64排螺旋CT肾癌灌注成像实验与临床应用研究
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摘要
利用64排螺旋CT灌注成像技术，通过对兔VX2肾移植瘤模型及肾癌进行CT灌注成像，测量各自的CT灌注值，与正常组比较并进行统计学分析，探讨肾癌的血流动力学变化规律；利用免疫组化技术，检测肿瘤血管生成量化指标微血管密度(MVD)及血管内皮生长因子(VEGF)，并与癌旁肾皮质比较，探讨肾癌CT灌注参数与MVD、VEGF等量化指标的相关性，从而评价CT灌注成像反映肾癌血管生成的临床应用价值。
第一部分兔VX2肾移植瘤模型64排螺旋CT灌注成像研究
研究目的
建立兔VX2肾移植瘤模型，通过对兔VX2肾移植瘤模型的64排螺旋CT灌注成像，探讨兔VX2肾移植瘤血流动力学变化规律，为研究人类肾癌提供理论依据。
材料与方法
1主要仪器与试剂
Brilliance 64排螺旋CT扫描仪(荷兰PHILIPS公司)，配置有Mxview(带有肾脏灌注软件)工作站，双筒高压注射器(美国MEDRAD公司)；非离子型对比剂典比乐(370mgI／ml)。
2实验资料
2．1实验对象分组
20只新西兰大白兔，体重2-2.5kg，雌雄不限。随机将实验动物分为实验组和正常对照组。
2．2兔VX2肾移植瘤模型的建立
荷瘤VX2新西兰种兔一只，将瘤细胞接种兔后腿的皮下成瘤并传代，制成种兔。选取2周左右种兔切除肿瘤，取瘤块边缘生长旺盛组织，制成1-2mm~3大小碎块。采用切开后腹壁暴露肾脏瘤块直接包埋法种植，建立兔VX2肾移植瘤模型。饲养16-20天进行实验。
2．3 CT灌注成像技术与方法
先常规全肾轴位平扫，层厚2.5mm，间距2.5mm，再行宽范围同层动态增强扫描，设定最宽扫描范围40mm，使用双筒高压注射器，注射速率1.5ml／s；先注射非离子型对比剂370mgI／ml典必乐4ml，后注射生理盐水2ml，注射后延迟1秒钟开始扫描，共扫描50次，总时间50s。灌注扫描主要参数有：探测器排列64×0.625mm，无间隔连续扫描，层厚2.5mm，电压120KV，电流150mAs，360度旋转时间0.5s。
感兴趣区(ROI)的选取：包括主动脉、肾静脉、肿瘤实质及肾皮质。主动脉和肾静脉可为圆形或椭圆形，肿瘤实质及肾皮质可为不规则形。ROI尽可能大，不能小于50个像素，以减少量子噪声，但不能达到器官的边缘以避免部分容积效应的影响，实质区ROI尽量不包括‘肾窦脂肪，病灶则应避开坏死区。3图像后处理、数据收集及观察
在Mxview工作站上，应用自带的灌注软件进行图像后处理。腹主动脉代替肾动脉被确定为输入动脉，肾静脉被确定为输出静脉。划出腹主动脉、肾静脉、肾皮质感兴趣区(region of interest，ROI)，软件自动测量出各ROI的CT值，并绘制出时间—密度曲线(time density curve，TDC)和灌注彩图，根据TDC，软件自动生成显示灌注指标，包括。肾脏血流量(blood flow，BF)、肾脏血容量(blood volume，BV)及对比剂的平均通过时间(mean transit time，MTT)等。研究期间所有的测量均重复2次，取其平均值。
观察TDC形态并收集、记录实验组和对照组BF、BV、MTT等灌注参数。
4病理学观察
常规苏木素-伊红(HE)染色，显微镜下观察正常肾及肿瘤的组织结构。
5统计学分析
采用SPSS13.0软件包进行统计学分析。各项灌注参数指标以平均数±标准差表示，数据采用两独立样本t检验，P＜0.05，被认为显著性差异。
结果
1兔VX2肾移植瘤模型大体形态及病理学观察
10只新西兰大白兔均成功长出肿瘤，大小范围0.6-1.4cm，最小者直径0.6cm，边缘光滑，密度均匀；最大者直径1.4cm，边缘大部分光滑，小部分欠光整，中央部见小片坏死区，部分瘤体突出于肾包膜之外。
VX2移植瘤组HE染色见肿瘤细胞核大、异型、深染，呈混乱簇状及乳头状排列；对照组肾组织HE染色可见基本正常的肾小球及肾小管，结构基本规则，细胞形态良好。
2时间-密度曲线(TDC)特征
主动脉TDC：依据先后分为基线段、上升段、下降段及水平段4阶段。基线段较平直；然后骤然上升形成波峰，上升段和下降段表现为骤升骤降；然后是较平直并缓慢下降的水平段。
肾静脉TDC：基线段较平直，维持时间较长；然后大约在主动脉峰值之后上升形成波峰，并下降较快，表现为速升速降；波峰明显低于主动脉，时间跨度长于主动脉；之后缓慢下降，也可见到小的再循环峰。
正常对照组肾脏TDC：基线段较主动脉的TDC稍长，上升段较陡，但陡度小于主动脉，达到峰值后缓慢上升一段时间再缓慢下降，表现为速升速降，然后是较平直并缓慢下降的水平段。
移植瘤组TDC：与正常组相似，但开始上升时间向后推移，上升速率减慢，上升段明显平缓，幅度减低，且峰值出现时间迟于正常肾皮质。3正常对照组、移植瘤组BF、BV、MTT灌注参数比较
正常对照组BF、BV、MTT分别是(114.2±58.6)ml／(min.100mg)、(107.9±99.1)ml／100mg、(22.4±19.1)s，移植瘤组BF、BV、MTF分别是(24.6±14.3)ml／(min．100mg)、(106.7±64.5)ml／100mg、(40.9±13.1)s。两组BF、MTT比较，参考值P＜0.05，被认为显著性差异；两组BV比较、参考值P＞0.05，统计学无显著性意义
结论
