In vitro three-dimensional cancer metastasis modeling:Past,present, and future

Metastasis is the leading cause of most cancer deaths, as opposed to dysregulated cell growth of the primary tumor.Molecular mechanisms of metastasis have been studied for decades and the findings have evolved our understanding of the progression of malignancy. However, most of the molecular mechanisms fail to address the causes of cancer and its evolutionary origin, demonstrating an inability to find a solution for complete cure of cancer. After being a neglected area of tumor biology for quite some time, recently several studies have focused on the impact of the tumor microenvironment on cancer growth. The importance of the tumor microenvironment is gradually gaining attention, particularly from the perspective of biophysics. In vitro three-dimensional(3-D) metastatic models are an indispensable platform for investigating the tumor microenvironment, as they mimic the in vivo tumor tissue. In 3-D metastatic in vitro models, static factors such as the mechanical properties, biochemical factors, as well as dynamic factors such as cell–cell, cell–ECM interactions, and fluid shear stress can be studied quantitatively. With increasing focus on basic cancer research and drug development, the in vitro 3-D models offer unique advantages in fundamental and clinical biomedical studies.     

胶原纤维网络和癌细胞的力学微环境

文章以第一类胶原纤维网络为例, 着重分析了癌细胞三维微环境的多尺度结构及力学特征. 对于细胞与细胞外介质结合的蛋白集团、单个细胞以及细胞群体, 分别由单个纤维(或亚纤维)、纤维集束以及纤维网络整体来决定相应的力学环境. 文章同时也讨论了胶原纤维(及其类似材料) 的局限性.     

基于CEST机制的pH成像方法、原理和应用

化学交换饱和转移（Chemical Exchange Saturation Transfer,CEST）技术作为一种新型的磁共振成像（Magnetic Resonance Imaging,MRI）技术.它的原理为溶质池中被激发饱和的质子与游离水中未被饱和的质子间的化学交换,能够引起水质子磁共振信号的下降,从而获得组织内生物分子的相关信息.由于质子间的交换速率k_ex与组织微环境的pH值之间存在直接联系,因而可以通过溶质质子的CEST信号对活体组织进行pH成像.目前用于pH成像的溶质分子既包括内源性游离的蛋白质、多肽分子,还包括一系列的外源性小分子和金属螯合物.通过不同类型的比率法、内源性胺和酰胺浓度-独立检测（Amine and Amide Concentration-independent Detection,AACID）等成像方法,能够获得肾脏、中风脑组织以及肿瘤组织的pH图谱.本文详细总结了2000年以来利用CEST技术进行pH成像方面的研究进展,包括对比剂、成像方法和相关应用,展望了活体组织pH成像的发展趋势和应用前景.     

一种示踪干细胞的磁共振-荧光双模成像探针

本文设计并合成了Gd基磁共振-荧光双模成像探针——Gd-DOTA-PEG-GA,通过电穿孔的方式标记人源间充质干细胞（hMSCs）.电穿孔标记诱导细胞将探针组装成团簇状纳米粒子进入细胞质,显著延长其与细胞结合的时间,并呈现出明显的T₂信号减弱效应,且信号减弱效应可以持续7天以上.在水溶液中,该探针的发射带集中在498 nm,并且荧光强度在一周内无明显衰减.该探针标记的细胞在荧光倒置显微镜下呈现绿色荧光.这些结果表明该探针可以作为磁共振-荧光双模成像探针用于干细胞示踪.     

