兔大腿VX2移植瘤模型磁共振扩散成像研究

采用大腿肌肉注射法建立兔VX2肿瘤模型,于造模后第7天、14天及21天进行磁共振成像(MRI)平扫、增强及扩散加权成像(DWI)检查,观察不同时期MRI表现。并于造模后第21天对照大体标本测量结果比较DWI及T2WI图像肿瘤最大径,同时比较肿瘤实体部分与髂窝内转移淋巴结的表观弥散系数(ADC)值。结果显示,造模后第7天,12只模型兔MRI常规及DWI图像均可见成瘤;第14天,2例肿瘤内出现坏死灶;第21天,12例肿瘤内均出现坏死,DWI图像可发现局部淋巴结转移灶,DWI及T2WI图像肿瘤最大径与大体标本测量结果差别无统计学意义。髂窝内转移淋巴结的ADC值与原发肿瘤实体部分ADC值间差别无统计学意义。DWI可以监测兔大腿VX2肿瘤生长,有效区分肿瘤早期坏死成分,并判断相应引流区域淋巴结的性质。     

小鼠Lewis肺癌原位模型的建立

目的利用Matrigel与Lewis制备细胞混悬液注射于小鼠左肺内,建立小鼠Lewis肺癌原位模型,评价其肿瘤生长情况、转移情况,以期建立更稳定、更接近于人肺癌生长情况的小鼠肺癌原位模型。方法将处于对数生长期的Lewis肺癌细胞混悬于Matrigel中,接种于C57BL/6近交系小鼠左肺内。分别于第4、7、10、13、16天各处死5只小鼠,观察其局部成瘤率、肿瘤生长情况、中位生存期及肿瘤转移情况,并对各阶段小鼠行肺部,肝脏,肾脏,脾脏病理切片检查。结果术后第7天解剖的5只小鼠中,3只小鼠肺上可见小的瘤结节形成,其余2只肺上未见肉眼成瘤,行病理HE染色检查在显微镜下可见2只小鼠肺脏有小的瘤结节形成。术后第10天以后处死的所有小鼠肺上均有肉眼成瘤,术后第13天,所有小鼠肺原位成瘤并伴有血性胸腔积液、胸腔内转移。术后第25天,有1只小鼠出现上述转移的同时还出现了心包膜转移及肾脏远处转移。5只小鼠生存期分别为17 d、20 d、22 d、22 d、25 d,小鼠中位生存期为21.2 d（17~25 d）。成瘤率100%。结论利用Matrigel法成功建立小鼠Lewis肺癌原位模型,稳定性好,成瘤率高,并具有远处转移的特性,更接近于人肺癌的发生、发展过程。     

兔VX2肝癌模型制作改良及影像学评价

目的 对兔VX2 肝癌模型制作进行改良,以用于介入治疗学研究,同时探讨瘤灶的CT 表现及CT 在检测瘤灶中的作用.方法 将VX2瘤细胞接种于兔皮下使其成瘤并传代;新西兰兔24只,以改良嵌插法建立移植性肝癌模型,于建模后7、14、21 d分别行超声、CT及血管造影检查,用于检测兔肝VX2 瘤灶,评估瘤灶生长变化;随后处死动物,进行尸解,评估影像检查结果.结果 24只（100%） 动物以改良嵌插法建立移植性肝癌模型全部成功.瘤灶以种植后2周CT显示最清楚和典型,直径1 cm～2 cm 左右,平扫呈低密度或等密度,动脉早期明显强化,门脉期呈低密度,与周围肝组织分界较清楚.肝动脉造影显示肿瘤富血供.而种植后超过3 周的肿瘤大部分发生坏死.结论 嵌插改良法是一种值得推广的建立移植性肝癌模型的方法;在对瘤灶进行影像学评价上应尽量选择CT检查,接种后1 周左右的瘤灶较小而难以观察;2 周左右呈肝动脉源性血供丰富的约1 cm～2 cm的实体瘤,造影征像为肿瘤血管与肿瘤染色;3 周以上瘤灶大多出现明显示坏死;因此对1～2cm大小的兔VX2 肝癌瘤体,最适合行血管造影检查.     

