【作者】 吴越；

【导师】 梁长虹；

【作者基本信息】 南方医科大学 ， 影像医学与核医学， 2010， 博士

【摘要】 第一部分兔VX2肝种植瘤模型的建立及该模型不同生长期的MSCT灌注特点目的：通过对兔肝脏种植VX2瘤株建立动物模型,为后续研究提供实验基础。并对该模型不同生长期的MSCT灌注参数进行研究,为MSCT灌注成像成为评价该肿瘤生长的影像学手段提供实验依据。方法：10只新西兰大白兔,采用动物后肢皮下注射接种传代保存瘤种。然后采用开腹瘤块包埋法将其种植于兔肝左叶上。分别于种瘤前、种瘤后第1wk、2wk和第3wk行MSCT灌注扫描,对比种瘤前肝实质与种瘤后肿瘤区域以及第2wk肿瘤区与非肿瘤区肝脏的肝血流量(HBF)、肝血容积(HBV)、平均通过时间(MTT)、毛细血管表面通透性(PS)和肝动脉分数(HAF)的变化规律。种瘤后第3wk于CT检查完成后处死实验兔,进行肝脏病理学检查。所有结果均进行统计学分析。结果：所有10只新西兰大白兔均成功建立了兔VX2肝种植瘤模型。瘤灶在CT平扫上表现为低密度,增强后病灶强化不明显,边界较清楚。肿瘤部位种瘤后1至3周HBF、PS、HAF、HBV值与种瘤前比较差异均有显著性意义(P<0.01)。种瘤后第2周,肿瘤区HBF、HBV、PS、HAF增高,与非肿瘤区肝组织比较差异有显著性意义(P<0.01)。肿瘤区MTT降低与非肿瘤区肝组织比较差异有显著性意义(P<0.01)。大体病理检查共发现10个单发肿瘤,其中8例中心出现坏死囊变区,与CT检查基本一致。光学低倍镜下见肿瘤细胞在肝内呈浸润性癌巢,与肝实质无明显边界,新生毛细血管丰富；高倍镜下可见瘤细胞体积大,呈不规则排列,核分裂像多见。结论：采用瘤块种植制备的兔肝VX2肿瘤模型,同时具有容易制备、生长周期短、成功率高及模型稳定等特点,是一种较理想的肝肿瘤实验动物模型,是我们进行基础与临床研究很好的选择。MSCT灌注成像能较好地评价兔VX2肝种植瘤模型的血流动力学变化,结合组织学的研究则可以提供更多的量化信息。第二部分兔VX2肝种植瘤索拉非尼治疗前、后的血管生成与细胞凋亡的实验研究目的：通过对兔VX2肝种植瘤模型应用索拉非尼前后的CT灌注参数与组织学进行研究,探索应用CT灌注成像这种影像学手段来评价肿瘤新生血管生成的可行性,同时为索拉非尼抑制肿瘤血管生成并促进肿瘤细胞凋亡的临床效果提供实验依据。方法：12只新西兰大白兔,采用开腹瘤块包埋法建立兔VX2肝种植瘤模型,2只建模后处死作为病理对照,其余10只于种瘤后第14d行多层螺旋CT灌注扫描,然后经口给予索拉非尼(20mg/kg/d) 7d,种瘤后第21d再次行CT检查,两次扫描肿瘤区所获得的参数值进行比较。检查完成后处死实验兔,进行细胞凋亡(Caspase-3)免疫组织化学染色。结果：12只兔均接种成功。两次扫描肿瘤区所获得的参数HBF、PS和HAF值比较差异有显著性意义(P<0.01),HBV、MTT值差异无显著性意义。Caspase-3阳性细胞主要分布于中央静脉及肝细胞坏死灶周围。Caspase-3表达强度明显高于对照组(P<0.05)。结论：CT灌注成像可以评价肿瘤新生血管的生成,而结合了组织学的研究则可以更加明确索拉非尼抑制肿瘤血管生成并促进肿瘤细胞凋亡的作用。第三部分TACE联合索拉非尼治疗兔VX2肝种植瘤的MSCT灌注与病理及免疫组化对照研究目的：通过对兔VX2肝种植瘤模型应用TACE联合索拉非尼前后的CT灌注参数与组织学进行研究,探讨通过应用抑制肿瘤血管生成药物以进一步提高TACE疗效方法的可行性。方法：30只新西兰大白兔建立兔VX2肝种植瘤模型,将模型兔随机分成A、B、C三组,每组10只。A组为生理盐水对照组,B组为TACE治疗组,C组为TACE+索拉非尼联合治疗组。VX2肝种植瘤模型兔于治疗前(治疗时间选择接种VX2肿瘤组织2wk)及治疗后第1、2、3wk行CT平扫观察肿瘤生长情况。于治疗前与治疗后第3wk行CT灌注扫描并记录肿瘤的肝血流量(HBF)、肝血容积(HBV)、平均通过时间(MTT)、毛细血管表面通透性(PS)和肝动脉分数(HAF)等参数值。然后处死实验兔,进行病理以及血管内皮生长因子(vascular endothelial growth factor, VEGF)与CD34免疫组织化学染色。检测VEGF与CD34的表达情况,并在高倍视野下(×400)计算肿瘤细胞VEGF染色阳性率及微血管密度(micro vessel density, MVD)。结果：治疗后不同时期各组两两比较肿瘤大小与肿瘤生长率均有显著性差异(P<0.01)。VEGF染色阳性呈棕褐色,多见于细胞质内,呈均匀或不均匀的颗粒状或团块状；随着肿瘤细胞增生活跃,阳性细胞数量增多,染色也增强。肿瘤组织中CD34均有表达,切片中肝癌血窦内皮细胞均见不同程度的黄染,癌旁可见个别血管染色。结论：索拉非尼与TACE联合应用能够抑制肿瘤生长,并提高TACE治疗的疗效,通过应用多层螺旋CT灌注成像对肝VX2移植瘤血流灌注参数进行定量测定,以及与免疫组化进行对照研究,提示索拉非尼具有抗兔VX2肿瘤生长及抑制肿瘤血管生成的作用。第四部分：索拉非尼治疗原发性肝癌的MSCT灌注与临床应用研究目的：探讨MSCT灌注成像在评价索拉非尼治疗原发性肝癌中的临床价值。方法：6名原发性肝癌患者在应用索拉非尼前与3个疗程后(3 months)行MSCT灌注及实验室常规检查,各项指标均进行统计学分析。结果：原发性肝癌患者应用索拉非尼后,CT灌注参数HBF、HAF与PS显著下降(P<0.01)。其临床实验室常规检查(血常规、肝功能、肾功能)均无显著改变(P>0.05)。结论：MSCT灌注成像可以评价索拉非尼治疗原发性肝癌的血液动力学变化。索拉非尼这种抗肿瘤药物临床副作用较小,是治疗原发性肝癌患者一个不错的选择。更多还原

【Abstract】 Part one:Establishment of the rabbit VX2 liver tumor model and evaluation of the characteristics in different growth period on MSCT perfusion imagingPurpose:To establish experimental rabbit VX2 liver model by implanting VX2 tumor cells into the rabbit liver, and to evaluate their perfusion parameter changes during different growth period.Methods:The VX2 tumor was passaged through successive subcutaneous injection into the hind limb of the New Zealand white rabbits. Tumor cells were implanted in the left lobe of the rabbit liver (n=10). The MSCT perfusion scan was performed before and 7th day 14th day and 21th day after VX2 tumor cells implantation. The perfusion parametes including HBF, HBV, MTT, PS and HAF were measured in the tumor and non-tumorous regions. All the rabbits were executed for pathological examination after MSCT examnination at the third bearing-tumor week. The data were