结肠癌裸鼠模型活体近红外荧光成像

目的 探讨人结肠腺癌裸鼠皮下微小移植瘤的近红外荧光成像表现及应用近红外荧光成像测量移植瘤大小的可行性.方法 应用小动物分子成像仪和非特异性探针Cy5.5对20只裸鼠行近红外荧光成像,进而得到关于肿瘤大小和荧光强度的信息;活体成像后裸鼠处死取瘤,游标卡尺测量肿瘤大小.结果 20只裸鼠皮下微小移植瘤早期成像清晰,荧光测量瘤体的平均径线为3.093 mm×2.188 mm,荧光强度均值为85219.40 PC.近红外荧光成像与游标卡尺测算移植瘤体积所得2组数据呈线性相关, r=0.915,P<0.0001.结论 近红外线光学成像具有敏感性高的特点,可在早期发现微小移植瘤, 并且可较准确直观地反映晚期移植瘤的形态和大小.     

ICG纳米探针在早期结肠癌诊断中的应用及其价值

 

目的 评估ICG纳米探针在结肠癌荧光分子成像中的靶向性、荧光效应及其早期诊断价值。方法 建立裸鼠结肠癌皮下移植瘤模型,应用人血清白蛋白(HSA)包裹的ICG纳米探针,并以Folate RsenseTM680、吲哚菁绿作为对照组,经裸鼠结肠癌皮下移植瘤模型尾静脉注射探针后,进行活体荧光分子成像,观察HAS-ICG纳米探针的成像效果,量化分析肿瘤部位的荧光信号强度。结果 活体荧光分子成像结果显示:探针HAS-ICG、Folate RsenseTM680均在实验瘤鼠皮下肿瘤部位出现浓聚,浓聚高峰分别在注射后1 h、24 h。与对照组相比,HAS-ICG纳米探针比商业探针有较好的靶向标记性和信噪比。结论 HSA-ICG纳米探针具有很好的标记HCT116结肠肿瘤细胞的能力,可通过荧光分子成像技术诊断早期裸鼠结肠癌变。

     

契伦科夫光分子成像技术对胃癌裸鼠移植瘤诊断的可能性探讨

 目的 采用契伦科夫光分子成像(Cerenkov luminescence imaging,CLI)技术对胃癌裸鼠移植瘤进行放射性核素光学分子成像,探索契伦科夫光诊断胃癌的可行性。方法 2只胃癌裸鼠移植瘤模型尾静脉分别注射1.67×10⁷ Bq ¹⁸F-FDG和3.34×10⁷ Bq ¹⁸F-FDG,1 h后采集契伦科夫光,选取相同大小的ROI进行信号强度分析。成像结束后解剖出移植瘤组织和肝、肾、心脏、胃肠道、肺脏再次成像。然后进行CLI动态成像:1只胃癌裸鼠移植瘤模型尾静脉注射1.67×10⁷ Bq ¹⁸F-FDG,分别于注药后40、60、80、100、120、140、160、180 min采集移植瘤部位的光学信号并进行对比。结果 移植瘤部位出现契伦科夫光信号的浓聚,契伦科夫光信号强度与尾静脉注射的¹⁸F-FDG的活度成正比,注射3.34×10⁷ Bq ¹⁸F-FDG和1.67×10⁷Bq ¹⁸F-FDG的胃癌移植瘤小鼠移植瘤部位光子数值分别为8.432×10⁵ p/s和3.172×10⁵ p/s。随时间的延长,移植瘤摄取¹⁸F-FDG的契伦科夫光信号逐渐减弱。结论 CLI可以实现胃癌裸鼠移植瘤的放射性核素光学成像,具有实现光学分子成像诊断胃癌的潜力。     