VX2肿瘤侵犯兔下腔静脉的生物学行为观察

目的建立VX2肿瘤侵犯新西兰兔下腔静脉(IVC)的动物模型,并观察相应的生物学行为。方法经腹直视下,将VX2肿瘤组织块分别种植在28只兔肾以下IVC附近的软组织内,然后随机分为A、B两组。A组术后每周在超声下观察肿瘤的大小、IVC和腹主动脉(AA)管径大小;DSA观察侧支循环建立。B组每周随机解剖4只,取IVC和AA做病理和免疫组化(PCNA)检查,并观察转移情况。结果 1.超声显示:所有肿瘤均种植成功,肿瘤面积大小与生长时间呈明显正相关(r=0.879),IVC管径与肿瘤面积大小显著负相关(r=-0.8295);4周时25%的IVC管腔闭塞,5周时达到91.7%。2.病理和免疫组化显示:3周时IVC管壁中见大量肿瘤细胞;5周时AA管壁中未见肿瘤细胞。3.DSA显示:IVC慢性受侵犯过程中侧枝循环建立良好。4.解剖观察:模型动物在第4周开始出现肝、肺和腹腔转移。结论 VX2肿瘤成瘤率高,其生长变化、对IVC的侵犯和远处器官转移均随生长时间而变化明显。     

介绍一种兔VX2肿瘤的传代和接种方法及其应用经验和体会

目的:介绍一种兔VX2肿瘤的传代和接种方法及其应用经验和体会,从而更好的利用此模型进行生物医学研究。方法:从荷瘤新西兰大白兔取活性良好的肿瘤组织块,制备肿瘤细胞悬液,过滤后接种于健康成年新西兰兔左后肢肌肉内。通过一般观察、MRI和大体及病理学切片对肿瘤进行验证。结果:肿瘤传代和接种后生长良好,MRI、大体及组织学验证保持了VX2的肿瘤特点。结论:本研究介绍的兔VX2肿瘤的传代与接种方法稳定、可靠,值得大家推广和应用。     

兔VX2肝癌模型微波消融后磁共振灌注成像的可行性与应用价值

目的通过兔VX2模型探讨肿瘤消融治疗后动态变化过程中,磁共振灌注成像动态量化研究的可行性及其价值。方法16只新西兰大白兔分为实验组12只,对照组4只。实验组在兔肝脏种植VX2肿瘤后,观察肿瘤直径超过2.0 cm时行微波消融治疗。对比术后当天7、d、14 d及28 d实验组与对照组磁共振灌注成像量化指标—最大增强斜率（MSI）的动态变化差异,并与病理结果对照分析。结果对照组兔及实验组兔术后当天肝实质灌注MSI差异无显著性;实验组兔术前肿瘤与术后当天残留肿瘤的平均MSI差异无显著性;实验组兔残留肿瘤与良性强化组织的MSI差异有显著性。残留肿瘤的时间-信号强度曲线表现为快速上升型;良性强化组织的时间-信号强度曲线表现为缓慢上升型。结论磁共振灌注成像的动态量化研究是可行的,量化指标MSI与消融治疗后各种组织的病理结果相吻合,可更为准确地量化表达病变组织的病理状态的改变。     

循环血游离Shope病毒DNA含量与兔VX2肿瘤18F-FDG PET/CT影像表现的相关性分析

目的:探讨循环血中Shope病毒DNA含量与兔VX2肿瘤18F-FDG PET/CT影像学特征间的关系及其临床意义。方法:采用组织块接种法建立兔VX2肿瘤模型,并行18F-FDG PET-CT观测肿瘤大小及糖代谢相关值,实时定量荧光探针PCR法检测肿瘤组织及血浆中Shope病毒特征性DNA片段含量。结果:移植前外周血中未检测出Shope病毒特异性DNA片段;移植后2周,VX2肿瘤组织和循环血中均可以检测到特征性Shope病毒DNA片段。瘤体内DNA含量明显高于循环血中含量。循环血Shope病毒DNA含量与FDG-PET的最大标准摄取值(SUVmax)明显呈正相关(r=0.943,p=0.005),但与肿瘤体积相关性尚不明确(r=0.657,p=0.156)。结论:循环血Shope病毒DNA有望作为一种潜在的VX2肿瘤标志物,其廉价、无创的特性,有望在肿瘤的早期诊断和预后随访中发挥优势。     