1通过采用切开后腹壁直接包埋种植法，可获得稳定的兔VX2肾移植瘤模型。
2兔VX2肾移植瘤模型CT灌注参数BF低于正常肾皮质，MTT则高于正常肾皮质，CT灌注在一定程度上能反映出VX2移植瘤的血流动力学变化规律，为研究人类肾癌提供理论依据。
第二部分肾细胞癌64排螺旋CT灌注成像与MVD及VEGF的相关性研究
研究目的
利用64排CT灌注成像技术，探讨肾细胞癌血流动力学变化规律，重点为肾癌BF、BV、MTT等CT灌注参数与肿瘤微血管密度(MVD)及血管内皮生长因子(VEGF)之间的关系。
材料与方法
1主要仪器与试剂
同第一部分。
2研究对象
正常对照组10例：分左、右肾两组。
肾细胞癌组15例：均经手术病理证实为肾细胞癌，病理类型：透明细胞癌8例，颗粒细胞癌4例，乳头状癌3例；肾细胞癌组与同侧癌旁肾皮质(距离肿瘤边缘2.0cm以上)作为比较。
3扫描方案及参数
常规扫描：先常规轴位平扫中腹部，再行全肾灌注模式(按设定程序，床板循环往复移动)扫描，利用双筒高压注射器，注射速率5ml／s，先注射非离子型对比剂370mgI／ml典必乐40ml，后注射生理盐水20ml，注射后延迟8秒钟开始扫描，扫描15次。扫描参数主要有：探测器排列64×0.625mm，层厚5.0mm，间距5.0mm，Pitch1.156，120KV，100mAs，360度旋转时间0.4s，扫描间隔时间4.7-5.6s(默认最小)，总时间为85-90s。
4图像后处理及数据收集
同第一部分。注意个别受检者因呼吸影响出现图像漂移时，要用微调通过手工进行逐层校正，以保证TDC的正确和完整。
5病理检查和免疫组化观察方法
5.1病理制作准备
肿瘤组织的取材部位与CT灌注图像所选的ROI一致。每个组织块连续切片3张，层厚为4μm，一张HE染色用于普通病理诊断，一张用于MVD免疫组化染色，另一张用于VEGF免疫组化染色。
5.2 MVD计算方法
参照Weidner等的判断标准，首先在100倍光镜下浏览全片，寻找肿瘤血管高密度区，选定4个血管密度最高区在高倍镜下(200倍)观察，在目镜网格测微尺0.25μm~2的范围内计数，将4个视野血管数目进行平均，取其平均值。5.3 VEGF检查的标本的制作及结果分析标准
血管内皮生长因子检测采用链霉素-生物素-过氧化酶连接法(streptavidin-biotin-peroxidase，SP法)进行免疫组化染色，二甲基氨联苯胺(diamino benzidine tertrahydrochloride，DAB)显色试剂盒，进行VEGF的检测和量化分析，所用试剂盒均购自Roche公司。DAB显色，苏木素复染，常规脱水、透明、封片，用已知阳性标本做阳性对照，对照组由磷酸缓冲液(PBS)代替一抗。
VEGF表达结果的判断采用兼顾VEGF阳性染色的强度和阳性细胞所占的百分比作为判断标准。VEGF阳性表达定位于肿瘤细胞浆中，首先将细胞浆染色强度打分，0分为无色，1分为淡黄色，2分为棕黄色，3分为棕褐色，再将阳性细胞所占的百分比打分：0分为阴性，1分为阳性细胞≤10％，2分为11％～50％，3分为51％～75％，4分为＞75％。染色强度与阳性百分比的乘积＞3分为免疫反应阳性。并按乘积分数分为4个等级：-(0，1，2分)、+(3，4分)、++(6，8分)、+++(9，12分)。
6统计学分析
所有计数资料数据用均数±标准差(x±s)表示，经SPSS13.0统计软件分析，两两样本均数的比较用t检验；多个样本均数的比较用方差分析。等级资料的比较用非参数检验，对于肾癌实质BF与MVD和VEGF表达之间的关系分别采用Pearson相关分析和Spearman’s相关分析法。P＜0.05，统计学有显著性差异。
结果
1 TDC形态
1.1主动脉及肾静脉TDC形态：同第一部分，形态较实验兔肾高耸。
1.2正常肾脏TDC形态表现
肾皮质TDC呈速升缓降型，峰值出现在主动脉峰值之后，上升段的形态与主动脉类似，但较主动脉平缓，幅度低，下降段在快速轻度下降后，维持缓慢下降的平台期。
1.3肾癌TDC形态表现
依据肾癌CT灌注的TDC形态不同将15例肾癌分为富血供和乏血供两型，其中8例为富血供肾癌，判断标准为：瘤体TDC形态类似于主动脉TDC，曲线上升幅度及陡峭程度均稍低于腹主动脉，上升段呈较陡的上升型曲线，而下降段不明显，水平段延长；7例为乏血供型肾癌，判断标准为：肿瘤实质TDC曲线与腹主动脉TDC明显不同，曲线上升幅度及陡峭程度明显低于腹主动脉，上升段较缓慢，下降段不明显，有的可呈波浪状。
2正常左、右侧肾皮质灌注参数比较
左侧肾皮质BF(182.99±25.89)ml／(min．100mg)、BV(109.74±13.07)ml／100mg、MTT(9.50±3.13)s；右侧肾皮质BF(190.68±33.68)ml／(min．100mg)、BV(122.84±19.75)ml／100mg、MTT(8.70±3.30)s。左、右肾皮质灌参数BF、BV、MTr比较，P＞0.05，统计学无显著性差异。
3肾癌组与同侧癌旁肾皮质灌注参数比较
肾癌组BF(133.0±29.9)ml／(min．100mg)、BV(107.2±23.1)ml／100mg、MTF(15.6±6.5)s，同侧癌旁肾皮质组BF(166.1±26.0)ml／(min．100mg)、BV(84.8±31.6)ml／100mg、MTT(11.3±3.3)s，两组间P＜0.05，统计学有显著性差异。
