内皮抑素对小鼠Lewis肺癌移植瘤抑瘤作用的分子影像研究

目的　利用绿色荧光蛋白 (greenfluorescentprotein ,GFP)标记的小鼠Lewis肺癌 (LLC)移植瘤模型 ,观察内皮抑素对LLC的作用。方法　将GFP标记的小鼠LLC细胞种植于 15只C5 7小鼠体内 ,分为空白对照组、尾静脉注射内皮抑素组、局部注射内皮抑素组。采用活体荧光成像 ,测定抑瘤率、微血管密度及测定VEGF和Bcl 2水平的方法评价治疗效果。结果　试验组抑瘤率分别为2 1 6 %和 2 7 7% ,较对照组增高 (P <0 0 5 )。尾静脉注射组MVD为 (13 77± 4 4 1)个 /高倍镜视野 ,局部注射组MVD为 (14 13± 4 0 5 )个 /高倍镜视野 ,明显低于空白对照组 (2 7 76± 8 87)个 /高倍镜视野 (P <0 0 1)。微血管计数 (MVD)密度降低 (P <0 0 5 ) ,试验组VEGF和Bcl 2强阳性较对照组少(P <0 0 1)。结论　GFP标记的小鼠LLC模型荧光显像清晰稳定。可在活体观察肿瘤细微的变化过程 ;内皮抑素对小鼠Lewis肺癌移植瘤有明显的抑制作用。     

荧光素酶活体发光技术在肺癌分子影像学研究中的初步应用

目的 建立稳定表达荧光素酶报告基因的人肺癌细胞系,并将该肺癌细胞接种于严重联合免疫缺陷(SCID)小鼠的皮下,构建异种移植动物模型,为下一步进行肺癌进展、转移以及药物敏感性等相关的分子影像学研究奠定基础. 材料与方法 利用表达重组荧光素酶基因的慢病毒载体(Lenti-Luc)感染人肺癌细胞系NCI-H460,获得稳定表达荧光素酶基因的肺癌细胞,将该细胞接种于SCID小鼠的皮下.肿瘤细胞接种2周后,利用生物发光成像系统及MRI对肿瘤进行检测,确定肿瘤的大小和位置,并进行组织病理学检查验证. 结果 接种肺癌细胞的4只SCID小鼠中,均生长出肿瘤.生物发光检测和MRI的结果 相符,病理证实小鼠皮下肿块为癌组织. 结论 成功建立了稳定表达荧光素酶报告基因的肺癌细胞系及异种移植动物模型.荧光素酶活体发光是一种进行肿瘤分子影像学研究的重要手段.     

USPIO标记脐带间充质干细胞靶向小鼠肺癌MR、病理对照研究

目的 将USPIO-PLL标记人脐带间充质干细胞(MSCs)移植入肺癌LLC种植瘤的小鼠,利用3.0T MR对MSCs活体示踪;运用免疫组化SABC法,探讨MSCs与血管内皮细胞生长因子(VEGF)表达及肿瘤血管的关系;综合分析移植MSCs对肺癌的影响.方法 培养人脐带MSCs;以多聚赖氨酸(PLL)为转染剂,用USPIO对MSCs-PLL进行磁性标记;将磁标记的MSCs通过鼠尾静脉植入LLC种植瘤小鼠体内,分别于移植前、移植后1 d、10 d行MRI观察;组织块行免疫组化.结果 (1)移植后1 d MRI可监测到MSCs到达肿瘤区,10 d到达肿瘤区MSCs增多,MRI信号降低,差异有统计学意义(P<0.05);移植1 d、10 d抑瘤率均为正值;(2)移植后10 d,MSCs移植组VEGF表达阳性率为86.67％,MVD值为44.22±12.36;NS组VEGF表达阳性率为26.67％,MVD值为20.29±8.47;VEGF阳性表达及微血管密度(MVD)值组差别均有统计学意义(P<0.05).结论 3.0T MR可以有效进行干细胞移植后的活体示踪;干细胞对肺癌具有双向作用:既可定向趋化分化抑制肿瘤生长,也可一定程度增强VEGF表达及微血管形成.     

