兔VX2肝癌模型制作技术改良

目的探讨制作兔VX2肝癌模型的新技术、新方法。方法将新西兰兔30只随机等分为3组,分别为嵌插组、改良嵌插组和经皮穿剌组。分别按不同方法建立肝癌模型,于建模后第1、2、3周分别进行CT扫描,评估肿瘤大小。3周后处死动物进行解剖,观察肝脏成瘤及转移情况,比较各组转移率,并行常规病理组织学检查。结果 30只动物建模成功28只,成功率93%,经皮穿剌组有2只肝脏未见种植灶,嵌插组和改良嵌插组全部种植成功。改良嵌插组腹壁转移率显著低于嵌插组和经皮接种组。结论改良嵌插法3周内不会形成接种部位腹壁以及远处的转移,是一种理想的移植性肝癌模型建模方法。     

肝动脉造影和碘油CT 对小肝癌的诊断( 附15 例报告)

目的：探讨肝动脉造影和碘油CT对小肝癌诊断和临床应用价值。材料和方法：临床诊断肝癌术后复发、超声、平扫厦增CT检查、MRI检查肝内病灶最大径≤1cm患者15例。经股动脉插管肝动脉造影观察肿瘤染色后再注入碘油4～12ml。1—4周后作CT扫描,再根据CT检查结果作出诊断。结果：15例共发现42个病灶。在41个小肝癌病灶中五种影像方法检出率分别为超声70．6％,CT60％,MRI66％,血管造影88．5％,碘油CT99％。肝动脉造影和碘油CT均明显为优。结论：本研究显示肝动脉造影和碘油CT是目前诊断小肝癌最敏感和准确的影像方法之一。     

兔VX2肺外周肿瘤超声造影特征与CD31和CD34微血管密度的相关性研究

摘要 目的：探讨兔VX2肺外周肿瘤超声造影特征与CD31和CD34微血管密度的相关性研究。方法：15只雌性新西兰白兔进行VX2 肺肿瘤模型，总共有30个VX2 癌植入15只兔子的肺中。通过高倍镜分析不同期VX2肿瘤的MVD。通过双重免疫荧光化学染色分析不同生长期VX2肿瘤Ki67和CD31阳性表达。分析肿瘤形态学组织病理学结果与微血管分布和CEUS结果之间的相关性。结果：随着生长周期的进展，VX2肿瘤的MVD增大（P<0.05）。随着生长周期的进展，VX2肿瘤内Ki67和CD31的阳性表达率增加（P<0.05）。CEUS显示VX2 肿瘤在早期动脉期有明显的外周增强和短暂的内部增强。肺外周VX2 肿瘤中 PI和CD31 MVD值之间呈显著正相关性（r=0.734，P<0.05）。CD31 MVD和CD34 MVD之间呈负相关（r=-0.691，P<0.05）。PI和CD34 MVD值之间不存在显著相关性（r=-0.412，P>0.05）。结论：与CD34 MVD 相比，CD31 MVD 可以更有效地评估肿瘤血管生成。CEUS作为一种无创成像方法，可以有效评估兔周围型肺癌的肿瘤血管生成情况。     

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。     

兔脑种植VX2肿瘤动物模型的建立

目的建立兔VX2肿瘤脑内种植动物模型,观察其生长特性。方法采用兔脑内VX2肿瘤组织块种植法将VX2肿瘤组织块种植入24只成年New Zealand大白兔右侧大脑皮质内,用B超检测肿瘤的生长情况,在实验兔在肿瘤种植后第13、171、9、21、232、5天取材,进行组织学观察。并观察实验兔在种植VX2肿瘤后的生存期及出现厌食、偏瘫等神经系统体征和死亡的时间。结果VX2肿瘤组织块种植入脑内后荷瘤兔的中位生存期为24.5 d,平均生存期为24.8 d,肿瘤体积随种植后的时间在对数坐标中接近一条直线。VX2肿瘤种植入兔脑17 d后血供较丰富、呈鱼肉状生长,与正常脑组织边缘界限不清楚,第17~19天肿瘤中心出现坏死并出现腹腔内转移。光镜下从VX2肿瘤种植后第17天开始肿瘤细胞向正常脑组织浸润,并形成瘤巢,瘤周脑组织形成水肿带。结论在缺乏兔源性脑肿瘤的情况下,采用兔VX2肿瘤组织块颅内种植能较好模拟颅内肿瘤生长,为对脑肿瘤的某些实验研究提供条件。     

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。     

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。     

放射性粒子125I联合TACE治疗兔VX2肝肿瘤的实验研究

目的:通过研究不同活度的125I粒子以及联合TACE治疗兔VX2肝移植癌的疗效及其病理基础,探讨125I粒子组织间植入治疗肝癌的有效性。方法:建立兔VX2肝癌模型。60只肝癌模型兔随机分成5组,对照组(A组)植入空白剂量(0mCi)125I粒子,B组植入1.0mCi125I粒子,C组植入0.7mCi125I粒子,D组植入0.4mCi125I粒子,E组植入0.7mCi125I粒子+TACE。观察植入前后各组肿瘤体积并计算抑瘤率,切除肿瘤组织及周围正常组织进行常规病理检查。结果:各治疗组肿瘤大小在治疗前后比较具有统计学差异(P<0.01),均小于同期对照组(A组)(P<0.01)。在不同观察时期抑瘤率差别均有统计学意义(P<0.05),各个组间抑瘤率差异在治疗后2周最为明显(P<0.01),但均高于D组(P<0.01)。治疗后6周病理提示D组部分组织内仍可见少量肿瘤细胞,而其余各治疗组均未见明显的肿瘤细胞残存,B组对周围肝组织损伤较大,C组、E组适中。总体疗效E组优于其余各治疗组。结论:125I粒子联合TACE治疗肝癌效果明显优于单一的治疗方案,是肝癌目前较为理想的治疗方案,其中单个粒子活度以0.7mCi左右较为适宜。     