4 CT灌注参数与MVD、VEGF的关系
4.1肾癌和同侧癌旁肾皮质VEGF的表达
15例肾癌组织中VEGF表达阳性者12例，同侧癌旁肾皮质组织则无VEGF阳性表达，经非常参数检验，两组间P＜0.05，统计学有显著性差异。
4.2肾癌和同侧癌旁肾皮质中CD34的表达
肾癌组织与同侧癌旁肾皮质MVD在200倍视野下测量值分别为：122.2±25.4条、28.2±14.7条。经独立样本t检验，两组间P＜0.05，统计学有显著性差异。
5 CT灌注成像参数值、VEGF及MVD的相关性
5.1 VEGF的表达与MVD之间的相关性
VEGF表达阳性的。肾癌病人的MVD值明显高于VEGF表达阴性者，经Spearman相关分析VEGF表达和MVD之间呈正相关(r=0.870，P=0.000)。
5.2肾癌CT灌注参数值BF、MTT与MVD的相关性
对肾癌组中MVD和BF、MTT的采用Pearson相关分析，发现肿瘤实质的BF值与MVD呈正相关(r值为r=0.565，P=0.028)，MTT与MVD呈负相关(r值为r=-0.878，P=0.000)。
5.3肾癌组CT灌注参数值BF与VEGF的相关性
对肾癌组中的VEGF和BF经Spearman相关分析，(P=0.06＞0.05)，统计结果表明VEGF表达和灌注参数值BF之间尚不能认为有相关性。
结论
1肾细胞癌CT灌注参数与癌旁肾皮质比较BF减低，而BV增高，MTT延长。CT灌注参数可以反映出肾癌组织血流动力学与正常肾皮质明显不同。
2肾癌CT灌注参数BF值与MVD存在正相关，MTT与MVD呈负相关；而VEGF在肾癌组织中表达较高，在癌旁肾皮质中表达较低。因此CT灌注成像可以在一定程度上反映肾癌的血管生成状况。
64-slice spiral CT was applied to perform CT perfusion imaging (CTPI) in rabbit VX2 renal carcinoma model and renal cell carcinoma (RCC), measuring the CT perfusion (CTP) value respectively, comparing with that in the normal group and making the statistical analyses, and studying the hemodynamic changes of RCC. The quantitative indexes of tumor angiogenesis including microvessel density (MVD) and vascular endothelial growth factor (VEGF) of RCC and peritumoral renal cortex by immunohistochemical techniques were compared to explore the correlations between CTP parameters of RCC and the quantitative indexes of tumor angiogenesis, and consequently, to evaluate the clinical value of CTPI in RCC angiogenesis evaluation.
PartⅠThe study of CTPI with 64-slice spiral CT on rabbit VX2 renal carcinoma model
Objective
To investigate the rule in hemodynamic changes of rabbit VX2 renal carcinoma model and provide theoretical bases for research in human renal tumor, through the establishment of rabbit VX2 renal carcinoma model and CTPI with 64-slice spiral CT on rabbit VX2 renal carcinoma model.
Materials and Methods
1 Major equipment and reagents
Brilliance 64-slice spiral CT scanner (Holland PHILIPS), with Mxview (with renal perfusion software) workstation and double-syringe power injector (US MEDRAD); non-ionic contrast agent, iopamidol (370mgl/ml).
2 Experimental data
2.1 Experimental groups
A total of 20 New Zealand white rabbits, weight ranging from 2-2.5kg, male or female, were randomly divided into the experimental group and the normal control group.
