【作者】 范义；

【导师】 张雪林；

【作者基本信息】 南方医科大学 ， 影像医学与核医学， 2009， 博士

【摘要】 研究背景化疗栓塞病灶周边部的再血管化、存活癌细胞是肝癌TACE术后复发转移的主要病理基础。很长时期以来,CT扫描主要依靠肿瘤内部和周边的碘化油沉积情况来评价TACE的治疗效果。CT平扫可在某种程度上反映栓塞是否完全及栓塞后的瘤体变化,而CT双期增强扫描可反映存活瘤组织及新发病灶的血液供应情况。然而,碘化油与周围组织间较大的密度差所形成的放射状伪影与碘化油的高密度影响对栓塞病灶周边残余肿瘤的观察。MR自旋回波序列(SE)T1WI、T2WI与动态增强扫描的应用,对HCC的TACE术后随访价值较大,特别是SE序列T2WI,多数存活肿瘤在T2WI上为高信号,而TACE术后肿瘤内部的凝固性坏死为低信号。肝癌TACE术后在T1WI和T2WI上的信号多种多样,特别是T2W上,存活肿瘤、出血、液化坏死和炎性细胞的浸润均可表现为高信号,因此T2WI对判断肿瘤的存活有一定困难,而对凝固性坏死的判断十分可靠。介入治疗后残存肿瘤的强化较肝实质早而持续时间短,动态增强MRI早期呈结节状、片状强化,可以鉴别瘤内坏死、纤维组织与肿瘤中央的残存肿瘤,但难以鉴别包膜与包膜内残存肿瘤结节。如果为乏血供的HCC,就难以使用这种技术发现存活肿瘤。部分肿瘤患者进行了经皮肝穿酒精注射联合TACE治疗,肿瘤由低信号变成高信号,即便用动态扫描也很难区分肿瘤增强,从而无法判断存活肿瘤。原发性肝癌介入治疗术后的评价主要集中在CT和MR双期增强上,CT灌注成像定量分析组织灌注是评价肝癌动脉栓塞治疗后血流动力学变化的有效方法。但它的缺点也很突出,主要是需用大量的碘对比剂及具有潜在的过敏反应、中毒性肾损害等危险,同时病人也会受到较大剂量的辐射。MRI灌注图在检测TACE术后残存肿瘤组织的灌注方面很有价值。TACE的疗效可以通过比较术前术后的灌注差异而评估。DWI能够区分肿瘤中的坏死成分和存活成分,ADC值的改变早于T2WI上信号的改变,甚至早于组织学上的变化。因此,利用DWI对肿瘤组织不同时间平均ADC值的测量、对比,可以量化评价肝癌组织TACE术后的坏死、复发情况。本研究利用兔VX2肝癌模型,通过MR DWI、PWI成像观察肝癌栓塞前后坏死及血流灌注情况的变化规律,判断MR DWI、PWI成像在观察肝癌栓塞后坏死及血流灌注的临床应用价值,从而为肝癌介入治疗的最佳方法的选择及疗效观察提供了更有价值的影像诊断依据。第一章:兔VX2肝癌模型的制作及改进目的:将穿刺器械进行改良,采用肿瘤组织块直接穿刺种植肿瘤,并与肿瘤细胞悬液植入法、肿瘤组织块悬液种植法对比,观察种植成功率及肿瘤异位种植的情况,筛选更为简便、有效的肿瘤模型的制作方法。材料和方法:1.1兔VX2肝癌模型制作1.1.1实验动物新西兰大白兔30只,体重2.5～3.0kg,雌雄不限,由中山医科大学实验动物中心提供。随机分为3组,分别为肿瘤细胞悬液植入法组、肿瘤组织块悬液种植组、肿瘤组织块直接穿刺种植组(改进组),每组实验动物10只。1.1.2 VX2细胞株制备将瘤株接种于兔腹股沟区皮下或肌肉间,使之成瘤,三周后接种处可扪及一实质性包块。速眠新全麻后,无菌条件下剥离肿瘤,切取包块边缘生长旺盛的鱼肉样组织,置于盛有生理盐水的平皿中。1)配成2×10~6个细胞/ml浓度的VX2细胞悬液,用于第1组肿瘤的种植。2)取肿瘤组织用眼科剪将其剪成大小约1mm~3组织块。用于第2组肿瘤的种植。3)用眼科剪将肿瘤组织剪成约1mm×1mm×3mm大小瘤块用于第3组肿瘤的种植。1.1.3兔肝VX2肿瘤种植穿刺针的改进:使用18G腰穿针。取出穿刺针芯,用油石打磨去掉尖端,以细沙纸抛光以去除毛刺,使顶端平整光滑,即成为推针,与常规尖头针芯及套管配合使用,成为改良的穿刺用具。1.1.4兔肝VX2肿瘤的种植。动物术前禁食12h,用速眠新全麻,仰卧位,固定四肢于兔手术板上,常规剑突左侧腹部剪毛。CT扫描兔肝脏,确定肝左叶待接种部位,选定穿刺点、角度、深度。常规消毒铺巾,刀尖挑开穿刺点皮肤,用带尖头穿刺针芯的18G穿刺针穿入肝脏预定位置,拨出针芯,用注射器抽吸无回血。第1组动物注入VX2细胞悬液1ml,第2组注入肿瘤组织块悬液1ml,两组在退针时注入少量明胶海绵颗粒。第3组由助手固定好穿刺针,术者用眼科镊夹1块瘤块放入针鞘内,平头穿刺针芯将瘤块推入肝内,同理再分别放入2块组织块。如未成功,可再重复上述操作。将一小块明胶海绵用平头穿刺针芯推入肝内封堵针道。局部按压3分钟,并用创可帖封伤口。1.2 CT扫描种植后2周对新西兰白兔肝VX2瘤灶行CT扫描,四周再扫描一次,观察2周扫描是否有漏检病例。扫描条件:120kV,180mA,层厚3mm,间距3mm:延迟8～12s,30～40s行双期增强扫描。1.3观察指标及统计学处理分别观察不同种植方法的成功率,孤立病灶的成功率,异位种植情况。统计学处理采用SPSS13.0软件精确概率法进行统计分析,p＜0.05有统计学意义。结果:1、各组间种植成功率:瘤细胞悬液植入组30%,肿瘤组织块悬液种植组50%,肿瘤组织块直接种植组90%,经统计学分析p=0.036,可以认为不同种植方法成功率不同。2、不同种植方法孤立肿瘤种植的成功率:肿瘤组织块直接种植组最高(80%),依次为肿瘤组织块悬液种植组(20%)和瘤细胞悬液植入组(10%)。统计学分析有显著性差异(p=0.003)。3、不同种植方法异位种植发生数:瘤细胞悬液植入组1(3),肿瘤组织块悬液种植组1(5),肿瘤组织块直接种植组0(8),经统计学分析,无显著性差异(p=0.238)。结论:1、经皮穿刺肿瘤组织块直接种植法制作兔肝癌模型,成功率尤其孤立瘤灶发生率高,方法简单、易行,模型性质稳定,并发症及异位种植低,在肝癌的基础及临床研究中将得到越来越广泛的应用。2、兔VX2肝癌模型制备简单,是一种理想的肝癌研究动物模型。第二章:MR弥散加权成像在肝癌诊断的应用价值目的:研究兔肝癌不同时间在MR T1WI、T2WI及DWI的表现及ADC值的变化规律,从而探讨MR弥散加权成像在肝癌诊断中的作用。材料和方法:1.1动物分组及模型制作1、新西兰大白兔30只,体重2.5～3.0kg,雌雄不限,由广东省实验动物中心提供。分别在肿瘤种植后4天、1周、2周、3周对30只动物肝VX2瘤灶行MR扫描。2、采用“第一章:兔VX2肝癌模型的制作及改进”中“肿瘤组织块直接种植法”种植肿瘤。1.2 MR检查种植后4天、1周、2周、3周对兔肝VX2瘤灶行MR扫描,分别进行T1WI、T2WI、T2WI脂肪抑制序列和MR DWI成像。用Sense-Flex-M环形表面线圈。T2W_TSE_SENSE与T2W_TSE_SPIR序列扫描参数:TR 3544ms,TE 81ms,FOV150mm,层厚3mm,层间距-1mm,矩阵256×256,重建矩阵512×512,扫描时间1分14秒,NSA为2次。T1W_TSE SENSE序列扫描参数:TR407ms,TE 17ms,FOV 150mm,层厚3mm,层间距-1mm,矩阵256×256,重建矩阵512×512,扫描时间1分34秒,NSA为2次,EL=3。DWI采用单次激发平面回波成像(echo plannar imaging,EPI)序列,弥散因子b值取0 s/mm~2和300s/mm~2,TR 6000ms,TE 49ms,FOV 150mm,层厚3mm,层间距0mm,矩阵112×112,重建矩阵256×256,翻转角90°,扫描时间2分36秒,NSA为6次。