三维微纳米制造技术在癌症生物物理研究中的应用

癌症致命的主要原因是癌细胞在临床上的转移性. 癌细胞的侵袭和转移是一个非常复杂的三维过程, 但现有的癌症研究在活体上有诸多观测和操作上的困难. 而体外实验又通常在培养皿中进行, 其二维的生长环境已完全不能满足对癌细胞空间转移性的深入研究, 故在活体外构建出癌细胞侵袭和转移的三维物理模型具有十分重要的意义. 然而如何在体外尽可能真实地模拟体内癌细胞的生长微环境一直是困扰科学家的难题. 本文系统介绍了三维微纳米制造的几种主流技术, 探讨了它们在癌症生物物理研究中的应用和发展. 在此基础上为了在未来实现对体外三维模型的制造、观测和精确操作, 文章还创新性地提出了一种结合紫外线固化生物型水凝胶的三维成型技术、光片三维成像技术以及微纳米探针控制技术的一体化研究平台. 这些先进的技术和理念, 势必会逐步升级现有传统的癌症研究手段, 为未来理解和治疗癌症揭开全新的篇章.     

鼠S180肉瘤的体内31P MRS研究

利用表面线圈³¹P NMR研究了小鼠S180肉瘤生长过程中能量代谢和磷脂类变化的特点.结果发现:随着肿瘤体积的增大,(1)Pi和PME升高;(2)PCr降低,在肿瘤体积较大时常检测不到;(3)β-NTP(通常用来表示ATP的量)变化较小;(4)PDE波动性较大;(5)PCr/Pi和β-NTP/Pi比值均下降,且PCr/Pi比β-NTP/Pi下降得快;(6)PME/β-NTP比值升高;(7)肿瘤pH下降,且与PCr/Pi、β-NTP/Pi或(PCr+β-NTP)/Pi比值有相关性.讨论了与这些参数变化相关联的生物学意义.     

新型吡唑并[3,4-d]嘧啶-4-酮类衍生物的结构表征和镇痛活性

本文以2-氰基-3-乙氧基丙烯酸乙酯与3,4-二甲基苯肼为原料,通过多步反应合成了三种新型吡唑并[3,4-d]嘧啶-4-酮类衍生物（A~C）,通过核磁共振（NMR,包括¹H NMR、¹³C NMR）和液相色谱-质谱联用（LC-MS）技术表征确证了其结构,并完整归属了三种化合物的¹H NMR数据．对所合成的化合物1-(3,4-二甲基苯基)-6-甲基-5-[3-(哌啶-1-基)丙氧基]-1,5-二氢-4H-吡唑并[3,4-d]嘧啶-4-酮（A）,通过小鼠脑部质谱成像和福尔马林实验进行了初步的体内镇痛活性评价,我们发现化合物A能透过血脑屏障,并产生显著且剂量依赖的镇痛活性．本研究为以吡唑并[3,4-d]嘧啶-4-酮为骨架的镇痛药物的研发提供了结构和体内活性的基础研究数据．     

Quantitative and qualitative assessment of articular cartilage in the goat knee with magnetization transfer imaging

We investigated the role of collagen in the magnetization transfer (MT) effect in contrast to other macromolecules. By means of phantoms made of collagen, chondroitin sulfate (CS) and albumin, MR parameters have been optimized in order to reduce the acquisition time and improve the sensitivity, as well as to minimize the contributions from CS and albumin to the MT induced signal attenuation. The same method was used to study cartilage ex vivo (bovine articular and nasal cartilage plugs) and in vivo (goat knee femoral chondyle). In phantom samples, the MT signal attenuation depended on the collagen concentration while contributions from the other macromolecules were found to be minimal. In average, analysis of MT images revealed a 25%, 35% and 30% signal attenuation in 10% w/v type I collagen gels, cartilage plugs, and cartilage from the weight-bearing areas of the goat knee, respectively. Biochemical data revealed that treatment of cartilage plugs with bacterial collagenase led to collagen depletion and correspondingly to a decrease of the MT response. In contrast, trypsin-induced proteoglycan loss in cartilage plugs did not alter the MT effect. A significant correlation was observed between the collagen content in these plugs and their respective MT ratios and the rate constant k for the exchange process bound versus free water. Finally, data obtained from in vivo MT measurement of the goat knee demonstrated that intra-articular injection of papain might not only cause degradation of proteoglycans but also a change in collagen integrity in a dose-dependent manner. We conclude that in vivo measurement of MT ratios gives quantitative and qualitative information on the collagen status and may be applied for the routine evaluation of normal and abnormal articular cartilage.     

