利用荧光素酶标记的肿瘤细胞建立活体成像动物模型

目的建立稳定表达荧光素酶的人乳腺癌细胞株并构建适用于小动物活体成像系统观察的裸鼠皮下移植瘤模型。方法采用脂质体将携带荧光素酶基因的质粒转染到人乳腺癌细胞株MCF-7中,G418筛选出稳定表达荧光素酶的单克隆细胞株。扩增后接种于裸鼠,建立裸鼠皮下移植瘤模型,通过活体动物成像系统监测肿瘤的生长过程。结果获得了高水平稳定表达荧光素酶的乳腺癌单克隆细胞株,该单克隆细胞株与母细胞系MCF-7具有相似的生长特性。将稳定表达荧光素酶的克隆接种于裸鼠皮下可成瘤,小动物活体成像系统能准确监测肿瘤细胞在体内的生长过程。结论成功建立了稳定表达荧光素酶的乳腺癌单克隆细胞株。采用活体动物成像系统构建的裸鼠皮下移植瘤模型是拓展肿瘤体内生长、转移及治疗相关研究的理想模型。     

活体成像下评价脾脏注射法大肠癌肝转移动物模型

目的:建立可用于活体成像的小鼠大肠癌肝转移移植瘤模型。方法:利用慢病毒将荧光素酶表达载体转染至人肠癌LoVo细胞株,经嘌呤嘧啶筛选获得稳定表达荧光素酶的细胞克隆。进行裸鼠脾脏注射,采用脾脏注射切脾法和保脾法两种方法制造结肠癌肝转移模型,比较小鼠肝转移情况,并对肝脏组织进行HE染色,分析肿瘤细胞的生长分布特点。结果:建立了肠癌细胞株的荧光素酶基因稳定表达的亚克隆,两组经脾注射方法肝转移率均为65%以上,具有可操作性,切脾组具备更多的肝脏转移数及更高的肝脏成瘤率。结论:成功构建了可用于活体成像的小鼠肠癌肝转移移植瘤模型,确定了脾脏注射切脾法制造小鼠结肠癌肝转移模型是较好的造模方法。     

人胃癌裸鼠原位移植瘤模型的建立及其活体荧光成像检测

目的:建立可动态实时监测的人胃癌裸鼠原位移植瘤模型.方法:将稳定表达荧光素酶的人胃癌细胞SGC-7901fLuc+注入裸鼠后肢根部皮下形成皮下移植瘤.待皮下移植瘤瘤块长至直径0.5 cm时,剥取肿瘤组织,应用叠合法将瘤块原位移植于裸鼠胃小弯处,形成原位移植瘤模型.利用小动物活体荧光成像系统,每4日监测移植瘤的进展情况.荷瘤3周后,解剖荧光成像阳性小鼠,利用H-E染色、光镜下观察移植瘤的形态.结果:应用小动物活体荧光成像系统,可在荷瘤裸鼠胃部检测到逐渐增强的荧光信号.荷瘤裸鼠胃部存在肿瘤包块贴附于胃壁,包块直径为0.5 ～1.0 cm,包块周围与相邻组织器官边界清晰,未见粘连,检查腹腔未见转移,未见腹水形成.应用叠合法构建人胃癌裸鼠原位移植瘤模型成功率为95％(19/20).对荷瘤胃组织H-E染色后,可见异常肿瘤细胞,肿瘤包膜完整,符合胃癌组织学特征.结论:成功建立了操作简便、成瘤率高的可动态监测的人胃癌裸鼠原位移植瘤模型,为研究胃癌发生机制、研发抗癌药物提供了理想的实验工具.     