1.3观察指标及统计学处理1、各种检查方法不同时间肿瘤检出率分别观察不同时间兔肝癌在MR T1WI、T2WI、T2WI脂肪抑制序列和MRDWI检出率。统计学处理采用SPSS13.0软件,种植后第四天各序列的种瘤检出情况比较,采用多个相关样本的非参数检验中的Cochran’s Q检验方法进行分析。各序列种植后四天和一周的检出率比较采用配对χ~2检验直接计算,p＜0.05有统计学意义。2、不同时间ADC值的计算在肿瘤种植后4天、1周、2周、3周ADC图上分别在中心区、边缘区随机选取感兴趣区,计算ADC值。用SPSS13.0的单个重复测量因素的方差分析(General Linear Model,repeated Measures)进行统计学分析,检验水准:α=0.05。结果:2.1不同MR序列的检出率2.1 1种植成功率:30个实验动物,肿瘤种植成功25例,成功率81.25%。2.1.2不同序列对兔肝VX2肿瘤的检出率种植后第四天各种检查方法即可显示肿瘤灶,在T1W上表现为边缘不清的等或低信号灶,在T2WI和T2WI SPIR和DWI序列上表现为高信号,边界欠清晰。一周后肿瘤在各序列中显示清楚,边界清晰,在肿瘤灶中心出现在T2WI、DWI低信号的液化坏死区。在种植后第四天,DWI、T1W、T2WI、T2WI SPIR检出率分别为80%、60%、72%和72%。经统计学分析Cochran’s Q=10.2,p=0.017,差异有统计学意义。种植后第一周各检查系列均检查出病变25例。种植后4天和一周的检出率比较:DWI组无显著性差异(χ~2=3.24,p＞0.05);而T1w、T2WI和T2WI SPIR组有统计学差异(p＜0.05)。2.2肿瘤种植后不同时间ADC值的变化周边区在种植后第4天、1周、2周、3周ADC值分别为1862.58±150.97、1636.70±5152.96、1273.43±173.59及1005.68±192.00,差异有显著性(F=153.997、p=0.000)。而中心区第4天、1周、2周、3周ADC值分别为1498.81±264.41、1078.92±153.58、662.751±174.27、1553.19±179.01(F=149.442、p=0.000)。随着时间的增加ADC值降低,但在第三周ADC值升高,甚至超过了第四天,表明病灶中央出现了坏死灶。结论:1、DWI是检查肝癌比较敏感技术,可较早期检出小肝癌,在肝脏肿瘤的诊断中将得到越来越广泛的应用。2、MR弥散加权成像不但对肿瘤的定性诊断有很大帮助,通过ADC值,可大致判断肿瘤的坏死情况和发展趋势。第三章:肝癌化疗栓塞前后MR弥散和灌注成像研究。目的:通过MR DWI、PWI成像观察肝癌栓塞前、后肿瘤坏死及血流灌注情况的变化规律,并与病理和微血管密度对比,判断MR DWI、PWI成像在观察肝癌栓塞后坏死及血流灌注的临床应用价值。材料和方法:1.1实验动物分组及模型制备肝癌模型新西兰大白兔30只,体重2.5～3.0kg,雌雄不限,由广东省实验动物中心提供。实验动物随机分为3组,分别为TACE术前、术后三天、术后一周组,每组10只动物。采用“第一章:兔VX2肝癌模型的制作及改进”中,“肿瘤组织块直接种植法”制作肿瘤模型。1.2兔肝癌的化疗栓塞肿瘤种植3周,常规MR扫描发现病灶,进行肝动脉化疗栓塞。兔用速眠新全麻,取仰卧位固定于兔手术板上。常规消毒、铺巾。用手术刀切开腹股沟下皮肤,用止血钳分离出股动脉,用带有塑料套的穿刺针刺入到股动脉内。拔出针芯,见有血液喷出,送入黑泥鳅导丝,撤出塑料套,通过导丝引入4F超滑导管进入腹主动脉达腹腔动脉干开口处。用手推注造影剂造影,观察肿瘤血管和肿瘤染色。撤出导丝,引入3F微导管入腹腔干,越过胃动脉,将导管超选入肿瘤供血动脉,注射碘油、MMC(1mg)及表阿霉素(1mg)混合物1-3ml,至碘油沉积良好、肿瘤血管消失、动脉内碘油滞留停止。拨管,右侧股动脉结扎。局部皮肤缝合、消毒、包扎。1.3 MR弥散和灌注加权成像1.3.1扫描条件各组在TACE术前、术后三天、术后一周分别进行MR弥散和灌注成像。应用Sense-Flex-M环形表面线圈。DWI扫描条件:见第二章。PWI成像:采用TFE序列,灌注成像参数:矩阵89×256,TR 4.3 ms,TE1.4 ms,激励次数1,视野355 mm×75 mm,层厚5mm,间距1mm,NSA为1次。扫描层面数4层,每层反复激励65次,总扫描时间为1分20秒。高压注射器经通过家兔耳缘静脉注射对比剂钆特酸葡胺注射液(多它灵,377mg/ml),剂量为0.2mmol/kg,注射流速为2ml/s。生理盐水3ml以同样速度注射冲管,团注开始即进行扫描。1.3.2 MR灌注图像后处理将原始图像输入到PHILIPS后处理工作站(VIEWFORUM),自动生成MR灌注图。分别在肿瘤中心区、边缘区设置感兴趣区,分别分析时间—信号强度曲线(time signal-intensity curve,TIC)及肿瘤中心区、边缘区最大增强斜率(maximum slop of increase,MSI)的变化规律。1.4病理观察3组动物MR弥散和灌注成像之后,用过量硫喷妥钠处死。根据MR图像引导切取肿块所在部位肝脏,进行大体病理观察,HE染色并进行免疫组化,计算其中心区、边缘区MVD值。1.5观察指标及统计学处理肿瘤中心区、边缘区在TACE前、后4天、后一周的ADC值、SMI值及MVD值。用SPSS13.0进行统计分析,采用计量资料的单因素的方差分析(One-Way ANOVA)。检查水准:a=0.05。MVD与ADC值相关分析、MVD与MSI值相关分析采用双变量相关分析(Bivariate)。结果:2.1化疗栓塞前后DWI结果1、化疗栓塞前后DWI成像改变及与病理对照化疗栓塞前MR成像在肝左叶可见T1低信号、T2高信号、DWI高信号的肿瘤。大体病理示肿瘤为白色,剖面呈鱼肉状,光镜下细胞体积大,胞浆丰富,淡红染色,核肥大,核分裂像多见,可见少量坏死,周边区和中央区未见明显区别。栓塞后3d在DWI上出现斑片状低信号区,为凝固性坏死的表现。大体病理示肿瘤为白色,肿瘤中心可见豆腐渣样坏死组织,光镜下出现大量核碎裂、核溶解,肿瘤坏死较多。栓塞后1w,病灶进一步进展,病灶中心在T2、DWI上出现了大面积低信号的坏死区。大体病理肿瘤中心坏死组织增多,光镜下肿瘤坏死增多。2、化疗栓塞前后ADC值的改变边缘区ADC值在栓塞前、栓塞后3d、栓塞后1w分别为1089.31±107.24、1865.38±157.28、1860.97±194.46,有显著性差异(F=80.875,p=0.000)。中心区ADC值在栓塞前、栓塞后3d、栓塞后1w分别为1595.92±197.51、1633.38±193.82、1848.14±183.01,有显著性差异(F=5.049,p=0.014)。2.2 MR灌注结果1、MR灌注图改变栓塞前HBV图、HBF图清楚显出肿瘤灶,并可见病灶中央高灌注区。病灶中心区强化曲线,表现为速升速降;边缘区强化曲线强化峰值比病灶中心区高。栓塞后3天强化曲线缓慢升高,缓慢下降。栓塞后1W表现与栓塞后3天相似,中心区伪彩进一步下降,甚至消失。边缘区强化曲线缓慢升高,缓慢下降。2、栓塞前后不同时间MSI变化边缘区在栓塞前、栓塞后3d、栓塞后1w的MSI值分别为7.35±1.20、8.70±0.96、2.85±0.96,有统计学差异(F=85.986,p=0.000)。术后第三天到一周组间变化较大。中心区在栓塞前、栓塞后3d、栓塞后1w的MSI值分别为2.41±0.59、1.50±0.38、1.33±0.46,统计学分析有显著性差异(F=14.414,p=0.000)。