Confirmation of the protective effect of cysteamine in in vitro ultrasound exposures

Armour and Corry (Radiat. Res. (1982) 89 369–380) reported that ultrasound-induced damage to in vitro Chinese hamster ovary cells was significantly reduced in the presence of cysteamine. The objective of this study was to attempt verification of this result. Four series of experiments were undertaken using in vitro cell suspensions, namely: (1) determination of the effect of cysteamine concentration on cell growth; (2) determination of the temperature dependence of ultrasonically induced cell damage; (3) determination of a dose-response relationship for the cytotoxicity of cysteamine; and (4) assessment of cell integrity and reproductive capacity in the presence or absence of cysteamine during ultrasonic exposure. Ultrasound parameters included a resonance frequency of 1 MHz, a continuous wave exposure duration of 5 min, and intensities from 0 to 21.6 W cm⁻². The results indicated a dependence of ultrasound's efficacy on the medium's temperature during insonation and a significant reduction of ultrasound efficacy in compromising cellular integrity in the presence of cysteamine.     

体内31P NMR表面线圈探头的研制

报道一种为进行活体小动物核磁共振实验而研制的体内³¹P NMK表面线圈探头.该探头工作频率为161.83MHz,无载Q值为300,可无损伤地研究生命体内各部位的性质.本文给出了用该探头所测得的小鼠体内肝脏、大腿肌肉、大脑以及大脑缺氧状态下³¹P NMR谱图实例.     

基于嗓音源变换函数的声门发声效率估计方法

在澄清嗓音源概念的基础上提出了一种以嗓音源变换函数为基础的声门发声效率估计方法。将嗓音源变换函数定义为声门上声学嗓音源与声门气流体积速度波在频域上的比值。通过精心设计的活体犬喉及人体发声实验分别在不同元音、压紧嗓音、气声、假声和典型喉病变条件下。采用稳态元音发声方式对这一新的声门发声效率估计方法实验研究;并与其他发声效率方法综合比较。结果表明,该方法能消除上声道传输与共鸣作用的影响,能反映不同发声方式的差异;频域平均声门发声效率反映了物理定义上的声门发声效率的变化,而声门基频发声效率的变化与AC/DC比值变化一致。     

Confocal microscopy of whole mount embryonic cartilage: Intracellular localization of F-actin, chick prolyl hydroxylase and type II collagen mRNA

We show that whole mount preparations of embryonic chick sterna can be analyzed with confocal laser scanning microscopy (CLSM). This technique replaces the traditional sectioning of cartilage or culturing of chondrocytes. Whole ‘chunks’ of cartilage can be stained with dyes, used for immunohistochemistry or in situ hybridization. Although other stains have been used, the stains presented include phalloidin and propidium iodide which stain filamentous actin (F-actin) and the DNA and RNA of cells, respectively. Collagen secreting endoplasmic reticulum (ER) was localized with a primary antibody to chick prolyl hydroxylase (CPH) that was detected with a secondary antibody conjugated to FITC. The intracellular localization of type II collagen mRNA was analyzed using in situ hybridization. The cDNA probe specific for the C-propeptide region of the 1 type (II) collagen mRNA was nick translated and labeled with biotin-16-dUTP. Biotin labeled probes were visualized with avidin-FITC. Depending on the intensity of the stain, we were able to analyze approximately 3–10 layers of chondrocytes. Stains penetrated into the cartilage better than antibodies and biotin-avidin labeled cDNA probes. The F-actin was located as bands of filaments in the superficial layers of the cartilage and was associated with the membranes that marked cell boundaries as deep as 10 layers of chondrocytes. The ER stained with anti-chick prolyl hydroxylase was prominent in perinuclear regions of the cells, but the antibody was only able to penetrate 4–5 cell layers. Single label in situ hybridization studies show that chondrocytes are positive for type II collagen mRNA. Similar to the immunohistochemistry, in situ hybridization probes were only able to penetrate 4–5 cell layers. The type II collagen mRNA appeared perinuclear in the chondrocytes, similar to the ER staining pattern.     