裸鼠胃癌移植瘤的近红外荧光成像

目的：通过对非特异性探针Cy5.5在裸鼠体内分布及显像研究,探讨近红外荧光成像对胃癌的早期诊断及动态监测价值。方法：用MGC-803细胞株建立胃癌动物模型,进行早期、实时活体及离体显像实验。结果：近红外荧光成像可检测早期胃癌移植瘤的平均大小为2.807mm×3.045mm,与游标卡尺测得的肿瘤大小呈直线相关,r=0.924,P〈0.05。Cy5.5主要分布在肿瘤组织,主要代谢器官为肾脏;注入探针30min后,裸鼠肿瘤部位成像清晰,荧光寿命、荧光强度均高于对照部位（P〈0.05）。60min后,肿瘤区的荧光强度始高于血液（P〈0.05）。90min时达峰值。肿瘤部位的平均荧光寿命为（3.1376±0.9894）ns,明显高于对侧部位（P〈0.05）。结论：近红外荧光成像可用于胃癌的早期诊断及瘤体的动态监测。     

DCE-MRI对裸鼠胃癌原位移植瘤的可行性研究

目的 评估动态增强磁共振成像(DCE-MRI)用于裸鼠胃癌原位移植瘤模型研究的可行性.方法 建立裸鼠胃癌原位移植瘤模型,15只采用钆喷酸葡胺注射液为对比剂行DCE-MRI,扫描后取出移植瘤瘤体检测微血管密度(MVD).并对比裸鼠胃癌原位移植瘤模型MVD与正常胃黏膜组织MVD的差异.结果 15只裸鼠胃癌原位移植瘤模型成功行MRI平扫及DCE-MRI,裸鼠胃癌原位移植瘤微血管转运常数(Ktrans)值为(2.11±0.44) min-1,反流速率常数(Kep)值为(4.59±0.93) min-1, 血管外细胞外容积分数(Ve)值为0.46±0.06.裸鼠胃癌原位移植瘤中MVD显著高于正常胃黏膜组织(χ2=16.205,P<0.001).结论 DCE-MRI可以无创定量评估胃恶性肿瘤血管参数.     

小鼠Lewis肺癌移植瘤模型活体分子成像研究

目的建立能进行光学活体分子成像的lewis肺癌(LLC)移植瘤模型，探讨其在观察研究肿瘤的生长、转移、肿瘤血管生成及治疗效果等方面的价值。方法利用pLEIN-CMV-GFP逆转录病毒载体将绿色荧光蛋白(green fluorescent protein，GFP)报告基因转入小鼠Lewis肺癌(LLC)肿瘤细胞，体外稳定高效表达后，种植于小鼠双侧腹股沟，在不同时间利用光学活体成像仪成像，观察并用病理学证实。结果所有动物均成功制成荷GFP基因LLC移植瘤模型，GFP基因稳定高效表达，可见随肿瘤生长，绿色荧光团块增大。结论该模型稳定可靠，能够使我们利用光学分子成像的方法，实时、无创、连续地观察肿瘤生长，为研究肿瘤提供了新的思路和方法。     

人胃癌裸鼠原位移植瘤P53、c-erbB-2癌基因蛋白免疫组化和超微结构的研究

为探讨胃癌的分子发病机制及为实验治疗提供理想动物模型，采用显微外科原位移植技术，将47例人胃癌标本移植裸鼠胃粘膜层，观察原位移植成瘤、侵袭和转移情况，及P53、c-erbB-2、raSP21癌基因的表达和形态学特征。从47例胃癌标本中筛选出的人胃腺癌、鳞癌、鳞腺癌三株裸鼠原位移植模型，对三种癌蛋白均呈阳性表达，并与肿瘤生长方式、侵袭浓度和淋巴结转移有关，超微结构观察发现移植瘤与来源人胃癌细胞相似。此模型可用于研究胃癌的分子发病机制、侵袭、转移及实验治疗。     