2.3病理学观察2.3.1光镜下病理表现肿瘤组织在低倍镜下可见肝内浸润性癌巢,与肝实质无明显边界。肿瘤细胞分散排列,可见纤维间隔,新生毛细血管丰富。高倍镜下可见瘤细胞体积大,胞浆丰富,淡红染色,核肥大,核分裂像多见。化疗栓塞后瘤细胞团内可见大片坏死区及多个片状或灶性坏死,大部分瘤细胞核固缩及核碎裂。2.3.2免疫组化病理1、免疫组化表现肿瘤血管被染成棕色、褐色,化疗栓塞前后肿瘤中心区、边缘区随着时间进展肿瘤血管减少。2、化疗栓塞前后MVD改变边缘区MVD值在栓塞前、栓塞后3d、栓塞后1w分别为51.14±19.71、26.88±12.76、11.07±6.52,经统计学分析:F=20.582,p=0.000,有显著性差异。中心区MVD值在栓塞前、栓塞后3d、栓塞后1w分别为13.49±5.17、8.52±3.64、8.32±5.11,有显著性差异(F=3.899,p=0.033)。2.4 DWI、PWI与微血管密度相关性MVD与ADC值间存在负相关(r=-0.534,p=0.000);MVD与MSI值间存在正相关(r=0.694,p=0.000)。结论:1、栓塞前病灶中心区和边缘区强化曲线,表现为速升速降,边缘区强化峰值比病灶中心区高。栓塞后强化曲线缓慢升高,缓慢下降,随着时间发展,上升幅度降低。2、肿瘤中心区、边缘区MVD值在栓塞前、栓塞后3d、栓塞后1w的比较,均有统计学差异,化疗栓塞后肿瘤的微血管明显减少。3、肿瘤中心区、边缘区MSI值在栓塞前、栓塞后3d、栓塞后1w的比较中有统计学差异,随着时间发展MSI降低。MVD与MSI值呈正相关,MVD的减少使单位时间、单位组织内肝血流量增加,是MSI降低的病理基础。4、MRI灌注成像能在活体判断肝癌的血管生成程度及血供情况,MR灌注值改变可揭示肿瘤新生血管的血流动力学变化,为研究肝癌的血流动力学改变提供了一种新的方法,对判断栓塞后肝癌的灌注水平变化及疗效评估也很有价值。5、中心区和边缘区ADC值在栓塞前、栓塞后3d、栓塞后1w有显著性差异,说明TACE治疗3天后组织弥散增加。MVD与ADC呈负相关。ADC值作为可靠的动态监测治疗前后肿瘤坏死情况的方法,可较好地反应TACE后肿瘤的转归。更多还原

【Abstract】 BackgroundTranscatheter arterial chemoembolization（TACE） is considered to be an effective treatment for hepatocellular carcinoma.However,angiogenesis,the process leading to the formation of new blood vessels from preexisting vessels,plays a central role in the survival of tumor cells,in local tumor growth,and in the development of distant metastasis and reduces therapeutic effectiveness.Early detection of residual tumor and vascularity after interventional treatments is critical before TACE and during follow up.Generally,computed tomographic（CT） images could be considered as a routine modality to judge the efficacy of TACE.The pattern and distribution of iodized oil in the tumor are useful for assessing the therapeutic effects of TACE.A greater amount of accumulation of iodized oil within tumor indicates a greater area of necrosis.During follow-up,the focal defect or washout of iodized oil in the mass with the contrast-enhanced area suggests the presence of a viable tumor and that additional treatment is needed.The contrast-enhanced CT is a reliable method for the assessment of therapeutic efficacy.On contrast-enhanced CT,necrotic tissues were unenhanced and viable tumors were enhanced.But it was difficult to judge the viability and necrosis of the tumors correctly due to the inhomogeneous deposition,because lipiodol negative areas could not actually represent the viability of the tumors.It can also be difficult to evaluate contrast enhancement in a tumor with partial retention of iodized oil on contrast-enhanced CT because of the beam-hardening artifacts produced by the high attenuation of iodized oil.MR imaging was valuable in the evaluation of therapeutic efficiency of TACE, especially on T₂WI,most of viable tumors were hyperintense and the coagulative necrosis within the tumors considered as a positive response to TACE was hypointense. But a signal intensity of the tumors after TACE was variable on SE T₁WI and T₂WI,all of viable tumors,hemorrhage,liquefied necrosis and inflammatory infiltration could also result in hyperintensity on the T₂WI.Therefore,it was difficult to assess the viable tumors of HCC after TACE by conventional SE imaging.Dynamic contrast scanning also has a great value