the Tet-On regulating HSV-tk/GCV suicide gene system in MCF-7 cells

Ganciclovir (GCV) affects the molecular mechanism of cell death and DNA damage by the rAAV (recombinant adeno-associated virus)-mediated Tet-On/HSV-tk/GCV suicide gene system in human breast cancer cell line MCF-7. A rAAV/TRE/Tet-On/HSV-tk combining a Tet-On regulating system and a suicide gene HSV-tk was used to transfect human breast cancer cell line MCF-7, and therapeutic effects on this system were studied. Afterwards, we used RT–PCR, western blotting, and a modified comet-assay to explore the potential mechanism of the HSV-tk/GCV suicide gene system in breast cancer treatments. MTT assay has shown that the cell number of GCV + rAAV + Dox group was significantly decreased compared with that of other groups after treatment and flow cytometric analysis detected that there was a substantial increase of S phase cells in this group, which means the HSV-tk/GCV suicide gene system probably works on the cell cycle. RT–PCR detected the expression level of p21 increased and PCNA had an opposite trend. Western blotting detected the protein expression of p21 and p53 increased and PCNA, CDK1, cyclin B decreased in GCV + rAAV + Dox group. The modified comet-assay shown that the very small extra fragments generated by the GCV + rAAV + Dox group treatment are visible as a small cloud extending from the comet in the direction of electrophoresis. The therapeutic mechanism of the HSV-tk/GCV suicide gene system on human breast cancer cell line MCF-7 is probably by upregulating the expression of p21 through a p53-dependent DNA damage signalling pathway, leading the decrease of protein expression of PCNA, cyclin B, CDK1 in MCF-7 cells and promoting the cell cycle arrest at G1/S phase. In summary, the HSV-tk/GCV suicide gene system arouses the death of MCF-7 cells from blocking the cell cycle and DNA damage.