【作者】 易峰涛；

【导师】 张永学；

【作者基本信息】 华中科技大学 ， 影像医学与核医学， 2009， 博士

【摘要】 目的：在建立兔VX2移植瘤的基础上，进行¹⁸F-FDG PET／CT显像研究，探索PET／CT显像的最佳时间点；了解移植瘤的发生、发展及转移的过程；观察兔VX2移植瘤氩氦刀治疗前后PET／CT显像变化，并与病理学变化进行比较，寻找氩氦刀治疗后疗效评价的方法。方法：将肿瘤细胞悬液注射到兔大腿肌肉内或将组织碎块包埋于兔的肝脏，观测局部肿瘤的生长情况，建立兔VX2移植瘤模型。在接种后不同的时段及注射¹⁸F-FDG后不同时间进行PET／CT扫描，测定病变部位SUVave，继之进行病理特征和免疫组化的染色检测，获得兔VX2移植瘤动物模型的最佳PET／CT显像时机；将该时机兔VX2移植瘤的氩氦刀治疗术前PET／CT显像，与治疗后不同时间的PET／CT显像进行对比，同时与组织学结果进行对照，以阐明VX2移植瘤氩氦刀治疗后的变化过程及PET／CT在氩氦刀治疗后疗效监测的价值。1．VX2移植瘤动物模型制作将带有VX2肿瘤的荷瘤兔（荷瘤兔由华中科技大学同济医学院附属同济医科大学放射科赠与）麻醉后，行局部消毒，取出肿瘤组织，用剪刀将组织剪成碎沫，制成肿瘤组织混悬液备用。将健康日本大白兔麻醉后，分别在肝脏左右叶、双侧大腿及左前腿，各注入VX2肿瘤组织混悬液0.1 ml。观察不同部位如肝脏（血液供应丰富的器官）、大腿（血液供应一般部位）和前腿（血液供应贫乏的部位）肿瘤细胞的生长。2．兔VX2移植瘤¹⁸F-FDG PET／CT显像研究将日本大白兔随机分组（本研究第一部分48只大白兔分为4组；第二部分36只大白兔分为6组）；分别在肿瘤种植后不同时段（肿瘤种植后第2、4、6、8 w），注射¹⁸F-FDG后不同时间（注射放射性核素后20、40、60、80、100、120 min）进行PET／CT显像。经耳缘静脉建立静脉通路，注入2 ml生理盐水，检查静脉通路是否通畅，确认通畅后根据大白兔体重注入¹⁸F-FDG 0.75 mCi／kg，最后再注入2ml生理盐水冲管，拔出针头，压迫止血。先行CT透射扫描，然后进行PET扫描。检测肿瘤的大小，并经影像经迭代法处理和重建，分别获得CT、PET及两者融合图像。用Xeleris图像工作站对PET／CT断层图像进行分析，获得横断面数据，利用CT图像准确选取肿瘤最大切面，勾画肿瘤边缘，获得肿瘤组织的SUVave和SUVmax；勾画肿瘤旁开2cm处相等范围的正常组织，获取正常组织的SUVave和SUVmax，并和肿瘤组织的结果进行对照。3．病理学研究PET／CT显像后处死大白兔，取出肿瘤区组织，4％甲醛固定，石蜡包埋。进行常规HE染色，显微镜观察肿瘤细胞形态。单克隆抗体VEGF标记血管。依Weidner技术测量肿瘤微血管密度：先在低倍镜下（10×）寻找肿瘤血管密集区，然后用高倍镜视野（40×）计算5个视野的微血管数目，取平均值作为肿瘤微血管密度。结果1．VX2移植瘤动物模型建立的结果VX2肿瘤株容易在兔体内生长，肿瘤移植后肉眼或触诊可以观察局部肿瘤的生长情况，实验结果显示，肿瘤混悬液接种容易成活，成瘤速度快，于2～8周生长最旺盛；8周后生长速度稍减缓。8周后发现肿大淋巴结。种植瘤成瘤大小不一，生长速度有快有慢，总体来说，前腿移植瘤生长速度慢，瘤体较小；后腿及肝脏移植瘤生长较快，瘤体较大，坏死也多。总成活率80％。2．PET／CT显像结果肿瘤组织对¹⁸F-FDG有较好的摄取和滞留，注射¹⁸F-FDG后40～100分钟，肿瘤显像很清晰。小肿瘤呈结节样浓聚，较大的肿瘤可见类环状浓聚影，液化坏死区无放射性核素浓聚，与周围组织对比度好。注射¹⁸F-FDG后20、40、60、80、100min和120min的SUVave均值分别为：0.81、1.86、2.03、2.02、1.66和1.15；其中40、60和80min的SUVave与20和120min的SUVave相比差异有显著性意义，P＜0.01。种植VX2肿瘤细胞后2、4、6、8周的肿瘤组织和肿瘤旁开2cm正常组织的SUVave分别为：1.39、1.70、1.90、1.36和0.46；肿瘤组织和肿瘤旁开2cm的正常组织的SUVave进行统计学比较，差异均有显著性意义（P＜0.01）。氩氦刀治疗前后PET／CT对比，冷冻治疗后3天，放射性浓聚较治疗前明显减少，SUVave降低（治疗前的均值为2.13；治疗后为0.68），中间可见明显的放射性缺损；冷冻治疗后1周，在治疗区可见斑片状的放射性浓聚；冷冻治疗后1个月，治疗区仅现少许点片装放射性浓聚影；冷冻治疗后2个月，治疗区未见放射性浓聚影。治疗后SUVave即刻呈现出明显降低，随后上升，其后缓慢下降的趋势。3．病理学检查结果氩氦刀治疗前病理学检查提示：肿瘤大小的增加值、病理性核分裂像、平均肿瘤微血管密度与注射¹⁸F-FDG后60 min的SUVave密切相关，相关系数r=0.991、0.975、0.966，P=0.009、0.025、0.034。氩氦刀治疗前后病理学变化：冷冻治疗后第1天，绝大部分肿瘤细胞已经死亡，红染无核，细胞轮廓不清楚，主要为细胞核碎片及红、白细胞。与周围正常的肌肉组织交界分明，坏死的细胞与正常的肌肉细胞直接相邻，几乎没有过渡形态的变化。治疗后1周，坏死物质周围形成炎性反应带，坏死物质中出现大量炎性细胞浸润；治疗后1月，血管周围出现纤维母细胞，原冷冻区结构紊乱，淋巴细胞及巨噬细胞活跃其中。小血管增多。治疗后2月坏死物质消失，可见少许淋巴细胞残留，原肿瘤区形成纤维结节。充分展现了肿瘤细胞冷冻治疗后表现为肿瘤细胞的不可逆的坏死、炎性细胞浸润、机化这一过程。结论在兔VX2移植瘤动物模型建立的基础上，进行了¹⁸F-FDG PET／CT显像，研究氩氦刀治疗前后PET／CT显像的变化及与病理学改变的关系，发现兔VX2肿瘤模型模拟临床实验一般在移植肿瘤后第4周左右进行治疗实验比较合适；PET／CT显像的时间应该是在注射¹⁸F-FDG后40～100分钟，其中60～80min图像最清晰；SUVave作为PET显像中半定量参数是恶性肿瘤诊断常用的判断指标，但在动物实验中肿瘤组织的值低于2.5；PET／CT还可以清楚地显示肿瘤的解剖部位、形态、大小和肿瘤细胞的代谢状况，及时发现转移，是一项可靠和有价值的检查手段。PET／CT显像也真实的显现了肿瘤组织氩氦刀治疗后的变化过程；是氩氦冷冻治疗后效果评价较理想的方法。更多还原

【Abstract】 Purpose: The current study employed combined positron emissiontomography/computed tomography （PET/CT） imaging analysis on rabbit VX2 tumormodel, to explore optimal timing for PET/CT imaging, and to understand the initiation,development and metastasis of transplanted tumor. PET/CT imaging evaluation andpathologic examinations were also conducted after Argon-Helium cryoablation treatment,to establish protocols for evaluating clinical effectiveness and outcome.Materials and Methods: In order to establish VX2 transplanted tumor model in rabbitsand observe tumor growth, VX2 carcinoma cell suspension was injected into the rabbitleg muscle or tumor blocks were inoculated into the rabbit liver. ¹⁸F-FDG PET/CT scanswere performed at different stages after tumor inoculation to acquire PET/CT images.Standardized uptake values （SUVave） were measured at the lesion sites, along withpathological