in the evaluation of therapeutic efficacy of TACE.The residual viable tumors were shown as rapidly enhanced portions within the lesions,when necrotic portions had no enhancement at the contrast early phase scanning.At the late phase scanning,the enhancement of the most lesions became hypointense,and just a few lesions showed a persistent enhancement.Both viable tumors and inflammatory infiltration could present such changes,so the contrast early phase scanning was not more reliable in the evaluation of viable tumors.Existing diagnostic imaging techniques provide limited evaluation of tissue characteristics beyond morphology.Perfusion imaging ultimately may have a role in the earlier identification of tumor recurrence if subtle differences in enhancement of benign versus malignant tissue can be captured earlier.CT perfusion imaging has the potential to provide both high-temporal-resolution and high-spatial-resolution imaging of the entire liver for the detection of HCC,however,the radiation doses must be considered.A whole-liver CT perfusion study would result in radiation doses that are approximately three to six times greater than those of a routine diagnostic CT examination.Perfusion MR imaging represents a promising technique for HCC surveillance.T1 weighted perfusion MRI has been used to assess tumor angiogenesis of uterine cervical carcinoma, and has a good correlation with microvessel density（MVD）.Diffusion-weighted imaging can provide an insight about water composition within a tumor and the degree of tumor viability.The apparent diffusion coefficients of malignant masses were significantly lower than those of benign masses.A study of an animal model of hepatocellular carcinoma showed that:diffusion-weighted imaging clearly distinguished the regions of tumor necrosis from viable tumor.So,DWI may offer significant utility in the longitudinal assessment of novel liver tumor treatments.The purpose of this study was to observe angiogenesis and necrosis of HCC before and after TACE by MR perfusion Imaging and diffusion imaging,compared with microvessel density and histological evaluation,and to find out their feasibility and value in assessing therapeutic effectiveness of HCC during follow up.Chapter One:The establishment and improvement of VX2 Carcinoma modelObjective To establish suitable metastatic rabbit vx2 hepatic carcinoma model,to compare the success rate of different tumor implantation methods.Methods Thirty New Zealand white rabbits were randomly divided into three groups,10 rabbits in each group.Group 1:Solution of the VX2 carcinoma cells were injected into left liver by syringe needle.Group 2:Solution of the tumor tissue masses was transplanted into liver.Group 3（improvement group）:VX2 carcinoma masses were injected into the liver using percutaneous puncture inoculation method.The operation was guided by CT scanner in each group.CT scanning was performed in all experimental animals 2 week after implantation.Results The success rates of transplanted carcinoma were 30%,50%and 90%in group 1,group2 and group3 respectively,and the statistical difference was found.The respective ectopic implantation incidence at group 1,group2 and group3 