2.2 The establishment of rabbit VX2 renal carcinoma model
A tumor-transplanted VX2 New Zealand stud rabbit was prepared, tumor cells were inoculated in its hind leg subcutaneously to produce tumors and then the rabbit was sub-cultured to give birth to stud rabbits. Stud rabbits about two weeks were chosen and their tumors were removed; the vigorously-growing tissues surrounding the tumor masses were made into pieces about 1-2mm~3. Tumor masses transplantation by direct embedding was employed through the incisions in the posterior abdominal walls where kidneys were exposed, and the rabbit VX2 renal carcinomar model were established. Then these rabbits were fed for 16-20 days for subsequent experiments.
2.3 CTPI techniques and methods
First the conventional full renal axial plain scan was performed, CT slice thickness and spacing both being 2.5mm, and then dynamic enhanced CT scan in a wide range and at a single level was performed, the maximal range of scan being 40mm and the injection rate of double-syringe power injector being 1.5ml/s; 4ml of iopamidol (370mgl/ml), the non-ionic contrast agent was injected, then 2ml saline was injected and CT scan was performed is later after the injection, for 50 times, 50s in total. Parameters of perfusion scan were primarily made up of 64x0.625mm of the detector array, and in non-interval continuous scan, 2.5mm of CT slice thickness, 120KV of voltage, 150mAs of electric current and 0.5s of 360-degree rotation time.
Selection of ROI (region of interest), including the aorta, renal vein, tumor parenchyma and the renal cortex. The aorta and renal vein could be circular or elliptic, and the tumor parenchyma and renal cortex could be of the irregular shape. ROI should be as wide as possible to reduce the quantum noise, no fewer than 50 pixels; however, ROI could not reach the edge of the organ so as to avoid the impact of the partial volume effect (PVE); moreover, ROI of the parenchymatous area should not contain renal sinus fat and the lesion should avoid the necrotic area.
3 Image post-processing and data collection and observation
In Mxview workstation, renal perfusion software was employed for image post-processing. The abdominal aorta, taking the place of renal artery, was regarded as the afferent artery and the renal vein was regarded as the efferent artery. Regions of interest for the abdominal aorta, renal vein and renal cortex identified, the software would measure the CT value for each ROI automatically and the time-density curve (TDC) and the perfusion map were plotted; according to TDC, the software would generate and display perfusion indexes, including the renal blood flow (BF), renal blood volume (BV) and mean transit time (MTT) of the contrast agent and so on. All measurements should be executed twice during the period of study and their mean value was thus obtained.
The perfusion parameters of BF, BV and MTT etc in the experimental and control groups were collected and recorded, and TDC shapes were observed.
4 Pathological observations
Routine hematoxylin-eosin (HE) staining having been performed, the normal kidney and tumor tissues were observed under the microscope.
5 Statistical analyses
SPSS13.0 software package was applied for statistical analyses in this study. Each parameter index of peffusion was indicated by mean±standard deviation ((?)±s), data were indicated by two independent samples t-test and P＜0.05, was regarded as the significant difference.
Results
1 General shapes and pathological observation of tumor-transplanted models
The ten New Zealand white rabbits all gave birth to tumors, sizes ranging from 0.6-1.4cm; the diameter of the smallest tumor was 0.6cm, borders smooth and density uniform and the diameter of the largest was 1.4cm, most borders smooth, but a small part not smooth, due to the small flaky necrotic region observed in the center and some part of the tumor body protruding from the renal capsule.
HE staining for the VX2 renal carcinomar model group found that cell nucleus of tumors was large, abnormal or deep dyed, ranking in cluttered, clustered or mamillary state; by the HE staining of renal tissues, the comparatively normal glomerulus and renal tubule observed were regular in structure and relatively well in cell shape, and inflammatory cell infiltration could be observed in some part.
2 TDC features
Aorta TDC was divided into four segments, baseline segment, rising segment, declining segment and horizontal segment, based on the sequence. The baseline segment was relatively straight; then the curve suddenly rose to become wave crest, and the rising and declining segments showed sudden rise and sudden decline; in the end, the curve smoothly became the horizontal segment, declining gradually.
Renal vein TDC: the baseline segment was relatively straight, lasting for a long time; then the wave rose to form wave crest shortly after the formation of aorta wave crest, and declined fast, giving the impression of fast rise and fast decline; the crest value of renal vein was obviously lower than that of the aorta, but its time span was longer than that of the aorta; later, the curve declined gradually, when the small recycle crest could be seen.
Renal TDC in the normal control group: the baseline segment was longer than that in aortic TDC; the rising segment was comparatively steep, but gradient lower than that of the aorta; when the curve reached its crest value, it gradually rose for some period, later gradually declined, impressing us with fast rise and fast decline, and then it became the gradually declining horizontal segment, which was relatively smooth and straight.
TDC of the tumor-transplanted group: similar with that in the normal group, but the start point of rising was postponed; the rising speed slowed, so the rising segment was evidently smooth and the extent lowered; in addition, the crest value appeared later than that in the normal group.
