能谱CT评价兔VX2肝移植瘤抗血管生成的实验研究

发布时间：2018-01-24 15:49

  本文关键词： 兔VX2肝癌模型 能谱CT CT灌注成像 碘含量 血管内皮抑制素 肿瘤血管生成 成纤维细胞生长因子　出处：《郑州大学》2014年硕士论文　论文类型：学位论文

【摘要】：实验目的： 1分析兔VX2肝脏移植瘤在能谱CT灌注扫描的时间密度曲线、灌注参数特点。 2分析能谱成像扫描的单能量图像特点，能谱衰减曲线、碘含量的特点。 3分析重组入血管内皮抑制素（恩度）治疗后兔VX2肝移植瘤在灌注参数及能谱扫描碘含量的变化特点。 4用Western-blot检测所取组织的FGF2蛋白的表达并与CT灌注参数、标准化碘含量（NIC）进行相关性分析。 材料和方法： 由河南省实验动物中心提供健康新西兰大白兔52只（体重2.6～2.9Kg），实验得到郑州大学实验动物伦理委员会许可，采用开腹种植新鲜瘤组织块法建立肝移植瘤模型。 1抗血管生成治疗方案 模型建立中，由于麻醉意外，死亡1只。在移植瘤术后7天、14天经CT检查1只模型兔未见成瘤，剔除后续试验。从48只成功VX2肝脏移植瘤模型随机挑选2只，做病理检查HE染色。剩余46只进行分组，每组23只，对照组（A组）于移植瘤术后14天开始，每天静脉注射和治疗组相同剂量安慰剂，生理盐水，连续注射7天。治疗组(B组)于移植瘤术后第14天开始，每天静脉注射重组入血管内皮抑制素（恩度），（剂量为0.3mg/kg），连续注射7天。 2CT影像学检查 选用GE公司Discovery CT750HD对建模成功的兔VX2肝移植瘤在种植后7天、14天、21天（用药后7天）均行CT灌注扫描和能谱扫描（gemstone spectralimaging）。将重建图像数据传输到影像后处理工作站(Advantage Windows4.6, GEHealthcare)进行分析。 ①用CT灌注功能分析软件进行分析CT灌注成像：选择主动脉作为输入动脉，门静脉为输入静脉。在轴位上划感兴趣区，第一个感兴趣区选择整个肿瘤组织，第二个感兴趣区选择远离肿瘤的无瘤肝实质内。分别获得时间密度曲线（Time density cure，TDC）。自动生成灌注参数：血流量BF（blood flow）（ml/min/100mg），血容量BV（blood volume）（ml/100mg），平均通过时间MTT（mean transit time）（s），毛细血管表面通透性PS(permeability of the capillaryvessel surface)（ml/min/100mg）和肝动脉分数HAF（Hepatic arterial fraction），并得到相应的伪彩图。 ②用能谱成像（gemstone spectral imaging，GSI）后处理软件分析GSI图像：获得常规CT平扫图像，动脉期常规混合能量图像（conventional polychromaticimage，QC）并重建出单能量图像（monochromatic image，Mono）。对40keV水平、最佳信噪比（optimal CNR，OP）keV水平、常规混合能量（QC）水平三组图像的信噪比(contrast-to-noise ratio，，CNR)，图像噪声（niose）进行定量比较，并对三组图像的病灶显著性和总体图像质量进行评分（lesion conspicuity scores，LCS；overall image quality scores，OQS）；分析肿瘤区域以及无瘤肝组织的能谱曲线特征；在碘基图上定量测量肿瘤区域、无瘤肝组织、腹主动脉的碘含量，分别计算标准化碘含量（normalized iodine concentrations，NIC），比较治疗前后，肿瘤区域之间标准化碘含量的差异。 3灌注参数及标准化碘含量与分子生物学的相关性分析 每次CT扫描结束后，用CT引导下穿刺的方法取活体组织（肿瘤组织和远处无瘤肝组织）存放在-196℃的液氮中冷藏。western-blot检测组织内成纤维细胞生成因子（FGF1）蛋白的表达。按照蛋白样品的制备→SDS-PAGE→转膜→封闭→一抗杂交→二抗杂交→底物显色的实验流程，测量组织内FGF2蛋白的表达，分析灌注参数及标准化碘含量与FGF2表达的相关性。 结果 1兔VX2肝移植模型的CT表现及大体病理 ①正常兔肝脏，肝左叶大于肝右叶，增强扫描动脉期呈轻度强化，门静脉期强化程度增强。大体观：正常兔肝脏，位于腹腔前部，从肝脏腹面的裂沟可以将肝脏分为4部分，左侧两个肝叶肥大，右侧肝叶较小，左叶和右叶中间有狭窄的中央叶，在中央叶前方的肝门处还有尾状叶和乳头窦。新鲜的兔肝脏呈红褐色。重量约85～130g。显微镜下，可见肝细胞排列规整，细胞核形态规则，大小均匀，肝小叶、肝窦结构清晰。 ②种植成功的兔VX2肝移植瘤，CT平扫呈类圆形低密度影，增强扫描动脉期呈环形强化，环形壁较厚，门静脉期呈相对低密度，随着肿瘤增长，中心液化坏死增多。大体观：移植瘤呈类圆形，灰白色，包膜完整，中心切面可见液化坏死。低倍镜下可见瘤细胞呈巢状分布，肿瘤间质内有大量新生的毛细血管，高倍镜下，瘤细胞体积大，细胞形态不规则，大小不均，细胞核大、深染，可见异性型细胞核，核分裂像多见。 