were 1（3）,1（5） and 0（8）,no statistical difference was observed.The success rates of single nodule implanted tumor were the highest in group 3（80%） than in group 1（10%） and group 2 （20%）.The difference was statistically different（p=0.003）.Conclusion It is successful to improve the rabbit modes bearing VX2 metastatic hepatic carcinoma.And the method of percutaneous puncture carcinoma masses implanation guided by CT was a good method to establish the hepatic carcinoma models with a high success rates,especial rates of single nodule implanted tumor,low ectopic implantation rates and little damage of animals.Chapter Two:Observation of the value of MR Diffusion Weight Imaging in diagnosis of hepatocellular carcinomaObjective To investigate the value of MR DWI in diagnosing hepatic carcinoma by studying the appearances of rabbit vx2 hepatic carcinoma in MR T1 WI,T2WI and DWI series at different times after the tumors were transplanted.Methods The metastatic rabbit vx2 hepatic carcinoma models were presented in 30 New Zealand white rabbits.VX2 carcinoma masses were injected into the liver using percutaneous puncture inoculation method in guiding by CT scanner in each group.MR T1WI,T2WI,T2WI SiP and DWI were performed in all experimental animals at 4day,1, 2 and 3 weeks after implantation with a Sense-Flex-M.T2W_TSE_SENSE and T2W TSE_SPIR parameters:TR 3544ms,TE 81ms,FOV 150mm,slice thickness 4mm, intersection gap of-1 mm,matrix 256×256,restruction matrix 512×512,scan time 74s, NSA 2.The MR image parameters with T1WI SENSE were as follows:TR 407ms,TE 17ms,FOV 150mm,slice thickness 3mm,intersection gap of- 1 mm,matrix 256×256,restruction matrix 512×512,scan time 94s,NSA 2,EL=3.Diffusion-weighted images of the liver were acquired using an SE-EPI sequence with the following imaging parameters:b-values of 0,300/mm²,TR 6000ms,TE 49ms, FOV 150mm,slice thickness 3mm,intersection gap of 0mm,matrix 112×112,restruction matrix 256×256,flip angle 90°,scan time 156s,NSA 6.The diagnosis rates of different methods in different time were observed.The ADC values in cores and peri-areas were analyzed.Results Of 30 rabbits,25 were transplanted vx2 hepatic carcinoma.The success rate of carcinoma transplanted was 81.25%.The tumor lesion could be found in every examination series at 4 days after the implantation.The diagnosis rates of DWI,T1WI, T2WI and T2WI SPIR were 80%,60%,72%and 72%respectively.Statistical difference was found（p=0.007）.The diagnosis rates were 100%in all MR series at 1,2 and 3 week after implantation.In comparison of diagnosis rates between 4days and 1 to 3 week after the tumor implantation in different series,no significant difference was only observed on DWI series（p＞0.05） and significant difference on other series（p＜0.05）.The ADC values of peri-areas were 1862.58±150.97,1636.70±152.96, 1273.43±173.59,1005.68±192.00 respectively,at 4day,1,2 and 3 weeks after implantation.The significant difference was observed（F= 153.997,p=0.000）.The values decreased with the times developing from 4 days to 3 weeks after the implantation.The ADC values of cores were 1498.81±264.41,1078.92±153.58,662.751±174.27, 1553.19±179.01 respectively at 4day,1,2 and 3 weeks.There