光学功能成像技术在乳腺癌早期诊断中的应用

乳腺癌的发病率逐年上升,已经成为危害女性健康的第一大疾病,因而乳腺癌的早期发现和诊断对及时治疗和保证生活质量具有关键的意义。光学功能成像技术是乳腺肿瘤常用的检查手段之一,对肿瘤的良恶性能做出较准确的诊断。在过去的几十年中,光纤、光源、探测器、影像及计算机控制仪器的发展,对光学定量技术在临床诊断和治疗中的应用起到了史无前例的促进作用。在乳腺疾病的检测、诊断和监测上,     

活体动物体内成像技术及其在生物医学中的应用进展

活体动物体内成像是近年来新兴的检测活体动物体内基因表达及细胞活动的光学成像技术，具有操作简便，直观性强的特点。这项技术包括生物发光成像和荧光成像，采用报告基因产生的生物发光、荧光蛋白质或染料产生的荧光作为体内生物光源，与新型冷CCD成像相结合，实时探测活体动物体内生理或病理条件下的细胞和分子事件。本文简要综述了活体动物体内成像技术的原理、应用领域及发展前景。     

骨高转移人乳腺癌细胞株MDA-MB-231BM3及其动物模型的建立

目的:建立骨转移人乳腺癌细胞株MDA-MB-231BM3及其免疫缺陷小鼠骨转移动物模型.方法:将人乳腺癌细胞株MDA-MB-231改良后,接种于免疫缺陷小鼠左心室形成肿瘤骨转移,在放射性核素示踪下找到裸鼠骨转移病灶,然后切取病变骨组织进行体外培养,获得人乳腺癌骨转移细胞,用上述细胞重复以上体外-体内-体外循环3次,获得骨高转移人乳腺癌细胞株(MDA-MB-231BM3)及乳腺癌骨转移裸鼠模型.并将MDA-MB-231BM3与国外建株的人乳腺癌骨高转移细胞株MDA-MB-231BO在乳腺癌骨转移动物模型建立效率上进行比较.结果:获得以骨转移为主,兼以肺、肾上腺等组织转移的人乳腺癌细胞株MDA-MB-231BM3及其裸鼠骨转移动物模型.结论:MDA-MB-231BM3是以骨转移为主的高转移性人乳腺癌细胞株,该细胞株及其骨转移动物模型为乳腺癌转移的生物学研究提供了一个良好的技术平台.     

艾灸抑制小鼠皮下移植瘤生长的活体成像观察

目的：观察艾灸对小鼠皮下移植瘤的抑制作用。方法：通过慢病毒转染,构建能稳定表达荧光素酶基因的乳腺癌细胞株,建立小鼠皮下移植瘤模型后通过随机区组方法设为对照组和治疗组,治疗组又分为关元组、大椎组。治疗组艾灸隔天一次,每次10min,共7次,对照组不做处理。通过活体成像系统以及体外测量,监测肿瘤生长情况。治疗终点,处死小鼠,HE染色观察肿瘤的病理形态学变化。结果：建立能稳定表达荧光素酶基因的乳腺癌细胞株及小鼠皮下移植瘤模型,细胞数与荧光强度呈线性关系（ r=0.899）。活体成像显示：治疗组小鼠荧光强度较对照组明显减弱。体外测量,治疗组肿瘤体积及重量明显小于对照组（P〈0．05）,关元组和大椎组肿瘤体积和重量差异无统计学意义（P〉0．05）。与对照组相比,治疗组的肿瘤组织可见较多炎症细胞、嗜酸性增强。结论：艾灸对乳腺癌移植瘤生长有明显的抑制作用。活体成像技术可动态、连续地观察肿瘤生长变化,为临床用药提供理论依据。     

荧光蛋白活体成像系统在肿瘤研究中的应用

活体动物体内光学成像作为一项新兴的分子、基因表达的分析检测技术,已成功应用于生命科学、生物医学、分子生物学和药物研发等领域。荧光技术介导的活体动物光学成像在活体肿瘤研究中的应用已日趋深入,包括应用荧光蛋白对肿瘤内环境、肿瘤转移过程、休眠肿瘤细胞以及肿瘤治疗应答成像,本文对这些研究进展作一综述。     