3 Comparisons between the perfusion parameters of BF, BV and MTT in the normal control group and tumor-transplanted group
BF, BV and MTT in the normal control group was respectively (114.2±58.6)ml/ (min.100mg), (107.9±99.1)ml/100mg and (22.4±19.1)s, and in the tumor-transplanted group, (24.6±14.3)ml/(min.100mg), (106.7±64.5)ml/100mg and (40.9±13.1)s, respectively. P＜0.05, the reference value derived from the BF and MTT comparisons between the two groups suggested the significant difference; P＞0.05, the reference value obtained by comparing BV of the two groups suggested no evident significance.
Conclusions
1 Through transplantation by embedding method by incision in the posterior abdominal wall, steady rabbit VX2 renal carcinoma model can be obtained.
2 When the CT perfusion parameter of BF in rabbit VX2 renal carcinoma model, is lower in that of the normal control group, M'IT shall be higher than that in the normal group; CT peffusion parameters may reflect the hemodynamic changes of VX2 renal carcinoma model to some extent, providing theoretical bases for research in human RCC.
PartⅡThe study of correlations between CTPI with 64-slice spiral CT and MVD, VEGF in renal cell carcinoma
Objective
To investigate with CTP techniques the rule of hemodynamic changes for RCC, especially the correlations between CT perfusion parameters BF, BV and MTT etc, and MVD and VEGF.
Materials and Methods
1 Major equipment and reagents Ditto!
2 Subjects
Ten cases in the normal control group: divided into two subgroups by the left kidney and right kidney.
Fifteen cases in RCC group: all identified as RCC by surgical-pathological diagnoses, pathological types including eight cases of clear cell carcinoma, four cases of granulosa cell carcinoma and three cases of undifferentiated carcinoma; RCC was compared with ipsilateral peritumoral renal cortex (over 2.0cm away from the tumor border).
3 The scanning program and parameters
Routine scan: first the conventional middle abdominal axial plain scan was performed, then the full renal perfusion pattern scan was performed (by the set procedures, bed-board shifting in circulatory and reciprocating modes); the double-syringe power injector was employed for injection, rate being 5ml/s, first the injection of 40ml iopamidol (370mgI/ml), the non-ionic contrast agent and then the injection of 20ml saline, and 8s later after injection, scanning was started, 15 times in total. Parameters of scanning consisted of 64×0.625mm of the detector array, 5.0mm of CT slice thickness, 5.0mm of spacing, Pitchl.156, 120KV, 100mAs, 0.4s of 360-degree rotation time, 4.7-5.6s of scan interval (minimum being the default value) and 85-90s of total time.
4 Image post-processing and data collection
Ditto! Image drift would occur to individual subjects due to respiratory effects, so the images should be adjusted layer by layer manually through delicate adjustment, to ensure the correctness and integrity of TDC.
5 Pathological examination and immunohistochemical observation
5.1 Pathological preparations
The positions of tumor tissues derived were in accordance with that of CTPI ROI. Each tissue mass was sliced into 3 pieces and CT slice thickness was 4μm. One piece of HE staining was used for conventional pathological diagnosis, one for MVD rabbit immunohistochemical staining and the other for VEGF immunohistochemical staining.
5.2 MVD calculation methods
By the evaluation criteria of Weidner etc, first the whole tomogram was observed under 100xmicroscope, to detect the high-density regions of tumor blood vessels, then four highest-density regions of blood vessels were chosen for observation under the high power microscope (200x), enumerating within 0.25μm~2 of ocular mesh micrometer scale, averaging the number of blood vessels in the four fields of vision and obtaining the average value.
5.3 Standards for the preparation of specimens for VEGF examination and result analyzing
In detection of VEGFs, streptavidin-biotin-peroxidase (SP) method was applied for immunohistochemical staining, and the color development kit of diamino benzidine tertrahydrochloride (DAB) was applied for VEGF detection and quantitative analysis. All color development kits were purchased from Roche Company. DAB color development, hematoxylin re-staining, and routine dehydration, transparency and mounting were compared with existed positive specimens for positive contrast. In the control group, phosphate buffer solution (PBS) was applied to replace the first antibody.
The evaluation for results of VEGF expression applied the criterion of balancing the intensity of VEGF positive staining and the percentage of positive cells. First the staining intensity was graded, 0 point for colorless, 1 point for light yellow, 2 points for brown yellow and 3 points for brown and then the percentage of positive cells was graded, 0 point for negative, 1 point for (positive cells =10%), 2 points for 11%～50%, 3 points for 51%～75% and 4 points for (＞75%). The arithmetic product of staining intensity and positive percentage＞3 points, the immune reaction was positive. Results of VEGF expression were then classified into four degrees by arithmetic product scores: - (0, 1, 2 points), + (3, 4points), ++ (6, 8points) and +++ (9, 12points).
6 Statistical analyses
All enumeration data were indicated by ((?)±s), analyzed by SPSS13.0 software, and the comparison between mean values of each two samples underwent t-test; comparisons between mean values of multiple samples underwent variance analysis. The comparison between classified data underwent non-parametric test, the relationships between BF of RCC parenchyma and MVD as well as VEGF underwent Pearson correlation analysis and Spearman's correlation analysis. P＜0.05 suggested the significant difference.