携带绿色荧光蛋白的可诱导性真核表达载体的构建及其表达

目的　构建携带绿色荧光蛋白 (GFP)的可诱导性真核表达载体。方法　将GFPcDNA片段克隆到可诱导性真核表达载体pMD neo上 ,构建成携带绿色荧光蛋白的可诱导性真核表达载体pMD GFP ;再采用脂质体转染法 ,将GFP基因转入HCC 92 0 4细胞中 ,用荧光显微镜观察其表达情况。结果　成功构建了携带绿色荧光蛋白的可诱导性真核表达载体pMD GFP ;荧光显微镜观察显示 ,在转染GFP基因的HCC 92 0 4 /GFP细胞中 ,荧光均匀地分布于整个细胞。结论　携带绿色荧光蛋白的可诱导性真核表达载体的构建 ,为进一步观察活体内肿瘤的生长和转移提供了基础。     

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。

     

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.     

表达人HER2/neu及荧光素酶的小鼠乳腺癌模型的建立

目的建立表达HER2/neu表皮生长因子受体及萤火虫荧光素酶基因的小鼠乳腺癌细胞模型。方法将重组人HER2/neu原癌基因真核表达载体与荧光素酶报告基因依次转染小鼠乳腺癌4T1细胞系,用G418与潮霉素加压筛选阳性克隆,Western印迹及活体成像技术鉴定HER2/neu和荧光素酶在4T1细胞中的表达。在BALB/c小鼠前肢乳腺皮下注射该细胞1.5×106个,活体成像观察肿瘤的生长及转移。结果与结论经过两步转染获得抗G418及潮霉素4T1细胞,Western印迹结果显示,该细胞高表达HER2/neu。流式细胞术检测结果提示几乎所有细胞均有GFP的表达。用该细胞株接种小鼠后具有与野生型4T1细胞相似的成瘤性和转移能力。实验结果提示,我们已建立可表达HER2/neu表皮生长因子受体及荧光素酶基因的小鼠乳腺癌细胞株,为乳腺癌的研究提供一个良好的模型。     

表达人HER2/neu及荧光素酶的小鼠乳腺癌模型的建立

目的建立表达HER2/neu表皮生长因子受体及萤火虫荧光素酶基因的小鼠乳腺癌细胞模型。方法将重组人HER2/neu原癌基因真核表达载体与荧光素酶报告基因依次转染小鼠乳腺癌4T1细胞系,用G418与潮霉素加压筛选阳性克隆,Western印迹及活体成像技术鉴定HER2/neu和荧光素酶在4T1细胞中的表达。在BALB/c小鼠前肢乳腺皮下注射该细胞1.5×106个,活体成像观察肿瘤的生长及转移。结果与结论经过两步转染获得抗G418及潮霉素4T1细胞,Western印迹结果显示,该细胞高表达HER2/neu。流式细胞术检测结果提示几乎所有细胞均有GFP的表达。用该细胞株接种小鼠后具有与野生型4T1细胞相似的成瘤性和转移能力。实验结果提示,我们已建立可表达HER2/neu表皮生长因子受体及荧光素酶基因的小鼠乳腺癌细胞株,为乳腺癌的研究提供一个良好的模型。     

活体荧光成像技术评价X射线对小鼠乳腺癌移植瘤的治疗效果

目的 建立荧光素酶标记的小鼠乳腺癌细胞爪垫淋巴结转移模型,监测肿瘤早期淋巴结转移,并用荧光成像评价X射线局部治疗效果。方法 将表达荧光素酶的小鼠乳腺癌细胞系4T1-Luc接种至裸鼠爪垫皮下,建立爪垫皮下淋巴结转移模型。通过裸鼠活体荧光成像系统连续观察肿瘤细胞在淋巴结中的转移情况,并将有早期淋巴转移的荷瘤裸鼠按随机数字表法分为对照组和治疗组,活体荧光成像系统观察治疗效果,HE染色观察评价病理形态学变化。结果 成功建立了小鼠乳腺癌淋巴结转移模型,爪垫原发灶肿瘤体积与荧光光子数呈正相关性（r=0.958,P<0.001）,在肿瘤接种的第24天,治疗组爪垫肿瘤和腘窝处肿瘤部位的荧光光子数较对照组呈显著性降低（t=32.58,P<0.05）;用荧光光子数计算抑瘤率高达85%以上。HE染色观察到治疗组较对照组移植瘤坏死明显。结论 运用活体生物发光成像技术能够动态、客观、灵敏、可视化地评估X射线对小鼠乳腺癌肿瘤的抑瘤效应。     