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。     

89例巨块型原发性肝癌介入化疗栓塞治疗体会

目的探索巨块型原发性肝癌介入栓塞治疗的疗效。方法89例巨块型原发性肝癌患者,经皮股动脉穿刺插管至肝动脉,化疗栓塞治疗肝癌;碘油用量为20～50ml。再注入明胶海绵颗粒栓塞治疗。发现肝外肿瘤供血动脉,超选择捅管化疗栓塞后,注入适量明胶海绵栓塞治疗。结果本组病例中首次DSA造影发现11例存在肝外动脉供血;64例出现肝外供血动脉共计67支。术后甲胎球蛋白下降均〉50％。术后4～6周复查田,肿瘤最大直径缩小3．5～5．9cm。1、2、3年累计生存率分别为73．8％、48．3％和28．5％。结论巨块型原发性肝癌大剂量碘油栓塞联合肝外肿瘤供血动脉介入治疗,对于提高巨块型原发性肝癌的介入疗效具有重要意义。     

CT imaging with iopromide liposomes in a rabbit model

The aim of this study was to evaluate the computed tomography (CT)-imaging potential of iopromide-carrying liposomes (SPC/CH/SPG, 6:3:1) of approximately 200?nm in diameter in healthy rabbits and in rabbits with implanted liver tumors in an intraindividual comparison with iopromide. Normal rabbits and animals with VX2 tumors implanted into the liver received iopromide (600?mg of iodine/kg, bolus injection) and, 1 or 2 days later, iopromide liposomes (300?mg of iodine/kg, bolus injection or 10-minute infusion). CT imaging up to 1 hour after administration was performed, focusing on the aorta, vena cava, kidney, spleen, and liver. Pharmacokinetic parameters for CT enhancement were calculated. Detectability and delineation of liver lesions were assessed on a 4-grade scale, and differences were evaluated statistically. Using half the iodine dose, iopromide liposomes achieved similar blood-pool enhancement as iopromide. Detectability and delineation of liver lesions were easy/good in the arterial phase after iopromide injection, but poor in the venous and equilibration phases. Iopromide liposomes resulted in a long-lasting, good detectability and delineation of liver lesions similar or superior to that observed after iopromide in the arterial phase.     

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.     

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。     

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.     

兔肝VX2肿瘤射频消融术后残癌中基质金属蛋白酶9表达

目的:探讨兔肝脏VX2肿瘤射频消融术(radio-frequency ablation,RFA)后残余肿瘤组织中基质金属蛋白酶9(MMP-9)表达。方法:超声引导下将VX2肿瘤组织块接种于23只新西兰大白兔肝脏中,造模成功后随机分为5组,对照组(A组,n=3),不行RFA治疗;余20只为实验组行RFA治疗,在超声引导下射频针插入肿瘤偏心位置,展开电极致损毁范围最大为肿瘤总体积的2/3,人为造成残存肿瘤组织。根据治疗结束后不同时间点分为4组:0小时组(B组,n=5)、术后1周组(C组,n=5)、术后2周组(D组,n=5)、术后4周组(E组,n=5),行超声检查结束后处死大白兔,取肿瘤组织采取免疫组化法观察残存肿瘤组织中及未治疗肿瘤组中MMP-9的表达情况。结果:RFA术后0小时、1周、2周、4周残存肿瘤组织中MMP-9的表达均较对照组明显降低(P0.05)。结论:RFA治疗后残存肿瘤细胞中MMP-9水平表达减低。     

A Novel Minimally Invasive Technique to Create a Rabbit VX2 Lung Tumor Model for Nano-Sized Image Contrast and Interventional Studies

Background

The rabbit VX2 lung cancer model is a large animal model useful for preclinical lung cancer imaging and interventional studies. However, previously reported models had issues in terms of invasiveness of tumor inoculation, control of tumor aggressiveness and incidence of complications.

Purpose

We aimed to develop a minimally invasive rabbit VX2 lung cancer model suitable for imaging and transbronchial interventional studies.

Methods

New Zealand white rabbits and VX2 tumors were used in the study. An ultra-thin bronchoscope was inserted through a miniature laryngeal mask airway into the bronchus. Different numbers of VX2 tumor cells were selectively inoculated into the lung parenchyma or subcarinal mediastinum to create a uniform tumor with low incidence of complications. The model was characterized by CT, FDG-PET, and endobronchial ultrasound (EBUS). Liposomal dual-modality contrast agent was used to evaluate liposome drug delivery system in this model.

Results

Both peripheral and mediastinal lung tumor models were created. The tumor making success rate was 75.8% (25/33) in the peripheral lung tumor model and 60% (3/5) in the mediastinal tumor model. The group of 1.0×10⁶ of VX2 tumor cells inoculation showed a linear growth curve with less incidence of complications. Radial probe EBUS visualized the internal structure of the tumor and the size measurement correlated well with CT measurements (r² = 0.98). Over 7 days of continuous enhancement of the lung tumor by liposomal contrast in the lung tumor was confirmed both CT and fluorescence imaging.

Conclusion

Our minimally invasive bronchoscopic rabbit VX2 lung cancer model is an ideal platform for lung cancer imaging and preclinical bronchoscopic interventional studies.