were statistical difference in each group ADC values（F=149.442,p=0.000）,which decreased with the times developing before 2 weeks,but increased at 3 weeks.Conclusion MR DWI is a sensitive series in examination of liver tumor,,and will be used in diagnosis of liver tumor more and more.The values of ADC may help discriminate between the necrosis areas and normal areas,will play a important role in surgical operation and puncture biopsy.Chapter Three:MR diffusion and perfusion imaging of the liver before and after transcatheter hapatic chemoembolization for hepatocellular carcinoma Objective To observe angiogenesis and necrosis of HCC before and after TACE by MR perfusion imaging and diffusion imaging,compared with microvessei density and histological evaluation,and to find out their feasibility and value in assessing therapeutic effectiveness of HCC during follow up.Methods1.Animals and tumor models30 New Zealand white rabbits（weight:2.5-3.0kg） with VX2 liver tumors were randomly divided into three groups,10 rabbits in each group（group 1,pre-TACE;group2, 3 day post- TACE;group 3,1 week post- TACE）.The MR scan was performed before chemoembolization,at 3 day and 1 week after chemoembolization,respectively at group 1,2 and 3.The technique for tumor implantation was basically similar to that described in Group 3 of Chapter One.All three groups received interventional therapy after three weeks of the VX2 tumor was implanted.The laboratory animal center of Guangdong province provided all animals.2.ChemoembolizationEach rabbit was successfully catheterized with DSA.Conventional DSA was performed by using a C-arm unit guidance.Rabbits were initially anesthetized by using sumianxin（0.1-.02mg/kg）.Vascular access was achieved in the femoral artery through transfixion using a transfixion pin with plastic sleeve.A 4-F catheter was advanced over a guidewire into the celiac trunk.DSA of arteries was performed by using 2-3 ml manual injections of contrast agent.The 3-F catheter was advanced into the left hepatic artery.1-3 ml lipiodol emulsion（lipiodol mixed with 1mg MMC and 1mg pharmorubicin） were superselectively delivered into tumor vessel by manual injection.Follow-up angiography was performed and confirmed the complete stasis of antegrade blood flow to the liver tumors.After chemoembolization,the catheter was removed and the femoral artery was ligated.Then skin closure was finished.3.MR imagingMR perfusion and diffusion imaging examination were performed in the supine position by using a clinical Sense-Flex-M coil.The scans were done before chemoembolization,at 3 day and 1 week after chemoembolization,at group 1,2 and 3 respectively.MR diffusion imaging parameters has been described in Chapter Two. The MR perfusion imaging protocol consisted of T1-weighted fast field echo（FFE） sequences with a field of view of 355 mm×75 ram,matrix 89×256,TR/TE of 4.3/1.4 milliseconds,slice thickness of 5 mm,intersection gap of 1 mm,NSA of 1.This sequence was repeated 65 times,in4 slice（in total,80 seconds）,a dose of 0.2 mmool/kg of Gadoteric Acid contrast medium was injected after the start of the scan.Injection of Gadodiamide was performed through a 22-gauge needle via ear