小鼠肺癌模型的建立与MR成像

目的:观察应用小动物线圈对小鼠移植型肺癌模型MR成像的效果.方法:采用1.5T MRI仪结合小鼠专用线圈对23只小鼠肺癌A549细胞株模型进行成像.结果:①肿瘤大小:直径2～3mm 16只,直径3～4mm7只.②肿瘤数量:单个瘤体21例,两个瘤体2例.③肿瘤部位:腋下21例,侧腹壁2例.④肿瘤强化:轻度～中度强化23例.结论:小鼠肺癌模型适合用小动物线圈MR成像,对小肿瘤或早期肿瘤的研究有价值.     

Diagnostic and therapeutic evaluation of 111In-vinorelbine-liposomes in a human colorectal carcinoma HT-29/luc-bearing animal model

Colorectal carcinoma is a highly prevalent and common cause of cancer in Taiwan. There is still no available cure for this malignant disease. To address this issue, we applied the multimodality of molecular imaging to explore the efficacy of diagnostic and therapeutic nanoradiopharmaceuticals in an animal model of human colorectal adenocarcinoma [colorectal cancer (CRC)] that stably expresses luciferase (luc) as a reporter. In this study, an in vivo therapeutic efficacy evaluation of dual-nanoliposome (100 nm in diameter) encaged vinorelbine (VNB) and (111)In-oxine on HT-29/luc mouse xenografts was carried out. HT-29/luc tumor cells were transplanted subcutaneously into male SCID mice. Multimodality of molecular imaging approaches including bioluminescence imaging (BLI), gamma scintigraphy, whole-body autoradiography (WBAR) and in vivo tumor growth tracing, histopathology and biochemistry/hematology analyses were applied on xenografted SCID mice to study the treatments with 6% polyethylene glycol (PEG) of (111)In-NanoX/VNB-liposomes. In vivo tumor growth tracing and BLI showed that tumor volume could be completely inhibited by the combination therapy with (111)In-VNB-liposomes and by chemotherapy with NanoX/VNB-liposomes (i.e., without Indium-111) (P<.01). The nuclear medicine images of gamma scintigraphy and WBAR also revealed the conspicuous inhibition of tumor growth by the combination therapy with (111)In-VNB-liposomes. Animal body weights, histopathology and biochemistry/hematology analyses were used to confirm the safety and feasibility of radiopharmaceuticals. A synergistic therapeutic effect on CRC xenografted SCID mice was proven by combining an Auger electron-emitting radioisotope (Indium-111) with an anticancer drug (VNB). This study further demonstrates the beneficial potential applications of multimodality molecular imaging as part of the diagnostic and therapeutic approaches available for the evaluation of new drugs and other strategic approaches to disease treatment.     

Serial in vivo imaging of the lung metastases model and gene therapy using HSV1-tk and ganciclovir.

Noninvasive imaging in lung metastatic tumor models is used infrequently because of technical limitations in detecting metastases. We have previously used 2'-fluoro-2'-deoxy-5-iodo-1-beta-d-arabinofuranosyluracil labeled with (131)I ((131)I-FIAU) and demonstrated the applicability of noninvasive imaging for monitoring cancer gene therapy in an experimental animal model of herpes simplex virus type 1 thymidine kinase (HSV1-tk)-expressing tumor xenografts. We have now used the same animal model to effectively and noninvasively monitor the location, magnitude, and duration of therapeutic gene expression over time for the lung metastases model. METHODS: To improve the detectability of lung metastases, an experimental blood-borne lung metastases model in mice was established using intravenously administered HSV1-tk-expressing NG4TL4 fibrosarcoma cells (NG4TL4-TK) and simulated the clinical application of HSV1-tk plus ganciclovir (GCV) prodrug activation gene therapy. The efficacy of noninvasively monitoring the sites of development of lung metastatic lesions and their GCV-induced regression were assessed by SPECT with (131)I-FIAU. RESULTS: The results of this study showed that the lung metastases model of NG4TL4-TK cells could be successfully detected as early as 24 h after intravenous injection of tumor cells radiolabeled with (131)I-FIAU and also subsequently detected by extended monitoring with the intravenous injection of (131)I-FIAU on day 10. In mice treated with GCV, gamma-camera imaging demonstrated a significant growth inhibition of NG4TL4-TK cells of primary tumors and lung metastases on day 7 after initiating treatment. CONCLUSION: We conclude that this in vivo imaging approach will be useful for future studies of the lung metastases model and for the assessment of novel anticancer and antimetastatic therapies.     