Results
1 TDC shapes
1.1 TDC shapes of the aorta and renal vein: in the same TDC segment, higher than that of experimental rabbits.
1.2 Normal renal TDC shapes
Renal cortex TDC rose fast and declined fast, its crest value occurred after that in the aorta; the shape of rising segment was similar with that of the aorta, but smoother and lower in amplitude; the declining segment first declined fast and slightly, and then it turned into plateau period, maintaining its steady declining.
1.3 RCC TDC shapes
According to the CTP TDC of RCC, the 15 cases with RCC were classified into two types with rich and poor blood supply, respectively, among which eight cases had rich blood supply RCC, criteria as below: the tumor body TDC shape was similar with the aorta TDC, and the amplitude and steep degree of the curve were lower than that of the abdominal aorta; seven cases had poor blood supply RCC, criteria as below: the tumor parenchyma TDC was evidently different from that of the abdominal aorta, and the amplitude and steep degree of the curve were lower than that of the abdominal aorta obviously, with slow rising segment and indistinct or even wavy declining segment.
2 Comparisons between perfusion parameters in normal left and right renal cortexes BF, BV and MTI" of the left renal cortex were respectively, (182.99±25.89)ml/ (min.100mg), (109.74±13.07)ml/100mg and (9.50±3.13)s, and in the right renal cortex, (190.68±33.68 (ml/(min.100mg), (122.84±19.75)ml/100mg and (8.70±3.30)s, respectively. Comparisons between BF, BV and MTT of the left and right renal cortexes indicated P＞0.05, no significant statistical difference.
3 Comparisons between perfusion parameters of the RCC group and ipsilateral peritumoral renal cortex group
In the RCC group, BF (133.0±29.9)ml/ (min.100mg), BV (107.2±23.1)ml/100mg and MTT (15.6±6.5)s were obtained, and in the ipsilateral peritumoral renal cortex group, BF (166.1±26.0)ml/ (min.100mg), BV (84.8±31.6)ml/100mg and MTT (11.3±3.3)s were obtained, P＜0.05, indicating significant statistical difference.
4 Comparisons between MVD as well as VEGF and perfusion parameters
4.1 VEGF expressions in RCC and ipsilateral peritumoral renal cortex
In the RCC group with 15 cases, 12 cases had positive VEGF expression, but in the ipsilateral peritumoral renal cortex group, no positive VEGF expression. After non-parametric test, we found that P＜0.05, suggesting significant statistical difference.
4.2 CD34 expressions in RCC and ipsilateral peritumoral renal cortex
The measurements of MVD for the RCC and ipsilateral peritumoral renal groups under 200×microscope were respectively, 122.2±25.4 stripes and 28.2±14.7 strips. After independent sample t-test, we found that P<0.05, indicating significant statistical difference.
5 The correlation between CTPI parameters and VEGF as well as MVD
5.1 The correlation between VEGF expression and MVD
MVD value of RCC patients with positive VEGF expression was evidently higher than that with negative VEGF expression; Spearman correlation analysis showed that VEGF expression was positively correlated with MVD (r=0.870, P=0.000).
5.2 Correlations between RCC CTP parameters, BF and MTT, and MVD
Pearson correlation analysis for MVD and BF as well as MTT in the RCC group found that BF value of tumor parenchyma was positively correlated with MVD (r=0.565, P=0. 028) and MTT was negatively correlated with MVD (r=-0.878, P=0.000).
5.3 The correlation between RCC CTP parameter BF and VEGF
Spearman correlation analysis for RCC VEGF and BF found that (P=0.06＞0.05), indicating that the correlation between VEGF expression and the perfusion parameter BF was unclear.
Conclusions
1 Among RCC CTP parameters, BF were lower than that of the normal renal cortex, but MTI" and BV increased. CTP parameters may reflect the evident differences lying between hemodynamics of RCC tissues and the normal renal cortex.
2 Among RCC CTP parameters, BF Was positively correlated with MVD and MTT was negatively correlated with MVD; in addition, VEGF had a high expression in RCC tissues, but had a low expression in the peritumoral renal cortex. Therefore, CTPI might reflect the angiogenesis situation of renal cell carcinoma.