2兔VX2移植瘤灌注结果 ①肿瘤区域BF、BV、PS、HAF高于无瘤肝组织，肿瘤区域MTT低于无瘤肝组织。分别进行统计学分析具有统计学意义（p＜0.05）。②术后7天到14天随着肿瘤的生长，肿瘤组织CT灌注参数BF、BV、HAF呈增高的趋势，MTT、PS呈减低的趋势，其中，BF、MTT、PS差异具有统计学意义（p＜0.05），而BV、HAF差异没有统计学意义（p＞0.05）。无瘤肝组织从术后7天到14天各个灌注参数值随肿瘤生长无明显变化（p＞0.05）。③重组人血管内皮抑制素（恩度）治疗后，治疗组BF、BV、PS、HAF较对照组减低，MTT较对照组增高，分别进行统计学分析，BF具有统计学意义（P＜0.05），其余参数无统计学意义（p＞0.05）。④各灌注参数与血管生成因子FGF2蛋白进行相关分析，BF与FGF1蛋白之间有相关性，相关系数为r=0.932其他各灌注参数与FGF1蛋白之间无相关性。 3能谱CT扫描结果 ①三组图像评估：OP组CNR（2.63±1.59）低于40keV（2.81±1.74）两者进行统计学分析无统计学意义（p＞0.05），OP组CNR高于QC组（1.95±1.55）进行统计学分析有统计学意义（p＜0.05），40keV组和QC组之间的差异有统计学意义（p＜0.05）。图像噪声，OP组（7.89±1.35）低于40keV组（9.12±1.28）高于QC组（6.67±1.36）分别进行统计学分析差异有统计学意义（p＜0.05）。病灶显著性评分（LCS），OP组（3.86±0.68）与40keV组（3.77±0.57）之间的差异没有统计学意义（p＞0.05），两组分别都高于QC组（3.29±0.49）之间的差异有统计学意义（p＜0.05）。总体质量评分（OQS），OP组（3.85±0.58）高于40keV组（3.10±0.25）和QC组（3.38±0.55）之间的差异有统计学意义（p＜0.05）。 ②能谱曲线分析：计算能谱曲线上不同单能点的斜率，计算公式为Kx=(HU40keV-HUXkeV)/(X-40)，其中Kx为不同单能量点所对应的斜率，HU40keV为40keV的CT值，HUXkeV为不同单能量所对应的CT值。在各个单能量点，肿瘤组织的斜率均大于无瘤肝组织，进行统计学分析，有统计学意义（p＜0.05）。 ③标准化碘含量（NIC）：7天、14天肿瘤组织的NIC大于无瘤肝组织，之间的差异具有统计学意义（p＜0.05）。重组人血管内皮抑制素恩度治疗后，治疗组肿瘤区域NIC小于对照组，二者之间的差异有统计学意义（p＜0.05）。NIC与FGF2蛋白进行先关性分析有良好的相关性，相关系数为r=0.957。 结论： 1采用开腹种植瘤块法建立兔VX2肝移植瘤模型，成瘤率高，成瘤时间短，移植瘤形态规则，转移率低，其病理学表现及生物学行为接近人类原发性肝细胞肝癌，该模型可广泛应用于相关的影像学及血流动力学的基础研究。 2CT灌注成像及能谱成像影像，分别通过生成灌注参数和碘基值可以提供肝移植瘤的血流灌注及能谱成像信息，反映肿瘤的血流动力学。能谱成像在最佳单能量水平更有助于对肿瘤的检出。 3FGF2蛋白在肿瘤组织中处于高表达状态，可以反映肿瘤的血供，在临床上，FGF2可以反映提供有关肿瘤的血管生成的相关信息。为观察肿瘤生成及治疗提供可参考的生物学指标。 4重组人血管内皮抑制素（恩度）表现出明显的抑制肿瘤血管生成作用，在化疗的过程可用于CT灌注成像及能谱成像观察肿瘤血管生成。可通过CT灌注参数及能谱扫描评价抗血管治疗的效果，这项无创检查技术值得进一步推广应用于临床的肝癌的诊断及疗效判定的研究。
[Abstract]:Objective:
1 the time density curve and perfusion parameters of VX2 liver transplantation tumor in rabbit were analyzed by CT perfusion scan.
2 the characteristics of the single energy image, the spectral attenuation curve and the iodine content of the spectral imaging scan are analyzed.
3 the changes of the perfusion parameters and the content of iodine in the rabbit VX2 liver transplantation tumor after recombinant endostatin (endostatin) were analyzed.
4 Western-blot was used to detect the expression of FGF2 protein in the tissue and to analyze the correlation between the CT perfusion parameters and the standardized iodine content (NIC).