Pulsed high-intensity focused ultrasound enhances systemic administration of naked DNA in squamous cell carcinoma model: initial experience

PURPOSE: To determine whether exposures to pulsed high-intensity focused ultrasound can enhance local delivery and expression of a reporter gene, administered with systemic injection of naked DNA, in tumors in mice. MATERIALS AND METHODS: The study was performed according to an approved animal protocol and in compliance with guidelines of the institutional animal care and use committee. Squamous cell carcinoma (SCC7) tumors were induced subcutaneously in both flanks of female C3H mice (n = 3) and allowed to grow to average size of 0.4 cm(3). In each mouse, one tumor was exposed to pulsed high-intensity focused ultrasound while a second tumor served as a control. Immediately after ultrasound exposure, a solution containing a cytomegalovirus-green fluorescent protein (GFP) reporter gene construct was injected intravenously via the tail vein. The mouse was sacrificed 24 hours later. Tissue specimens were viewed with fluorescence microscopy to determine the presence of GFP expression, and Western blot analysis was performed, at which signal intensities of expressed GFP were quantitated. A paired Student t test was used to compare mean values in controls with those in treated tumors. Histologic analyses were performed with specific techniques (hematoxylin-eosin staining, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling) to determine whether tumor cells had been damaged by ultrasound exposure. RESULTS: GFP expression was present in all sections of tumors that received ultrasound exposure but not in control tumors. Results of signal intensity measurement at Western blot analysis showed expressed GFP to be nine times greater in ultrasound-exposed tumors (160.2 +/- 24.5 [standard deviation]) than in controls (17.4 +/- 11.8) (P = .004, paired Student t test). Comparison of histologic sections from treated tumors with those from controls revealed no destructive effects from ultrasound exposure. CONCLUSION: Local exposure to pulsed high-intensity focused ultrasound in tumors can enhance the delivery and expression of systemically injected naked DNA.     

IBP对口腔鳞癌增殖作用的体内研究

目的 观察体内IRF-4结合蛋白（IBP）对口腔鳞癌（OSCC）细胞Tca8113生长的影响.方法 利用先前构建的高表达IBP的OSCC细胞株（Tca8113/IBP）及其空质粒对照组细胞株（Tca8113/C1）进行裸鼠成瘤试验,采用活体成像技术检测两组裸鼠接种瘤体的绿色萤光蛋白（GFP）信号.在处死裸鼠后,将接种的肿瘤组织取出并测量体积.结果 Tca8113/IBP组裸鼠显示的GFP信号区域较Tca8113/C1组显著增大,肿瘤体积也较Tca8113/C1组显著增大（P＜0.05）.结论 上调IBP表达在裸鼠体内能促进OSCC细胞的增殖.     

蒲黄水提物对小鼠Lewis肺癌的抑制作用

目的探讨蒲黄水提物对Lewis肺癌荷瘤小鼠的抑瘤作用。方法建立Lewis肺癌小鼠模型，随机分为阴性对照组，氟尿嘧啶注射液组，蒲黄水提物50、100、200rag·kg^-1·d^-1 3个剂量组，每组8只，连续治疗14d后，剥取胸腺、脾脏、肿瘤，称取重量，计算免疫器官指数及抑瘤率，并用流式细胞仪检测肿瘤细胞凋亡率及细胞周期。结果连续灌服给药14d后蒲黄水提物3个剂量，对Lewis肺癌移植瘤的生长具有明显的抑制作用，其中100、200mg·kg^-1·d^-1剂量组有显著性差异（P〈0．05）；流式细胞仪检测各剂量组均出现不同程度的细胞周期阻滞，诱导肿瘤细胞凋亡率分别为：5．73％、20．76％、4．55％。结论蒲黄水提物对Lewis肺癌小鼠移植瘤的生长有一定的抑制作用，在肿瘤的预防及综合治疗中具有一定的应用价值。     

Molecular imaging with 123I-FIAU, 18F-FUdR, 18F-FET, and 18F-FDG for monitoring herpes simplex virus type 1 thymidine kinase and ganciclovir prodrug activation gene therapy of cancer.