第一部分兔VX2肾移植瘤模型64排螺旋CT灌注成像研究
研究目的
建立兔VX2肾移植瘤模型，通过对兔VX2肾移植瘤模型的64排螺旋CT灌注成像，探讨兔VX2肾移植瘤血流动力学变化规律，为研究人类肾癌提供理论依据。
材料与方法
1主要仪器与试剂
Brilliance 64排螺旋CT扫描仪(荷兰PHILIPS公司)，配置有Mxview(带有肾脏灌注软件)工作站，双筒高压注射器(美国MEDRAD公司)；非离子型对比剂典比乐(370mgI／ml)。
2实验资料
2．1实验对象分组
20只新西兰大白兔，体重2-2.5kg，雌雄不限。随机将实验动物分为实验组和正常对照组。
2．2兔VX2肾移植瘤模型的建立
荷瘤VX2新西兰种兔一只，将瘤细胞接种兔后腿的皮下成瘤并传代，制成种兔。选取2周左右种兔切除肿瘤，取瘤块边缘生长旺盛组织，制成1-2mm~3大小碎块。采用切开后腹壁暴露肾脏瘤块直接包埋法种植，建立兔VX2肾移植瘤模型。饲养16-20天进行实验。
2．3 CT灌注成像技术与方法
先常规全肾轴位平扫，层厚2.5mm，间距2.5mm，再行宽范围同层动态增强扫描，设定最宽扫描范围40mm，使用双筒高压注射器，注射速率1.5ml／s；先注射非离子型对比剂370mgI／ml典必乐4ml，后注射生理盐水2ml，注射后延迟1秒钟开始扫描，共扫描50次，总时间50s。灌注扫描主要参数有：探测器排列64×0.625mm，无间隔连续扫描，层厚2.5mm，电压120KV，电流150mAs，360度旋转时间0.5s。
感兴趣区(ROI)的选取：包括主动脉、肾静脉、肿瘤实质及肾皮质。主动脉和肾静脉可为圆形或椭圆形，肿瘤实质及肾皮质可为不规则形。ROI尽可能大，不能小于50个像素，以减少量子噪声，但不能达到器官的边缘以避免部分容积效应的影响，实质区ROI尽量不包括‘肾窦脂肪，病灶则应避开坏死区。3图像后处理、数据收集及观察
在Mxview工作站上，应用自带的灌注软件进行图像后处理。腹主动脉代替肾动脉被确定为输入动脉，肾静脉被确定为输出静脉。划出腹主动脉、肾静脉、肾皮质感兴趣区(region of interest，ROI)，软件自动测量出各ROI的CT值，并绘制出时间—密度曲线(time density curve，TDC)和灌注彩图，根据TDC，软件自动生成显示灌注指标，包括。肾脏血流量(blood flow，BF)、肾脏血容量(blood volume，BV)及对比剂的平均通过时间(mean transit time，MTT)等。研究期间所有的测量均重复2次，取其平均值。
观察TDC形态并收集、记录实验组和对照组BF、BV、MTT等灌注参数。
4病理学观察
常规苏木素-伊红(HE)染色，显微镜下观察正常肾及肿瘤的组织结构。
5统计学分析
采用SPSS13.0软件包进行统计学分析。各项灌注参数指标以平均数±标准差表示，数据采用两独立样本t检验，P＜0.05，被认为显著性差异。
结果
1兔VX2肾移植瘤模型大体形态及病理学观察
10只新西兰大白兔均成功长出肿瘤，大小范围0.6-1.4cm，最小者直径0.6cm，边缘光滑，密度均匀；最大者直径1.4cm，边缘大部分光滑，小部分欠光整，中央部见小片坏死区，部分瘤体突出于肾包膜之外。
VX2移植瘤组HE染色见肿瘤细胞核大、异型、深染，呈混乱簇状及乳头状排列；对照组肾组织HE染色可见基本正常的肾小球及肾小管，结构基本规则，细胞形态良好。
2时间-密度曲线(TDC)特征
主动脉TDC：依据先后分为基线段、上升段、下降段及水平段4阶段。基线段较平直；然后骤然上升形成波峰，上升段和下降段表现为骤升骤降；然后是较平直并缓慢下降的水平段。
肾静脉TDC：基线段较平直，维持时间较长；然后大约在主动脉峰值之后上升形成波峰，并下降较快，表现为速升速降；波峰明显低于主动脉，时间跨度长于主动脉；之后缓慢下降，也可见到小的再循环峰。
正常对照组肾脏TDC：基线段较主动脉的TDC稍长，上升段较陡，但陡度小于主动脉，达到峰值后缓慢上升一段时间再缓慢下降，表现为速升速降，然后是较平直并缓慢下降的水平段。
移植瘤组TDC：与正常组相似，但开始上升时间向后推移，上升速率减慢，上升段明显平缓，幅度减低，且峰值出现时间迟于正常肾皮质。3正常对照组、移植瘤组BF、BV、MTT灌注参数比较
正常对照组BF、BV、MTT分别是(114.2±58.6)ml／(min.100mg)、(107.9±99.1)ml／100mg、(22.4±19.1)s，移植瘤组BF、BV、MTF分别是(24.6±14.3)ml／(min．100mg)、(106.7±64.5)ml／100mg、(40.9±13.1)s。两组BF、MTT比较，参考值P＜0.05，被认为显著性差异；两组BV比较、参考值P＞0.05，统计学无显著性意义
结论
1通过采用切开后腹壁直接包埋种植法，可获得稳定的兔VX2肾移植瘤模型。
2兔VX2肾移植瘤模型CT灌注参数BF低于正常肾皮质，MTT则高于正常肾皮质，CT灌注在一定程度上能反映出VX2移植瘤的血流动力学变化规律，为研究人类肾癌提供理论依据。
第二部分肾细胞癌64排螺旋CT灌注成像与MVD及VEGF的相关性研究