MSCT评价兔VX_2乳腺种植瘤抗血管生成治疗的研究

探讨多层螺旋CT(multi-slice spiral computed tomography,MSCT)灌注成像与肿瘤血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的相关性以评估兔VX2乳腺种植瘤抗血管生成治疗的疗效。将69只乳腺VX2瘤兔于肿瘤生长2周后随机分为对照组(生理盐水)、恩度组(Endostar)、CEF组[环磷酰胺(Cyclophosphamide C)、表阿霉素(Epirubicin E)和5-氟尿嘧啶(5-Fluorouracil F)]、联合治疗组(Endostar和CEF)。治疗2周后对瘤兔进行MSCT灌注扫描,获得血流量(blood fl ow,BF)、血容量(blood volume,BV)、平均通过时间(mean transit time,MTT)及表面通透性(permeability surface,PS)等灌注参数均值;随后取瘤组织进行免疫组化及Western blot检测VEGF蛋白表达情况。结果显示,对照组、CEF组、恩度组、联合治疗组BF、BV和PS均与VEGF表达结果呈正相关(R对照组=0.896、0.680、0.765,RCEF组=0.877、0.876、0.852,R恩度组=0.804、0.924、0.888,R联合治疗组=0.780、0.735、0.744;P0.05),MTT均与VEGF表达结果呈负相关(R对照组=–0.591,RCEF组=–0.678,R恩度组=–0.793,R联合治疗组=–0.687;P0.05)。MSCT灌注参数与VEGF蛋白表达具有相关性,MSCT灌注参数可以反映肿瘤治疗后免疫组化与分子水平VEGF表达的变化,MSCT可以在体无创评价兔VX2乳腺种植瘤抗血管生成治疗的疗效。     

胸腔原位种植与经左心室注射建立肺癌脑转移动物模型的比较

目的为研究肺癌脑转移机制提供一种可靠的造模方法。方法 18只BALB/c nude裸鼠随机分为2组,分别经胸腔原位种植与经左心室注射的方法,接种处于对数期生长的人肺腺癌PC-9细胞(1×106/0.1 m L),接种后观察裸鼠状态,在裸鼠出现严重恶液质时处死。解剖裸鼠,观察肺、脑、肝、肾转移情况;病理取材、HE染色观察。结果胸腔原位种植组:3周后,第4、6、9号裸鼠可见胸壁瘤结凸起形成,渐增大;裸鼠于第4~6周开始出现体重减轻,并逐渐出现恶液质,分别于第5~7周处死。开胸后见:胸腔广泛灰白色肿瘤结节、团块形成,双侧肋骨、胸膜、脊柱多发种植灶,双肺被侵蚀压缩,颜色苍白,形态改变。HE染色见:肺表面广泛种植瘤形成,与正常肺组织分界清楚;仅6号裸鼠出现脑转移。经左心室注射组:裸鼠于第3周开始出现体重下降,并逐渐出现恶液质,全部裸鼠于第4周处死。开胸后:除11、18号裸鼠胸壁见2~3个散在瘤结分布(直径约1~3 mm),其余胸腔视野正常;肺组织轮廓清楚,未见瘤结生成。HE染色见:9只裸鼠均出现大小不一的多发脑转移灶。胸腔原位种植组:脑转移率为11.1%;经左心室注射组:脑转移率为100%。结论经左心室注射建立肺癌脑转移动物模型的方法,较胸腔原位种植的方法保证了更高的脑转移率。     

对比剂动力学时间分辨成像及弥散加权成像技术活体动态监测兔VX2肌肉肿瘤生物学生长与血管生成

目的:应用对比剂动力学时间分辨成像(Time Resolved Imaging of Contrast Kinetics,TRICKS)技术增强磁共振血管成像(MRangiography,MRA)及弥散加权成像(Diffusion Weighted Imaging,DWI)技术活体动态监测兔VX2肌肉肿瘤生物学生长与血管生成,探讨肿瘤血管生成与肿瘤生长的关系。方法:30只新西兰白兔,每只均在右后腿肌肉内接种VX2肿瘤细胞1×107建立肿瘤模型。分别在肿瘤接种后第4、7、10、13、16天(每个时间点6只)分别进行T1WI、T2WI、DWI、TRICKS动态增强MRA及T1WI增强延迟扫描,活体监测兔VX2肌肉肿瘤血管生成,肿瘤标本HE及CD31免疫组化染色进行验证。两位医师双盲法分别测量不同生长点肿瘤的长、短径及体积,并与大体病理标本比较;测定TRICKS增强动态MRA所能显示肿瘤血管的最小直径及形态变化;观察ADC值变化与肿瘤生长的关系。结果(:1)ADC值随着肿瘤体积的长大而逐渐增大。(2)MRI活体测定肿瘤大小与病理大体标本所测算肿瘤体积的差异无显著性。(3)TRICKS增强MRA动态显示肿瘤血管的最小...     