The ability to monitor tumor responses during prodrug activation gene therapy and other anticancer gene therapies is critical for their translation into clinical practice. Previously, we demonstrated the feasibility of noninvasive in vivo imaging with 131I-5-iodo-2'-fluoro-1-beta-D-arabinofuranosyluracil (131I-FIAU) for monitoring herpes simplex virus type 1 thymidine kinase (HSV1-tk) cancer gene expression in an experimental animal model. Here we tested the efficacy of SPECT with 123I-FIAU and PET with 5-18F-fluoro-2'-deoxyuridine (18F-FUdR), 2-18F-fluoroethyl-L-tyrosine (18F-FET), and 18F-FDG for monitoring tumor responses during prodrug activation gene therapy with HSV1-tk and ganciclovir (GCV). METHODS: In the flanks of FVB/N female mice, 4 tumors per animal were established by subcutaneous injection of 1 x 10(5) cells of NG4TL4 sarcoma cells, HSV1-tk-transduced NG4TL4-STK cells, or a mixture of these cells in different proportions to model different efficacies of transfection and HSV1-tk gene expression levels in tumors. Ten days later, the animals were treated with GCV (10 mg/kg/d intraperitoneally) for 7 d. Gamma-Imaging with 123I-FIAU and PET with 18F-FUdR, 18F-FET, and 18F-FDG were performed before and after initiation of therapy with GCV in the same animal. RESULTS: Before GCV treatment, no significant difference in weight and size was found in tumors that expressed different HSV1-tk levels, suggesting similar in vivo proliferation rates for NG4TL4 and NG4TL4-STK sarcomas. The accumulation of 123I-FIAU at 24 h after injection was directly proportional to the percentage of NG4TL4-STK cells in the tumors. The 123I-FIAU accumulation at 4 and 7 d of GCV therapy decreased significantly compared with pretreatment levels and was proportional to the percentage of HSV1-tk-positive tumor cells. Tumor uptake of 18F-FUdR in all HSV1-tk-expressing tumors also decreased significantly compared with pretreatment levels and was proportional to the percentage of HSV1-tk-positive tumor cells. The accumulation of 18F-FET decreased minimally (about 1.5-fold) and 18F-FDG decreased only 2-fold after 7 d of GCV therapy, and the degree of reduction was proportional to the percentage of HSV1-tk-positive tumor cells. CONCLUSION: We have shown that gamma-camera imaging with 123I-FIAU was the most reliable method for prediction of tumor response to GCV therapy, which was proportional to the magnitude of HSV1-tk expression in tumor tissue. 123I-FIAU imaging can be used to verify the efficacy of elimination of HSV1-tk-expressing cells by therapy with GCV. PET with 18F-FUdR reliably visualizes proliferating tumor tissue and is most suitable for the assessment of responses in tumors undergoing HSV1-tk plus GCV prodrug activation gene therapy. PET with 18F-FDG or 18F-FET can be used as additional "surrogate" biomarkers of the treatment response, although these radiotracers are less sensitive than 18F-FUdR for monitoring tumor responses to prodrug activation gene therapy with HSV1-tk and GCV in this sarcoma model.     

Multimodal imaging analysis of tumor progression and bone resorption in a murine cancer model

OBJECTIVE: This study evaluates the use of multimodal imaging to qualitatively and quantitatively measure tumor progression and bone resorption in a xenotransplanted tumor model of human neuroblastoma. METHODS: Human neuroblastoma cells expressing a luciferase reporter gene were injected into the femur of nu/nu mice. Tumor progression with and without zoledronic acid treatment was monitored using radiographs, D-luciferin-induced luminescence, micro-computer tomography (CT) and micro-magnetic resonance imaging (MRI). RESULTS: We observed a gradual increase in D-luciferin-based bioluminescence concomitant with detectable osteolytic lesions. Tumor growth was inhibited (P=0.003-0.07) with zoledronic acid treatment. Micro-CT analysis in vivo provided a method to quantify bone loss, and its prevention by zoledronic acid. High-resolution MRI images allowed the observation of tumor cells within the bone marrow cavity, as well as distant metastasis. CONCLUSION: Multimodal imaging allows to measure tumor growth and bone resorption simultaneously in vivo and also proved useful in the detection distant metastasis.     

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。