USPIO-PEG-sLeX 分子探针在裸鼠鼻咽癌移植瘤模型 MRI 的初步研究

目的：通过超顺磁性氧化铁(USPIO)螯合唾液酸化酶 X(sLeX )形成靶向内皮细胞黏附分子-1(ELAM-1)的特异性磁共振成像的分子探针(USPIO-PEG-sLeX ),并探讨其在鼻咽癌移植瘤的应用价值。方法利用物理沉积方法合成 USPIO 纳米颗粒,通过 PEG 修饰形成 USPIO-PEG-sLeX 。裸鼠鼻咽癌移植瘤模型分为实验组和对照组,分别经尾静脉注入 USPIO-PEG-sLeX 和USPIO-PEG 前后进行 MR T2 mapping 成像,比较分析注射对比剂前后移植瘤的 T2值变化。结果USPIO-PEG-sLeX 具有良好表征。对照组和实验组的鼻咽癌移植瘤的平扫 T2值差异无统计学意义(P >0.05),增强扫描后2组的 T2值下降,2组的 T2值差异有统计学意义(P <0.05);实验组的强化率更低,2组的强化率差异有统计学意义(P <0.05)。实验组中瘤体与肌肉的强化率差异有统计学意义(P <0.05)。结论USPIO-PEG-sLeX 纳米磁性颗粒有望作为鼻咽癌 ELAM-1表达的靶向对比剂,在非创伤动态监测 ELAM-1的表达方面具有良好的潜在应用前景。     

组织仿体液中近红外荧光分子探针的3-D成像

目的 研制荧光分子断层成像实验系统,获取组织仿体内近红外荧光探针的3-D图像.方法 系统采用低温制冷CCD相机作为高灵敏弱光信号探测器,通过旋转目标物,得到各个角度的激发荧光场.组织内近红外光子传输遵循漫射近似理论,图像重建采用基于有限元的快速算法.结果 荧光检测系统可以获取全周高密度原始数据,重建图像可再现漫射介质内的荧光目标物的位置与3-D形状.结论 仿体液中荧光目标物重建结果良好,可作为在体动物光学断层成像平台.     

近红外荧光染料在肿瘤特异性成像中的研究

近年来,光学成像以其非侵袭性、实时、分辨率高等优势广泛应用于肿瘤研究领域,可对肿瘤进行早期诊断,反映肿瘤解剖学结构及代谢情况。近红外(near-infrared,NIR)荧光成像是目前光学分子成像领域研究的热点,以合适的荧光探针标记细胞、蛋白质分子或核酸,用特定波长的红光激发     

Optical imaging of spontaneous breast tumors using protease sensing 'smart' optical probes

OBJECTIVE: The objective of this study was to determine if spontaneous breast cancer lesions can be detected by fluorescence reflectance imaging (FRI) and fluorescence mediated tomography (FMT) using protease-sensing optical probes. MATERIALS AND METHODS: Transgenic (FVB/N-TgN (WapHRAS)69Lin Y)) mice, which spontaneously develop breast cancer, were injected intravenously with a cathepsin-sensing fluorescent imaging probe. FRI and FMT were performed 24 hours after probe injection and region of interest (ROI) analysis was performed. Magnetic resonance images were acquired for anatomic coregistration with the FMT data. Moreover, correlative immunohistochemistry and fluorescence microscopy were performed. RESULTS: All tumor nodules were clearly delineated by FRI showing an average signal intensity of 380 +/- 106 AU. Similarly, tumors were clearly detected by FMT imaging. Immunohistochemistry confirmed cathepsin-B expression of primary tumors and fluorescence microscopy revealed a strong Cy 5.5 deposition in the tissue. CONCLUSIONS: FRI and FMT using "smart" protease sensing probes permits detection of experimental spontaneous breast cancers. Because the expression levels of various proteases correlate with patient outcome, this technique may not only help to detect, but also to differentiate breast cancers noninvasively.     