Percutaneous Ultrasound Guided Implantation of VX2 for Creation of a Rabbit Hepatic Tumor Model

Creation of a VX2 tumor model has traditionally required a laparotomy and surgical implantation of tumor fragments. Open surgical procedures are invasive and require long procedure times and recovery that can result in post-operative morbidity and mortality. The purpose of this study is to report the results of a percutaneous ultrasound guided method for creation of a VX2 model in rabbit livers. A total of 27 New Zealand white rabbits underwent a percutaneous ultrasound guided approach, where a VX2 tumor fragment was implanted in the liver. Magnetic resonance imaging was used to assess for tumor growth and necropsy was performed to determine rates of tract seeding and metastatic disease. Ultrasound guided tumor implantation was successful in all 27 rabbits. One rabbit died 2 days following the implantation procedure. Two rabbits had no tumors seen on follow-up imaging. Therefore, tumor development was seen in 24/26 (92%) rabbits. During the follow-up period, tract seeding was seen in 8% of rabbits and 38% had extra-hepatic metastatic disease. Therefore, percutaneous ultrasound guided tumor implantation safely provides reliable tumor growth for establishing hepatic VX2 tumors in a rabbit model with decreased rates of tract seeding, compared to previously reported methods.     

Dynamical Observation on Biological Progression of VX2 Liver Tumors to Identify the Optimal Time for Intervention in Animal Models

Purpose

Based on practice guideline of “management of hepatocellular carcinoma (HCC): update” published by American Association for the Study of Liver Diseases (AASLD) and “Barcelona Clinic Liver Cancer staging system (BCLC),” this study investigated how to enroll the optimal VX2 liver tumor model for HCC researches by dynamically observing the biological progression of the tumor.

Materials

Thirty-two healthy New Zealand white rabbits were implanted VX2 liver tumor by cell suspension method (n=24) and tissue fragment method (n=8). All the rabbits underwent CT scans on day 7, 14, 21 and 28 after implantation to observe the size of the tumors, the time when metastases and ascites occurred and the survival time. Appropriate intervention times were estimated corresponding to different clinical HCC stages by using tumor diameter-time curve.

Results

The VX2 liver tumors grew rapidly within 28 days after implantation. And the tumors in the cell suspension group grew faster than those of the tissue fragment group. The appropriate intervention time corresponding to very early stage, early stage and intermediate stage were <11 days, 11–16.9 days and >16.9 days, respectively in the cell suspension group, and <19.9 days, 19.9–25.5 days and >25.5 days, respectively in the tissue fragment group.

Conclusion

Preclinical animal research needs to improve on different levels to yield best predictions for human patients. Researchers should seek for an individualized proposal to select optimal VX2 liver tumor models for their experiments. This approach may lead to a more accurate determination of therapeutic outcomes.     

Radiofrequency Ablation of Liver VX2 Tumor: Experimental Results with MR Diffusion-Weighted Imaging at 3.0T

Purpose

To evaluate the value of DWI in detecting the lesions of pre- and post-radiofrequency ablation (RFA) of the rabbit liver VX2 tumors.

Materials and Methods

Twenty-two New Zealand White rabbits were tested. The protocol was approved by the Committee on the Ethics of Animal Experiments. Twenty separate tumor fragments were implanted into the livers of 20 rabbits, the liver was exposed by performing midline laparotomy. 3.0T MR DWI (b = 0, 200, 400, 600, 800,1000 s/mm²) were performed 14–21 days after tumor implantation (mean, 17 days) in the 18 tumor-bearing animals. Then RFA was performed in the 18 tumor-bearing animals and in the two healthy animals. 3.0T MR DWI was performed 7–10 days after RFA (mean, 8 days). Pathology exam was performed immediately after the completion of post- RFA MR imaging. Analyzing the features of MRI and ADC values in the pre- and post- RFA lesions of the VX2 tumors, and histopathologic results were compared with imaging findings.