Materials and methods:
52 healthy New Zealand white rabbits (weighing 2.6 to 2.9Kg) were provided by Henan experimental animal center. The experiment was approved by the laboratory animal ethics committee of Zhengzhou University, and a liver transplantation tumor model was established by planting fresh tumor tissue in the open abdomen.
1 antiangiogenic treatment scheme
In the model, because of anesthesia accident death of 1. In the transplanted tumor after 7 days, 14 days CT 1 rabbit models were tumorigenic, excluding the follow-up test. From 48 successful VX2 liver tumor model of randomly selected 2 rats for pathological examination of HE staining. The remaining 46 groups, each group of 23 only, the control group (A group) in the beginning 14 days of transplantation tumor after operation, daily intravenous injection and treatment group the same dose of placebo, normal saline, continuous injection of 7 days. The treatment group (B group) on the fourteenth day after transplanted, every day into the intravenous injection of recombinant endostatin (Endostar), dose (0.3mg/kg), continuous injection of 7 days.
2CT imaging examination
The GE Discovery CT750HD for modeling the success of rabbit VX2 liver tumor in the field after 7 days, 14 days, 21 days (7 days after treatment) underwent CT perfusion scan and EDS (gemstone spectralimaging). The reconstructed image data to the image postprocessing workstation (Advantage Windows4.6, GEHealthcare) were analyzed.
The CT perfusion software to analyze CT perfusion imaging: the aorta as input artery, portal vein. The input for ROI in the axial region of interest, the first choice of the tumor, second regions of interest from tumor tumor free liver parenchyma. Obtain time density curve respectively (Time density cure, TDC). The blood flow perfusion parameters: automatic generation of BF (blood flow) (ml/min/100mg), blood volume (BV blood volume) (ml/100mg), mean transit time MTT (mean transit time) (s), permeability surface (permeability PS of the capillaryvessel (ml/min/100mg) and surface) hepatic arterial fraction HAF (Hepatic arterial fraction), and get the corresponding pseudo color.
The spectral imaging can be used (gemstone spectral imaging, GSI) analysis software GSI image postprocessing: conventional CT scan image, the arterial phase conventional mixed energy images (conventional polychromaticimage, QC) and reconstruct the single energy image (monochromatic image, Mono). The level of 40keV, the best signal-to-noise ratio (optimal CNR, OP the level of keV), conventional mixed energy (QC) SNR level of three sets of images (contrast-to-noise ratio, CNR), image noise (niose) for quantitative comparison, and were significant and the overall image quality of three groups of image score (lesion conspicuity scores overall image quality scores, LCS; OQS); analysis of tumor area and tumor free liver tissue spectrum curve characteristics; quantitative measurement of tumor in iodine kitu region, tumor free liver tissue, the iodine content of the abdominal aorta, the iodine content of standard were calculated (normalized iodine concentrat Ions, NIC), compare the difference of the standard iodine content between the tumor regions before and after the treatment.
The correlation analysis between 3 perfusion parameters and the standard iodine content and molecular biology
Every time after CT scan, and living tissue by CT method under the guidance of puncture (tumor tissues and distant tumor free liver tissue) stored in liquid nitrogen frozen -196 degrees of detection of.Western-blot in fibroblast growth factor (FGF1) protein expression. According to the experimental process of protein sample preparation, SDS-PAGE, transfer film, a closure, anti - two hybrid, hybrid anti chromogenic substrate, FGF2 protein expression measurement within the organization, analysis of the correlation between perfusion parameters and expression of normalized iodine content and FGF2.
Result
The CT manifestation and general pathology of 1 rabbit VX2 liver transplantation model
The normal rabbit liver, the left hepatic lobe is larger than the right lobe of the liver, enhanced scan and slight enhancement in the arterial phase, portal venous phase enhancement enhancement. General observation: normal rabbit liver, located in the abdominal part from the liver, ventral fissure can be divided into 4 parts on the left side of the liver, two hepatic lobe hypertrophy, right lateral lobe of the liver., with a narrow central lobe between right and left lobe, in front of the door at the central lobe of liver and caudate lobe and papillary sinus. Fresh rabbit liver is red brown. The weight is about 85 ~ 130g. under the microscope, visible liver cells arranged in neat nuclei in regular shape, uniform size, hepatic lobule, hepatic sinus clear structure.
The success of rabbit VX2 liver tumor. CT scan showed circular low density, enhanced scan showed ring like enhancement, the annular wall is thick, the portal venous phase showed relatively low density, with tumor growth, central liquefaction and necrosis increased. General observation: transplanted tumors were round, gray white, complete capsule the center section, visible necrosis. Under low magnification microscope nests of tumor cells, tumor stroma within a large number of new capillaries, at high magnification, the tumor cells were large and irregular in shape, uneven size, big nucleus anachromasis, visible type specific nuclei and mitotic more common.
The results of 2 rabbit VX2 transplanted tumor
鈶犺偪鐦ゅ尯鍩烞F,BV,PS,HAF楂樹簬鏃犵槫鑲濈粍缁

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