analysis and immunohistochemical staining, in order to pinpoint optimaltiming for PET/CT imaging. PET/CT imaging was performed on rabbits bearing VX2carcinoma at this selected time point and then at different time points after argon-heliumcryoablation treatment and these images compared. The images were also comparedwith the results of pathologic examination, to demonstrate the changes afterargon-helium cryoablation and to evaluate the role of PET/CT imaging in monitoring theefficacy of argon-helium cryoablation.1. Establishment of rabbit VX2 transplanted tumor modelVX2 carcinoma-bearing rabbits were anesthetized and subcutaneous tumor tissue was dissected free of surrounding fatty tissue. The tumor tissue was subsequently cut intosmall pieces by a pair of scissors in 0.9％normal saline, to make tumor cell suspension tobe used in transplantation. Healthy Japanese white rabbits were anesthetized, ventralabdomen shaved and disinfected, 1ml syringe with 50ml syringe needle was used toinject 0.1ml each of tumor cell suspension into the thigh muscle on both sides and the leftforeleg. Tumor growth was closely observed after inoculation.2. Imaging study on the rabbit VX2 transplanted tumorJapanese white rabbits were randomly divided into groups and inject ¹⁸F-FDG atdifferent time points after tumor inoculation to perform PET/CT scanning. 2mL normalsaline solution was injected via marginal ear vein of the rabbits to confirm venous access.Afterwards, ¹⁸F-FDG was injected at 0.75mCi/kg body weight, followed by another 2mLof normal saline solution to wash the catheter, and then withdraw the needle and appliedpressure to stop bleeding. CT scanning was performed, followed by PET scanning, tomeasure tumor size. Images were processed and reconstructed using iterativereconstruction algorism to obtain CT, PET and their fused images. Xeleris image fusionworkstation was used to view PET/CT cross-sectional images and obtain cross-sectionaldata. CT images were used to select maximal cross-sectional area of the tumor. Thetumor border was defined to acquire SUVave and SUVmax of tumor tissue; SUVave andSUVmax of normal tissue were calculated from region of interest 2cm away from tumortissue, for comparison.3. Pathologic studyEuthanize rabbits after PET/CT scanning. Tumor tissue was dissected and fixedwith 4％formadelhyde, followed by paraffin sectioning, H&E staining andmorphologically analysis under the light microscope. Vessels were labeled bymonoclonal antibody against Thy and VEGF. Intratumoral microvessel density wasmeasured by a method established by Weidner et al: find the area of high vessel densityunder low-magnification lenses, then switch to high magnification lenses （200x） tomeasure the number of microvessels in 5 fields and use the average number as a readout of tumor microvessel density.Results1. Results from establishing rabbit VX2 tumor modelVX2 tumor cell line grows easily in rabbit skeletal muscle and local tumor growthcan be observed or palpable after tumor cell transplantation. The present study indicatesthat VX2 tumor cell suspension inoculates easily and induces rapid tumor growth.Maximum tumor growth occurs at 2～8 weeks after inoculation and slows down after 8weeks, when swollen lymph nodes were found. Transplanted tumor mass varies in sizeand growth rate. In general, transplanted tumors in the foreleg grow at slower rate andare smaller; whereas tumors in the hind leg and liver grow faster, are bigger in size, andwith more necrosis. The overall survival rate was 80％.2. PET/CT imaging resultsTumor tissue has good uptake and retention of ¹⁸F-FDG. 40～100 