动物肿瘤模型的建立及其标准研讨

动物肿瘤模型，也是人类肿瘤的复制，对肿瘤发生、发展机制的研究及肿瘤预防和治疗等研究具有重要的意义．动物肿瘤模型的建立应注意选择动物的种系和致癌物的类型。动物种系间的差异很大，相同的致癌物对不同种系的动物可诱发不同的肿瘤，因此要诱发出台适的动物肿瘤模型，动物种系的选择极为重要．动物肿瘤模型分为动物自发瘤模型．诱发瘤模型和移植瘤模型．而移植瘤模型为本文讨论的重点。人类肿瘤移植瘤(指移植于免疫缺陷动物)的来源有肿瘤活检组织，手术切除的肿瘤标本和人类肿瘤细胞系。建立移植瘤的基本条件是：肿瘤标本的取材，应在无菌条件下取新鲜、无坏死、无包膜的瘤组织，手术标本的取材应在1—2个小时内完成．移植瘤受体动物(包括免疫缺陷动物)要求在4周龄左右，移植的最常用部位是背侧皮下。移植瘤建成的标准是：传代数应在15—20代(每代传3-4只动物)；最终移植成瘤率为100％；自发消退率减少到虽低限度(不一定完全达到零)；生长速度要稳定；宿主寿命相似(重复性强)；宿主反应性低(已适应受体动物体内生长)；瘤组织的组织学结构仍保持与原发瘤相似．符合以上标准即可称为移植性肿瘤模型。     

乳腺癌转移小鼠模型的建立与评价

乳腺癌转移是造成治疗失败并导致患者死亡的重要原因。虽然早期诊断的标记物和药物治疗在一定程度上限制了癌细胞的扩散,但对防止乳腺癌的复发却无能为力。小鼠肿瘤模型在很大程度上能模拟人体肿瘤的病理生理过程,乳腺癌转移小鼠模型的建立有助于揭示乳腺癌转移机制,采用活体成像技术可以动态的监测肿瘤细胞在动物体内的转移过程。本文概括了乳腺癌转移小鼠模型的建立方法和生物学特征以及转移灶的检测。     

Advancing animal models of neoplasia through in vivo bioluminescence imaging

Malignant disease is the final manifestation of complex molecular and cellular events leading to uncontrolled cellular proliferation and eventually tissue destruction and metastases. While the in vitro examination of cultured tumour cells permits the molecular dissection of early pathways in tumorigenesis on cellular and subcellular levels, only interrogation of these processes within the complexity of organ systems of the living animal can reveal the full range of pathophysiological changes that occur in neoplastic disease. Such analyses require technologies that facilitate the study of biological processes in vivo, and several approaches have been developed over the last few years. These strategies, in the nascent field of in vivo molecular and cellular imaging, combine molecular biology with imaging modalities as a means to real-time acquisition of functional information about disease processes in living systems. In this review, we will summarise recent developments in in vivo bioluminescence imaging (BLI) and discuss the potential of this imaging strategy for the future of cancer research.     

活体自发光成像监测长春瑞滨纳米胶束抗乳腺癌转移机制研究

  目的  乳腺肿瘤细胞的转移是乳腺癌致死的主要原因。淋巴转移作为血道和淋巴两大转移途径之一,在乳腺癌转移过程中至关重要。针对乳腺癌转移的特点,本研究组设计了具有淋巴富集特性的纳米载药系统,活体动态监测其对原位乳腺肿瘤生长以及肺脏和淋巴器官转移的抑制效果,旨在为临床用药提供科学依据。  方法  采用活体自发光成像实时动态地监测了聚乙二醇化磷脂（PEG-PE）包载的长春瑞滨纳米胶束抗乳腺癌体内转移的过程。  结果  与未包载的游离长春瑞滨（Free Vin）相比,PEG-PE包载的长春瑞滨胶束（NanoVin）增强了长春瑞滨的抗原位瘤活性,显著抑制了乳腺肿瘤细胞的淋巴及肺脏转移。  结论  通过静脉给药达到了血道转移和淋巴转移两条途径的双重清扫,为靶向药物设计提供了新思路。活体光学成像技术可动态监测肿瘤的转移,为临床应用影像技术检测药物疗效提供必要的技术手段。      

Treating triple-negative breast cancer by a combination of rapamycin and cyclophosphamide: An in vivo bioluminescence imaging study

Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, has been shown to inhibit the growth of oestrogen positive breast cancer. However, triple-negative (TN) breast cancer is resistant to rapamycin treatment in vitro. We set to test a combination treatment of rapamycin with DNA-damage agent, cyclophosphamide, in a TN breast cancer model. By binding to and disrupting cellular DNA, cyclophosphamide kills cells via interfering with their normal functions. We assessed the responses of nude mice bearing tumour xenografts of TN MDA-MB-231 cells to the combination of rapamycin and cyclophosphamide in both orthotopic mammary and lung-metastasis models. We tracked tumour growth and metastasis by bioluminescent imaging and examined the expression of Ki67, CD34 and HIF-1α in tumour tissues by immunohistochemistry and apoptosis index with TUNEL assay, and found that MDA-MB-231 cells are sensitive to rapamycin therapy in orthotopic mammary, but not in lung with metastasis. Rapamycin when combined with cyclophosphamide is found to have a more significant effect in reducing tumour volume and metastasis with a much improved survival rate. Our data also show that the sensitivity of TN tumours to rapamycin is associated with the microenvironment of the tumour cells. The data indicate that in a relatively hypoxic environment HIF-1α may play a role in mediating the anti-cancer effect of rapamycin and cyclophosphamide may prevent the feedback activation of Akt by rapamycin. Overall our results show that rapamycin plus cyclophosphamide can achieve an improved efficacy in suppressing tumour growth and metastasis, suggesting that the combination therapy can be a promising treatment option for TN cancer.     

In vivo magnetic resonance imaging of the estrogen receptor in an orthotopic model of human breast cancer

Histologic overexpression of the estrogen receptor α (ER) is a well-established prognostic marker in breast cancer. Noninvasive imaging techniques that could detect ER overexpression would be useful in a variety of settings where patients' biopsies are problematic to obtain. This study focused on developing, by in vivo MRI, strategies to measure the level of ER expression in an orthotopic mouse model of human breast cancer. Specifically, novel ER-targeted contrast agents based on pyridine-tetra-acetate-Gd(III) chelate (PTA-Gd) conjugated to 17β-estradiol (EPTA-Gd) or to tamoxifen (TPTA-Gd) were examined in ER-positive or ER-negative tumors. Detection of specific interactions of EPTA-Gd with ER were documented that could differentiate ER-positive and ER-negative tumors. In vivo competition experiments confirmed that the enhanced detection capability of EPTA-Gd was based specifically on ER targeting. In contrast, PTA-Gd acted as an extracellular probe that enhanced ER detection similarly in either tumor type, confirming a similar vascular perfusion efficiency in ER-positive and ER-negative tumors in the model. Finally, TPTA-Gd accumulated selectively in muscle and could not preferentially identify ER-positive tumors. Together, these results define a novel MRI probe that can permit selective noninvasive imaging of ER-positive tumors in vivo.     

乳腺癌动物模型研究进展

乳腺癌是严重危害女性健康的恶性肿瘤，其发病率逐年递增，可能跃居为女性恶性肿瘤的首位。在对其生物学行为的认识及发展新的治疗方法上，乳腺癌动物模型起着举足轻重的作用。乳腺癌动物模型的制作对进一步研究乳腺癌的病因、发病机理，从而对其进行有效预防、提高治疗效果将起到非常关键的作用，尤其在抗癌药物的药理研究中更为重要。一个理想的动物模型，首先应与人体内肿瘤病理生理过程相似，还要便于制作、观察及对各种处理方法进行监测和优选，目前关于乳腺癌动物模型的建立方法有以下几个方面。     

Rapid and quantitative assessment of cancer treatment response using in vivo bioluminescence imaging

Current assessment of orthotopic tumor models in animals utilizes survival as the primary therapeutic end point. In vivo bioluminescence imaging (BLI) is a sensitive imaging modality that is rapid and accessible, and may comprise an ideal tool for evaluating antineoplastic therapies. Using human tumor cell lines constitutively expressing luciferase, the kinetics of tumor growth and response to therapy have been assessed in intraperitoneal, and subcutaneous, and intravascular cancer models. However, use of this approach for evaluating orthotopic tumor models has not been demonstrated. In this report, the ability of BLI to noninvasively quantitate the growth and therapeutic-induced cell kill of orthotopic rat brain tumors derived from 9L gliosarcoma cells genetically engineered to stably express firefly luciferase (9LLuc) was investigated. Intracerebral tumor burden was monitored over time by quantitation of photon emission and tumor volume using a cryogenically cooled CCD camera and magnetic resonance imaging (MRI), respectively. There was excellent correlation (r=0.91) between detected photons and tumor volume. A quantitative comparison of tumor cell kill determined from serial MRI volume measurements and BLI photon counts following 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment revealed that both imaging modalities yielded statistically similar cell kill values (P=.951). These results provide direct validation of BLI imaging as a powerful and quantitative tool for the assessment of antineoplastic therapies in living animals.     