Near-infrared optical imaging of protease activity for tumor detection

PURPOSE: To build and test an optical imaging system that is sensitive to near-infrared fluorescent molecular probes activated by specific enzymes in tumor tissues in mice. MATERIALS AND METHODS: The imaging system consisted of a source that delivered 610-650-nm excitation light within a lighttight chamber, a 700-nm longpass filter for selecting near-infrared fluorescence emission photons from tissues, and a charge-coupled device (CCD) for recording images. The molecular probe was a biocompatible autoquenched near-infrared fluorescent compound that was activated by tumor-associated proteases for cathepsins B and H. Imaging experiments were performed 0-72 hours after intravenous injection of the probe in nude mice that bore human breast carcinoma (BT-20). RESULTS: The imaging system had a maximal spatial resolution of 60 microns, with a field of view of 14 cm2. The detection threshold of the nonquenched near-infrared fluorescent dye was subpicomolar in the imaging phantom experiments. In tissue, 250 pmol of fluorochrome was easily detected during the 10-second image acquisition. After intravenous injection of the probe into the tumor-bearing animals, tumors as small as 1 mm became detectable because of tumor-associated enzymatic activation of the quenched compound. CONCLUSION: Tumor proteases can be used as molecular targets, allowing visualization of millimeter-sized tumors. The development of this technology, probe design, and optical imaging systems hold promise for molecular imaging, cancer detection, and evaluation of treatment.     

胃癌荧光光谱特性初步研究

目的 应用激光荧光光谱解析法研究胃肿瘤组织中的荧光组成变化，从而分析胃肿瘤组织中荧光成分相对于正常组织中含量的变化，深入探讨诊断胃肿瘤的依据。方法 用氮分子激光器及OMA系统组成激光诱导荧光检测系统，检测并对照分析7例离体正常胃组织和肿瘤组织标本的荧光谱。采用1组高斯(gauss)函数作为基函数进行荧光谱线拟和，对离体正常和肿瘤胃组织荧光光谱进行解析。结果 在荧光350～650nm的特定光谱波段，胃肿瘤组织荧光光谱在420nm处与正常胃组织荧光光谱有较大差异，分析在胃癌组织中，该荧光峰所对应的成分含量低于正常组织中该成分的含量。这种差异为利用激光诱导荧光光谱分析方法诊断人体组织中的肿瘤提供了新的可能性。结论 胃肿瘤组织中缺少荧光峰在420nm附近的正常胃组织荧光成分。     

Cy5.5-DTPA-galactosyl-dextran: a fluorescent probe for in vivo measurement of receptor biochemistry

The high sensitivity of fluorescent reporters offers an opportunity to analytically probe the biochemistry of in vivo receptor systems with low target tissue concentration. We investigated the ability of an optical imaging system to acquire adequate signal for in vivo measurement of receptor biochemistry. The imaging system consisted of a small animal optical imager operating in the time domain (TD) and a fluorescent-labeled diagnostic probe of known receptor-binding properties. Optical imaging of mice (n = 4) using the targeted probe, Cy5.5-DTPA-galactosyl-dextran (2.2 Cy5.5, 4 DTPA, 68 galactose units per dextran, 124 kDa, 24 nmol/kg), demonstrated blood clearance and hepatic uptake. The mean and standard deviation for the time to reach 90% of the peak liver intensity were 15.4 +/- 1.6 min. Typical fluorescent intensities within a 10-pixel region-of-interest from a 30-s image acquired 30 min postinjection were in excess of 2.5 million counts. The nontargeted agent (Cy5.5-DTPA-dextran) did not demonstrate (n = 4) hepatic uptake. This uptake pattern was duplicated by nuclear imaging of rabbits using (99m)Tc-labeled Cy5.5-DTPA-galactosyl-dextran and Cy5.5-DTPA-dextran. This study demonstrated the feasibility of optically labeling a receptor-binding diagnostic probe and imaging in the TD with sufficient sensitivity and temporal resolution for pharmacokinetic analysis.     

Digital optical imaging of green fluorescent proteins for tracking vascular gene expression: feasibility study in rabbit and human cell models

PURPOSE: To investigate the feasibility of using a sensitive digital optical imaging technique to detect green fluorescent protein (GFP) expressed in rabbit vasculature and human arterial smooth muscle cells. MATERIALS AND METHODS: A GFP plasmid was transfected into human arterial smooth muscle cells to obtain a GFP-smooth muscle cell solution. This solution was imaged in cell phantoms by using a prototype digital optical imaging system. For in vivo validation, a GFP-lentivirus vector was transfected during surgery into the carotid arteries of two rabbits, and GFP-targeted vessels were harvested for digital optical imaging ex vivo. RESULTS: Optical imaging of cell phantoms resulted in a spatial resolution of 25 microm/pixel. Fluorescent signals were detected as diffusely distributed bright spots. At ex vivo optical imaging of arterial tissues, the average fluorescent signal was significantly higher (P <.05) in GFP-targeted tissues (mean +/- SD, 9,357.3 absolute units of density +/- 1,001.3) than in control tissues (5,633.7 absolute units of density +/- 985.2). Both fluorescence microscopic and immunohistochemical findings confirmed these differences between GFP-targeted and control vessels. CONCLUSION: The digital optical imaging system was sensitive to GFPs and may potentially provide an in vivo imaging tool to monitor and track vascular gene transfer and expression in experimental investigations.     