minutes afterinjection of ¹⁸F-FDG, tumor tissue was clearly visualized. Smaller tumors exhibitedpunctuate nodular shadows and bigger tumors exhibited circular, high-density shadows.No staining was observed in necrotic areas, which contrasted well with surroundingtissue. SUVave measurements at 60～80 min after ¹⁸F-FDG injection differedsignificantly from SUVave at 20, 40, 100 and 120 minutes （P＜0.05）. Tumor SUVave at2, 6 and 8 weeks after VX2 cells inoculation differed significantly from SUVave ofnormal tissue 2cm away from tumors （P=0.006; 0.001, 0.000; 0.007）.PET/CT images were compared before and after argon-helium cryoablation. Oneday after the treatment, radioisotope density was significantly lower with lower SUVavemeasurement and with apparent radioactive defect in the center. One week aftertreatment, patchy lesions were observed. One month after the treatment, there was noradioisotope uptake at the treated region. SUVave measurement was significant lowerimmediately after the treatment, followed by a rebound and then a slow decline.3. Pathological findings The increase in tumor size, the number of pathological karyokinesis and averagemicrovessel density were closely related to SUVavg （P=0.009; 0.025, 0.034 respectively;r=0.091, 0.975, 0.966 respectively）. Pathological changes before and after argon-heliumcryoablation: 3 days after the treatment, the vast majority of tumor cells had died andexhibited as anucleated, cytoplasmic red staining cells, with undefined cell margins. Inthe treated region, a few infiltrating inflammatory cells can be observed, along withfragmented nucleus, red blood cells and white blood cells. A clear demarcation wasobserved between the treated region and adjacent normal tissue. Necrotic tissue wasimmediately adjacent to normal muscle cells, without transitional morphology. One weekafter the treatment, an inflammatory zone was formed surrounding the necrotic tissue anda large number of infiltrated inflammatory cells were observed. One month after thetreatment, fibroblasts were observed surrounding vessels and cryoablated regionexhibited disorganized structure, with many active lymphocytes and macrophages.Increased angiogenesis was also observed. Necrotic tissues disappeared two months afterthe treatment and fibrous nodules were formed at the previous lesion site, with a fewresidual lymphocytes. The pathological changes fully demonstrated the cellular eventsafter cryoablation treatment: irreversible necrosis of tumor cells, inflammatory cellinfiltration and fibrosis.ConclusionsRabbit VX2 transplanted tumor model was established in this study and PET/CTimaging analysis was conducted on the tumor model. A comparative study betweenPET/CT imaging with pathological findings, before and after argon-helium cryoablation,was also performed. It was found that rabbit VX2 tumor model provided the bestclinical tumor model 4 weeks after tumor transplantation. PET/CT scanning should beconducted 40～100 min after ¹⁸F-FDG injection and the image resolution was optimal at60～80 min. SUVave is one of the semiquantative PET parameters to be used in malignanttumor diagnosis and a number lower than 2.5 is considered the threshold for diagnosing malignancy in animal experiments. PET/CT imaging is a reliable and valuable tool intumor diagnosis and can be used to reveal tumor anatomical site, morphology, size andtumor cell metabolism, therefore useful in monitoring metastasis in early phase.PET/CT imaging can faithfully demonstrate the changes after argon-helium cryoablationand can be used to evaluate the effectiveness of cryoablation treatment.更多还原