基于磁共振成像乳腺癌远处转移预测模型的研究

  目的  建立基于平扫磁共振成像（magnetic resonance imaging，MRI）和动态对比增强（dynamic contrast enhanced，DCE）-MRI影像特征参数的乳腺癌远处转移预测模型。  方法  回顾性分析2011年1月至2016年12月3 032例于天津医科大学肿瘤医院行乳腺MRI检查并经病理证实为乳腺浸润性癌患者的临床资料，根据纳入标准筛选出转移组93例和非转移组186例。分析转移组远处转移部位与分子分型的关系，同时对两组MRI影像特征进行单因素分析及多因素Logistics回归分析，获得独立预测因子并建立预测模型。  结果  转移组中Luminal型、HER-2过表达型、三阴性乳腺癌最常见远处转移部位分别为骨、肝脏、肺脏。单因素分析结果显示，两组间的病变类型、是否多发、T1WI和T2WI信号均匀度及病灶最大径进行比较差异具有统计学意义（P < 0.05）。多因素Logistics回归分析结果显示，病变类型、是否多发、T2WI信号均匀度及病灶最大径为独立预测因子。根据独立预测因子建立的预测模型准确率、敏感度、特异度和受试者工作特征曲线（receiver operating characteristic，ROC）下面积（area under receiver operat? ing characteristic curve，AUC）分别为82.8%、85.7%、75.0%和0.801。  结论  基于MRI影像特征的模型对预测乳腺癌远处转移具有潜在价值。      

基于免疫组化和生物信息学的乳腺癌预后预测模型的建立及应用

目的应用生物信息学方法从常用的免疫组化项目中筛选出用于预测乳腺癌预后的最佳项目组合并建立预后预测模型。方法应用支持向量机分析软件对15例预后不良和30例无瘤生存乳腺癌患者的免疫组化检测结果进行分析，筛选预测预后的最佳项目组合并建立模型。结果筛选出由孕激素受体（PR）、p53蛋白、表皮生长因子受体（EGFR）、组织蛋白酶D（Cathepsin D）、增殖细胞核抗原（PCNA）和人表皮生长因子受体2（c-erbB2）共6项组成的最佳预后预测模型，对预后不良组、无瘤生存组的预测准确率分别为80．0％和90．0％，总准确率86．7％。结论利用生物信息学方法对乳腺癌患者的免疫组化检测结果进行综合分析处理有助于判断其预后，值得进一步深入研究。     

Real-time bioluminescence and tomographic imaging of gastric cancer in a novel orthotopic mouse model

Gastric cancer is the second leading cause of cancer mortality worldwide. Understanding the multistep process of carcinogenesis of gastric cancer is pivotal to develop novel therapeutic strategies. Molecular imaging in preclinical cancer models bridges the gap of laboratory-based experiment and clinical translation. To this end, the human gastric cancer cell line SGC-7901 was established to stably express luciferase and GFP by lentiviral transduction (SGC7901-Luc-GFP). Preclinical models were developed by orthotopic transplantation of SGC-7901-Luc-GFP into the sub-serosal layer of the stomach of immunocompromised mice. Tumor progression and therapeutic responses were dynamically tracked by bioluminescence imaging (BLI). Bioluminescence tomography (BLT) was used to monitor stereoscopic morphological and signal changes during tumor progression. Good correlation between cell number and bio-luminescence/fluorescence intensity was observed (R(2)=0.9983/r(2)=0.9974) in vitro. Tumor progression and therapeutic response could be successfully followed directly by BLI. Importantly, BLT provided a more accurate spatial location and tomographic quantification of the internal lesion. In conclusion, our novel bioluminescence-based preclinical gastric cancer models enable superior, noninvasive monitoring gastric cancer progression and their drug responses. The BLT technique in particular, may have great potential for future oncological studies.