Regional lung microvascular permeability using dual isotope scintigraphy

We have extended the dual isotope probe technique of Basran et al. for the measurement of pulmonary microvascular permeability (PMVP) to include gamma camera data acquisition and functional imaging of the plasma protein accumulation index (PPA) throughout the lung fields. The study group consisted of 11 patients with possible increased PMVP following the drainage of a pleural effusion or the evacuation of air from a pneumothorax, and 11 control patients. The PPA was calculated (1) for probe data, (2) on a pixel by pixel basis for the camera data, the results being stored in a functional image and (3) for four pixel x four pixel regions of interest positioned over the lung fields using the functional image and raw data as a guide. Functional images of the control group showed uniformly low PPAs throughout the lung fields. Nine of the eleven patients in the effusion/pneumothorax group showed discrete areas of increased PPA on the functional images. In the effusion/pneumothorax group, the re-expanded lung four pixel x four pixel ROI PPA values were significantly higher than the nonexpanded lung ROI values (p less than 0.001). The re-expanded lung ROI values were also significantly higher than the mean of the left and right lung ROIs in the control group (p less than 0.01). Five of the eleven patients in the effusion/pneumothorax group had probe PPAs that differed significantly from zero. We believe that probe positioning problems in the absence of sufficient clinical guidelines were largely responsible for there being no overall significant difference in the probe PPAs between the two study groups.     

Fluorescent bacterial magnetic nanoparticles as bimodal contrast agents

OBJECTIVES: The purpose of this study was to assess whether fluorochrome-coupled bacterial magnetic nanoparticles can be used as bimodal contrast agent for both magnetic resonance imaging (MRI) and near-infrared fluorescence optical (NIRF) imaging of cultured macrophages. MATERIALS AND METHODS: Bacterial magnetic nanoparticles (magnetosomes, particle diameter: 42 nm) were harvested from Magnetospirillum gryphiswaldense and characterized by using MRI. After covalent coupling to the fluorescent dye DY-676 (lambdaabs./lambdaem.= 676 nm/701 nm, Dyomics, Jena, Germany), the fluorescent magnetosomes were analyzed by fluorescence-activated cell sorting. Subsequently, murine macrophages J774 were incubated with the bimodal contrast agent (3 hours) and examined by a whole-body near infrared small animal imaging system as well as by using a 1.5 T clinical MR system. Moreover, labeled cells were characterized using confocal laser scanning microscopy (CLSM) and ultrathin section transmission electron microscopy. RESULTS: Characterization of the nanoparticles by MRI revealed R1 and R2 relaxivities of 3.2 mMs and 526 mMs, respectively. Fluorochrome-coupled magnetosomes exhibited increased fluorescence intensities at wavelengths >670 nm. Macrophages that were incubated with the contrast agent showed a significant fluorescence emission in the near infrared range as imaged with a whole body NIR imaging system, FACS analysis and CLSM. Moreover, CLSM data showed the greatest fluorescence intensities within intracellular compartments and colocalized with the magnetosomes. With MRI, both T1 and T2 relaxation times were substantially shortened at concentrations greater than 600 cells/microL. DISCUSSION AND CONCLUSION: Macrophages could be labeled with fluorescent magnetosomes, and they were successfully imaged using both a 1.5 T MR scanner as well as with NIRF optical methods. The use of this bimodal contrast agent for diagnostic purposes may benefit from the excellent spatial resolution of the MRI and the high sensitivity of the fluorescence imaging.