vein at a flow rate of 2 ml/sec followed by injection of 3ml bolus of saline（NaCl 0.9%）.4.Histological evaluationEach animal was then sacrificed with overdose penthiobarbital sodium intravenous injection for pathology observation after MR examination.The livers were removed and fixed in 10%acetic formalin.A slice was cut at the same level as the slice imaged in MR and the general pathology was observed.Each VX2 tumor was divided into the periphery area and center area.The specimens of both areas were embedded in paraffin,and thinner sections of 3mm were cut and stained with hematoxylin-eosin-safran.These slices were examined with an optical microscope at 20 magnifications.Immumohistochemical staining was performed using the SP method.The specimens were observed under a low power microscope（×40）,then the sections with most dense area of microvessels were selected and observed under a high power（×400）.Microvessels were evaluated under a microscope,microvessel density in the center and rim of the lesion were calculated.5.Statistical analysisThe values of ADC,MSI and MVD in the center and rim of the lesion were calculated.All data were expressed as mean±SD,the statistical differences between different groups were analyzed by ANOVA,and the correlation between MSI and MVD, ADC and MVD was assessed by Spearman correlation analysis.Significance was accepted when p＜0.05. Results1.Lesion signal intensity characteristics in DWI images before and after TACEThe lesions assessed before TACE were hypo- intense relative to the surrounding liver parenchyma on T1-weighted images,and the most portion of tumors was hyperintense relative to the surrounding liver parenchyma on T2-weighted images and DWI images,and the heterogeneous signal intensity was observed when liquefied necrosis or fat degeneration occurred.At 3d after TACE,on DWI images,the heterogeneous hypo-intense was observed,which was coagulative necrosis confirmed by morphological observation.At 1w after TACE,the heterogeneous hypo-intense areas became larger.The coagulative necrosis progressed with the development of times.2.Changes of ADC valuesThe ADC values of peri-areas were 1089.31±107.24,1865.38±157.28 and 1860.97±194.46 respectively,at pre-TACE group,3 days post-TACE group and 1week post-TACE group.The significant difference was observed（F=80.875,p=0.000）.The ADC values of cores in each group were 1595.92±197.51,1633.38±193.82, 1848.14±183.01 respectively.Significant difference was found between pre-TACE and after TACE（F=5.049,p = 0.014）3.Characteristics on PWI images before and after TACEAll tumors lesions were depicted on HBF,HBV images.Time intensity curves derived from ROIs drawn in the peri-areas and cores of the tumor showed a rapid initial increase in signal intensity,representing hypervascularity before and after TACE.The peak values in peri-areas were higher than cores.The intensity curves in post-TACE groups showed less steep than pre-TACE group.4.Changes of MSI valuesThe MSI values of peri-areas were 7.35±1.20,8.70±0.96and 2.85±0.96 respectively at pre-TACE group,3 days post-TACE group and lweek post-TACE group.The significant difference was observed（F=85.986,p= 0.000）.The MSI values of