Results

The difference of ADC value between viable tumor and normal liver parenchyma was significant (P<.001). After RFA, when b = 200, 400, 600, 800, 1000 s/mm², the differences of ADC values of viable tumor, granulation tissue, necrosis, normal liver parenchyma were significant (P<.001). At the time the animals were sacrificed after RFA and MR imaging, histopathologic results of local viable tumors were found in 9 (50%) of the 18 treated tumors. Macroscopic viable tumors were found at the RFA sites in 3 (17%), all 3 macroscopic viable tumors were visualized at the periphery of the RFA areas.

Conclusions

3.0T MR DWI can be used to follow up the progress of the RFA lesion, it is useful in detecting different tissues after RFA, and it is valuable in the further clinical research.     

MSCT评价兔VX2乳腺种植瘤抗血管生成治疗的研究

探讨多层螺旋CT（multi—slice spiral computed tomography,MSCT）灌注成像与肿瘤血管内皮生长因子（vascular endothelial growth factor,VEGF）表达的相关性以评估兔VX2乳腺种植瘤抗血管生成治疗的疗效。将69R乳腺VX：瘤兔于肿瘤生长2周后随机分为对照组（生理盐水1、恩度组（Endostar）、cEF组[环磷酰胺（Cyclophosphamide C）、表阿霉素（EpirubicinE）和5-氟尿嘧啶（5．FluorouracilF）]、联合治疗CR（Endostar和CEF）。治疗2周后对瘤兔进行MSCT灌注扫描,获得血流量（bloodflow,BF）、血容量（bloodvolume,BV）、平均通过时间（meantransittime,MTT）及表面通透性（permeabilitysurface,PS）等灌注参数均值：随后取瘤组织进行免疫组化及Westernblot检测 VEGF蛋白表达情况。结果显示,对照组、CEF组、恩度组、联合治疗组BF、BV和Ps均与VEGF表达结果呈正相关（R对照组=0．896、0．680、0．765,RCEF组=0．877、0．876、0．852,R恩度组=0．804、0．924、0．888,R联合治疗组=0．780、0．735、0．744;P〈0．05）,MTT均与VEGF表达结果呈负相关（R对照组＝-0．591,RCEF组=0．678,R恩度组=0．793,R联合治疗组=-0．687;P〈0．05）。MSCT灌注参数与VEGF蛋白表达具有相关性,MSCT灌注参数可以反映肿瘤治疗后免疫组化与分子水平VEGF表达的变化,MSCT可以在体无创评价兔VX2乳腺种植瘤抗血管生成治疗的疗效。     

Tumor Angiogenesis after Heated Lipiodol Infusion via the Hepatic Artery in a Rabbit Model of VX2 Liver Cancer

Objectives

This study aimed to observe the changes in tumor angiogenesis after heated lipiodol (60°C) infusion via the hepatic artery in a rabbit model of VX2 liver cancer.

Materials and Methods

Twenty rabbits with VX2 hepatic tumors were randomly divided into 2 groups (10 rabbits in each group). Under anesthesia, a trans-catheter hepatic arterial infusion was performed, and lipiodol (37°C; control group) or heated lipiodol (60°C; treated group) was injected into the hepatic arteries of the animals. Then, changes in tumor angiogenesis were assessed using the following markers and methods. 1. Vascular endothelial growth factor receptor (VEGFR) and vascular endothelial growth factor (VEGF) expression levels in the tumor were assessed using western blotting and real-time quantitative polymerase chain reaction (PCR). 2. Proliferating cell nuclear antigen (PCNA) expression in the tumor was assessed through immunohistochemical staining. 3. The morphological changes in tumor vascular endothelial cells were observed using transmission electron microscopy (TEM).

Results

VEGFR and VEGF mRNA and protein expression levels were reduced in the treated group compared to the control group. PCNA protein showed reduced expression levels in the treated group compared to the control group. TEM indicated that the endothelial cell endoplasmic reticulum expanded, the chondriosome was swollen, and the endothelial cell microvilli were decreased after heated lipiodol infusion.

Conclusions

The tumor angiogenesis of rabbits with VX2 cancer was inhibited after arterial heated lipiodol infusion compared to lipiodol infusion.