Application of CR-39 for alpha-autoradiography and in vivo detection of B accumulations in tumor bearing mice

It is required to achieve an accurate measurement of ¹⁰B distributions and concentrations in the tumor cells for boron neutron capture therapy (BNCT). We employed a technique of neutron capture autoradiography (NCAR) of the whole body samples of tumor bearing mice using CR-39 plastic track detectors to the measurements of ¹⁰B accumulations in mice. In order to confirm the validity of the NCAR measurements, the differential REL spectrum for the neutron-induced particles emitted from the standard filter paper sheets was compared with that for ¹⁰B-compound solution. Differential REL spectrum for protons originated from the biogenically abundant nuclides through ¹⁴N (n, p)¹⁴C and ¹H (n, n′) ¹H reactions was measured in vivo using a small piece of CR-39 inserted into the tumor of mouse.     

Non-invasive and low-artifact in vivo brain imaging by using a scanning acoustic-photoacoustic dual mode microscopy

Photoacoustic imaging is a potential candidate for in vivo brain imaging, whereas, its imaging performance could be degraded by inhomogeneous multi-layered media, consisted of scalp and skull. In this work, we propose a low-artifact photoacoustic microscopy (LAPAM) scheme, which combines conventional acoustic-resolution photoacoustic microscopy with scanning acoustic microscopy to suppress the reflection artifacts induced by multi-layers. Based on similar propagation characteristics of photoacoustic signals and ultrasonic echoes, the ultrasonic echoes can be employed as the filters to suppress the reflection artifacts to obtain low-artifact photoacoustic images. Phantom experiment is used to validate the effectiveness of this method. Furthermore, LAPAM is applied for in-vivo imaging mouse brain without removing the scalp and the skull. Experimental results show that the proposed method successfully achieves the low-artifact brain image, which demonstrates the practical applicability of LAPAM. This work might improve the photoacoustic imaging quality in many biomedical applications which involve tissues with complex acoustic properties, such as brain imaging through scalp and skull.     

活体动物代谢过程的31P NMR研究——方法与初步结果

利用表面线圈探头测得了小鼠大脑,大腿肌肉和肝脏的体内³¹P NMR谱图。观察到PCr,ATP,PME,PDE,P_i等化合物的共振峰,并确定了各谱峰的化学位移。由大脑的P_i化学位移计算得到其细胞内pH值为7.15.用差谱方法解决了因定位不准而造成的肝脏谱图不纯。观察了小鼠大脑在缺氧状态下的代谢变化过程。当O₂浓度降至2%时,PCr开始下降,降到1%时,P_i明显升高。从空气变化到1%的O₂含量,致使小鼠死亡,小鼠大脑的pH值从7.16降到6.46。     

Ultrasonic imaging of in vivo bubbles in decompression sickness

Scanning ultrasonic imaging is used to monitor, in vivo, the development and location of microscopic bubbles formed in decompression sickness. Stationary or moving bubbles as small as 5 microns are detected. Methods of comparison of earlier reference images with real time images on an oscilloscope are described. Different image displays are presented, emphasising the usefulness of combined deflection and brightness scan modulation. Experiments show unmistakable decompression bubble echoes around fatty tissues in guinea pig legs, which disappear almost completely with recompression. Guinea pigs can be safely decompressed by keeping imaged bubble echoes below a threshold. Preliminary results of human studies are discussed.     