cores in each group were 2.41±0.59,1.50±0.38 and 1.33±0.46 respectively.Significant difference was found between pre-TACE and after TACE（F= 14.414,p= 0.000）5.Results of morphological observationUnder low magnification,HCC was seen as invasive carcinoma in the liver,there was no clear margin between neoplasm and the hepatic parenchyma,neoplastic cells distributed straggly,there was separating fibre with abundant capillary of newly developed;under high magnification,the volume of neoplasm was large with irregular morphology,cells distributed irregularly with abundant cytoplasm.Nucleus was hypertrophic with different size and shape,staining was non-uniform,phase of nucleous mitosis showed in many cells,a great number of lymphocytes and plasma cells could be seen infiltrating in the mesenchyma.After TACE lesions underwent un-complete necrosis,karyopycnosis and nuclear fragmentation was seen at the center of the lesions.6.Results of immunohistochemistryCD34 was expressed in HCC and various degree of yellow stain could be seen in the epithelial cells of hepatic sinusoid in the HCC section.But MVD of HCC tissues were lower significantly after TACE than before TACE.The MVD values of peri-areas were 51.14±19.71,26.88±12.76 and 11.07±6.52 respectively at pre-TACE group,3 days post-TACE group and lweek post- TACE group.The significant difference was observed（F=20.582,p=0.000）.The MVD values of cores in every group were 13.49±5.17,8.52±3.64,8.32±5.11 respectively.Significant difference was found between pre-TACE and after TACE（F=3.899,p=0.033）7.Correlate analysisThe values were analyzed by Pearson’s correlation coefficient.The results showed that there was negative correlation between ADC and MVD（r=-0.534,p=0.000）,positive correlation between MSI and MVD（r=0.694,p=0.000）. Conclusion1.Time intensity curves derived from ROIs drawn in the peri-areas and cores of the tumor showed a rapid initial increase in signal intensity.The peak values in peri-areas were higher than cores.The intensity curves in post-TACE groups showed less steep than pre-TACE group.2.Significant difference in MVD values of peri-areas and cores was found at pre-TACE group,3 days post-TACE group and 1 week post-TACE.That indicated capillary vessels decrease obviously after TACE.3.MVD values of peri-areas and cores decreased along the times of TACE developing. The statistical significant was found.There was a positive correlation between MSI and MVD.The hepatic blood flow decreased along with the MVD,which was the pathological basis of MSI values decreasing.4.MR perfusion imaging has a potential to evaluate the angiogenesis of liver tumors in vivo,and could demonstrate the haemodynamics variation of new vessels.It provides a new,valuable technology for hemodynamics study of HCC and evaluation of perfusion level and therapeutic effect after TACE.5.Significant difference of ADC values in peri-areas and cores was found between pre-TACE and after TACE,which demonstrated that the diffusion of tumor tissues increased after TACE.There was negative correlation between MVD and ADC values. Values of ADC could reflect the changes of tumor after TACE,was a reliable index to monitor the tumor necrosis.更多还原