statistically analyzed.Results:The rabbit VX2 liver tumor model was established successfully in this study. The tumors were demonstrated as low density with well-defined margin on the precontrast MSCT and were enhanced slightly on the postcontrast MSCT. The significant differences of the parameters of HBF, PS, HAF and HBV during the 1st-3rd week were found (P<0.01). There wre significant differences between the tumor area and non-tumorous area at the second week after tumor implantation The HBF, HBV, PS and HAF in the tumor area were increased after 2 weeks of tumor implantation and had stastistical differences as compared with non-tumorous area(P<0.01), while the MTT in the tumor area was decreased and had stastistical difference as compared with non-tumorous area(P<0.01). Ten solitary tumors, of which there was central cystic necrosis in 8 in pathological examination, were consistent with the imaging findings revealed by MSCT. Light microscope with low power revealed that the tumor was nest-like with newly-formed abundant capillary without clear margin between the tumor and liver parenchyma, and it revealed with high power that the tumor cells with abundant caryokinesis were bigger than those in normal liver cells, and they were distributed irregularly.Conclusion:Tumor-implanting method was a satisfactory method in animal liver tumor model construction with easy-going, short-growing and high-successful and stabling features. MSCT perfusion imaging could well evaluate the hemodynamics changes of the rabbit liver VX2 tumor.. MSCT could provide more quantification information while combined with its histology examination.Part two:Pre-and post-therapy of sorafenib for rabbit VX2 liver tumor:experimental study of neovascularization and cell apoptosisPurpose:To study the changes of MSCT perfusion imaging parameters and histology of pre-and post-therapy of sorafenib for rabbit VX2 liver tumor. To explore the feasibility of MSCT perfusion imaging in monitoring neovascularization, and to provide experimental basis to evaluate the effect of sorafenib on the inhibition of tumor angiogenesis and promotion of tumor cell apoptosis.Methods:The MSCT perfusion image was obtained in 12 New Zealand white rabbits at the second week after VX2 liver tumor implantation. Two of them were executed and served as the control group (for pathological examination), and the rest 10 rabbits were served as study group receiving oral administration of sorafenibat (20 m g/kg/d for one week) at 7-th day after tumor implantation. MSCT scans were performed respectively at 14-th and 21-th day after tumor implantation, and perfusion parameters (HBF, HBV, MTT, PS and HAF) in both groups were measured and compared in the tumor regions. Finally, all the rabbits were executed for Caspase-3 staining for detection of cell apoptosis.Results:Twelve (100%) rabbits were successfully implanted with VX2 tumor in the liver. The values of HBF, PS and HAF in tumor of the two groups of MSCT perfusions images were significant different (P<0.01). There were no significant difference in HBV and MTT between the two groups (P> 0.05). The positive cells of the Caspase-3 were observed around central vein and necrosis area of hepatocyte. The intensity expression of the Caspase-3 in tumor tissue was significantly higher than that of normal control group (P<0.05).Conclusion:MSCT combined with the histological examination could elucidate the therapeutic effects of sorafenib on the tumor angiogenesis inhibition and cell apoptosis promotion.Part three:MSCT perfusion imaging and pathological study on rabbit VX2 liver tumor treated with combinaion therapy of transcatheter arterial chemoembolization(TACE) and sorafenibPurpose:To study the changes of MSCT perfusion imaging and histology of rabbit VX2 liver tumor treated with combination therapy of TACE and sorafenib, and to explore the feasibility of improving the therapeutic effect of TACE by way of inhibition of tumor angiogenesis.Methods:Licer tumor modle was establised in 30 New Zealand white rabbits. They were randomly divided into three groups based on therapy stratigies:(A) 0.9% Sodium Chloride group; (B) TACE group; (C) TACE Plus sorafenib group. The MSCT scan was performed at 1st week to 3th week after post-therapy in order to measure the volume changes of tumor. Tumor growing situations were observed by MSCT at pre-therapy and post-therapy (at the 1st,2nd and 3th week). MSCT perfusion parameters of HBF, HBV, MTT, PS and HAF in the tumor regions were measured at pre-theraspy and the 3th week of post-therapy. All the rabbits with VX2 tumor were executed for routine pathological examination and VEGF and CD34 staining after 3 weeks of treatment. Expression of VEGF and CD34 was detected, and the percentage of positive tumor cells and micro vessel density (MVD) were calculated and comapred at high power field (400×magnification).Results:The size and growth rate of tumor were significant different between different groups. (P＜0.01). VEGF positive staining was mostly displayed homogeneous or inhomogeneous granule and clot with brown-yellow color in cytoplasm. The more tumor cells actively proliferated, the more abundant and strong staining positive tumor cells expressed. CD34 staining was expressed in tumor tissue, On light microscope, liver sinusoidal endothelial cells in the liver tissue were seen in varyied degrees of yellow dye. Individual vessel staining can be observed in adjacent cancer tissue.Conclusion:Sorafenib could inhibit tumor angiogenesis and improve the therapeutic effect of the TACE. Measurement of MSCT perfusion parameters and comparson with liver tumor immunohistochemistry findings reflected that sonafenib could prevent tumor growth and inhibition of tumor angiogenesis.Part four:Clinical application study of MSCT perfusion imaging in primary hepatic carcinomas treated with sorafenibPurpose:To study the changes of MSCT perfusion parameter and clinical indexes on primary hepatic carcinomas treated with sorafenib and evaluate the therapeutic effect of sorafenib.Methods:The MSCT perfusion and routine laboratory examination were performed on six patients with primary hepatic carcinomas before therapy and three months after treatment of sorafenib. All statistical data were done respectively.Results:There was significant decrease in HBF, PS and HAF post-treatment with sorafenib (P＜0.01). There was no significant difference in blood test, hepatic function and renal function (P>0.05)Conclusion:MSCT could evaluate the hemodynamic changes in patients with primary hepatic carcinomas after treatment with sorafenib. Sorafenb was the choice of treatment of primary hepatic carcinomas because of few side-effects.更多还原