脑科学研究中的质子磁共振波谱方法

核磁共振（NMR）波谱作为一种操作简单、高效且重复性良好的技术手段,在脑科学领域的应用日益广泛,尤其是离体核磁共振氢谱（¹H NMR）和活体核磁共振氢谱（¹H MRS）.¹H NMR和¹H MRS在实验设计、样品预处理、数据处理等方面各有优劣、各擅胜场.本综述主要从适用范围、样品预处理、数据处理分析等方面,对二者在方法学层面的研究现状进行总结和讨论,以期为脑科学领域的研究者进行大脑代谢相关研究时提供一定的参考和帮助.     

In vitro studies of cutaneous retention of magnetic nanoemulsion loaded with zinc phthalocyanine for synergic use in skin cancer treatment

In this study was developed a new nano drug delivery system (NDDS) based on association of biodegradable surfactants with biocompatible magnetic fluid of maguemita citrate derivative. This formulation consists in a magnetic emulsion with nanostructured colloidal particles. Preliminary in vitro experiments showed that the formulation presents a great potential for synergic application in the topical release of photosensitizer drug (PS) and excellent target tissue properties in the photodynamic therapy (PDT) combined with hyperthermia (HPT) protocols. The physical chemistry characterization and in vitro assays were carried out by Zn(II) Phtalocyanine (ZnPc) photosensitizer incorporated into NDDS in the absence and the presence of magnetic fluid, showed good results and high biocompatibility. In vitro experiments were accomplished by tape-stripping protocols for quantification of drug association with different skin tissue layers. This technique is a classical method for analyses of drug release in stratum corneum and epidermis+dermis skin layers. The NDDS formulations were applied directly in pig skin (tissue model) fixed in the cell's Franz device with receptor medium container with a PBS/EtOH 20% solution (10 mM, pH 7.4) at 37 °C. After 12 h of topical administration stratum corneum was removed from fifty tapes and the ZnPc retained was evaluated by solvent extraction in dimetil-sulphoxide under ultrasonic bath. These results indicated that magnetic nanoemulsion (MNE) increase the drug release on the deeper skin layers when compared with classical formulation in the absence of magnetic particles. This could be related with the increase of biocompatibility of NDDS due to the great affinity for the polar extracelullar matrix in the skin and also for the increase in the drug partition inside of corneocites wall.     

Intracellular pH mapping with SNARF-1 and confocal microscopy. I: A quantitative technique for living tissue and isolated cells

A fluorescent pH indicator in conjunction with confocal microscopy, was used to map intracellular pH in a variety of cells and tissues with high spatial resolution. The new pH-sensitive fluorescent probe SNARF-1 was excited with the 488 nm band of the argon ion laser of a Bio-Rad MRC-500 confocal microscope. Ratio images were created with pixel-by-pixel division, with the intensity of these images representing a function of pH, that is independent of dye concentration, photobleaching or path length. Cell cultures of rat aortic smooth muscle were loaded with 20 μм SNARF-1/AM for 20 min at 37°C. Intracellular pH levels were calibrated in situ by treatment of each cell with nigericin (20 μм) in solutions of known pH. The cytosolic pH of the majority of cells was uniform, however, pH gradients were evident between the cytosol and nuclear regions, indicating the ability of this technique to map intracellular and intraorganelle pH. Rat C6 glioblastoma spheroids were cultured then loaded with SNARF-1/AM at 10°C for 90 min. The pH values were calibrated in vitro, using SNARF-1 acid in buffered solutions of known pH. Ratio images of the bisected spheroids showed a marked gradient in pH from the outer cells compared with central necrotic cells. The degree of involvement of acidification in muscle fatigue was investigated by simultaneously determining force generation and intracellular pH in individual fibres of an intact rat muscle. The investigation was performed during a stimulation protocol which induced significant fatigue in the force response of the muscle. The fatigue protocol induced little change in cytosolic pH in the fibres. We show that the use of SNARF-1, in conjunction with confocal microscopy is a powerful technique for accurately mapping pH within single cells, multicellular tissues and intact organs, as well as for accurately recording dynamic changes in pH.