研究目的
利用64排CT灌注成像技术，探讨肾细胞癌血流动力学变化规律，重点为肾癌BF、BV、MTT等CT灌注参数与肿瘤微血管密度(MVD)及血管内皮生长因子(VEGF)之间的关系。
材料与方法
1主要仪器与试剂
同第一部分。
2研究对象
正常对照组10例：分左、右肾两组。
肾细胞癌组15例：均经手术病理证实为肾细胞癌，病理类型：透明细胞癌8例，颗粒细胞癌4例，乳头状癌3例；肾细胞癌组与同侧癌旁肾皮质(距离肿瘤边缘2.0cm以上)作为比较。
3扫描方案及参数
常规扫描：先常规轴位平扫中腹部，再行全肾灌注模式(按设定程序，床板循环往复移动)扫描，利用双筒高压注射器，注射速率5ml／s，先注射非离子型对比剂370mgI／ml典必乐40ml，后注射生理盐水20ml，注射后延迟8秒钟开始扫描，扫描15次。扫描参数主要有：探测器排列64×0.625mm，层厚5.0mm，间距5.0mm，Pitch1.156，120KV，100mAs，360度旋转时间0.4s，扫描间隔时间4.7-5.6s(默认最小)，总时间为85-90s。
4图像后处理及数据收集
同第一部分。注意个别受检者因呼吸影响出现图像漂移时，要用微调通过手工进行逐层校正，以保证TDC的正确和完整。
5病理检查和免疫组化观察方法
5.1病理制作准备
肿瘤组织的取材部位与CT灌注图像所选的ROI一致。每个组织块连续切片3张，层厚为4μm，一张HE染色用于普通病理诊断，一张用于MVD免疫组化染色，另一张用于VEGF免疫组化染色。
5.2 MVD计算方法
参照Weidner等的判断标准，首先在100倍光镜下浏览全片，寻找肿瘤血管高密度区，选定4个血管密度最高区在高倍镜下(200倍)观察，在目镜网格测微尺0.25μm~2的范围内计数，将4个视野血管数目进行平均，取其平均值。5.3 VEGF检查的标本的制作及结果分析标准
血管内皮生长因子检测采用链霉素-生物素-过氧化酶连接法(streptavidin-biotin-peroxidase，SP法)进行免疫组化染色，二甲基氨联苯胺(diamino benzidine tertrahydrochloride，DAB)显色试剂盒，进行VEGF的检测和量化分析，所用试剂盒均购自Roche公司。DAB显色，苏木素复染，常规脱水、透明、封片，用已知阳性标本做阳性对照，对照组由磷酸缓冲液(PBS)代替一抗。
VEGF表达结果的判断采用兼顾VEGF阳性染色的强度和阳性细胞所占的百分比作为判断标准。VEGF阳性表达定位于肿瘤细胞浆中，首先将细胞浆染色强度打分，0分为无色，1分为淡黄色，2分为棕黄色，3分为棕褐色，再将阳性细胞所占的百分比打分：0分为阴性，1分为阳性细胞≤10％，2分为11％～50％，3分为51％～75％，4分为＞75％。染色强度与阳性百分比的乘积＞3分为免疫反应阳性。并按乘积分数分为4个等级：-(0，1，2分)、+(3，4分)、++(6，8分)、+++(9，12分)。
6统计学分析
所有计数资料数据用均数±标准差(x±s)表示，经SPSS13.0统计软件分析，两两样本均数的比较用t检验；多个样本均数的比较用方差分析。等级资料的比较用非参数检验，对于肾癌实质BF与MVD和VEGF表达之间的关系分别采用Pearson相关分析和Spearman’s相关分析法。P＜0.05，统计学有显著性差异。
结果
1 TDC形态
1.1主动脉及肾静脉TDC形态：同第一部分，形态较实验兔肾高耸。
1.2正常肾脏TDC形态表现
肾皮质TDC呈速升缓降型，峰值出现在主动脉峰值之后，上升段的形态与主动脉类似，但较主动脉平缓，幅度低，下降段在快速轻度下降后，维持缓慢下降的平台期。
1.3肾癌TDC形态表现
依据肾癌CT灌注的TDC形态不同将15例肾癌分为富血供和乏血供两型，其中8例为富血供肾癌，判断标准为：瘤体TDC形态类似于主动脉TDC，曲线上升幅度及陡峭程度均稍低于腹主动脉，上升段呈较陡的上升型曲线，而下降段不明显，水平段延长；7例为乏血供型肾癌，判断标准为：肿瘤实质TDC曲线与腹主动脉TDC明显不同，曲线上升幅度及陡峭程度明显低于腹主动脉，上升段较缓慢，下降段不明显，有的可呈波浪状。
2正常左、右侧肾皮质灌注参数比较
左侧肾皮质BF(182.99±25.89)ml／(min．100mg)、BV(109.74±13.07)ml／100mg、MTT(9.50±3.13)s；右侧肾皮质BF(190.68±33.68)ml／(min．100mg)、BV(122.84±19.75)ml／100mg、MTT(8.70±3.30)s。左、右肾皮质灌参数BF、BV、MTr比较，P＞0.05，统计学无显著性差异。
3肾癌组与同侧癌旁肾皮质灌注参数比较
肾癌组BF(133.0±29.9)ml／(min．100mg)、BV(107.2±23.1)ml／100mg、MTF(15.6±6.5)s，同侧癌旁肾皮质组BF(166.1±26.0)ml／(min．100mg)、BV(84.8±31.6)ml／100mg、MTT(11.3±3.3)s，两组间P＜0.05，统计学有显著性差异。
4 CT灌注参数与MVD、VEGF的关系
4.1肾癌和同侧癌旁肾皮质VEGF的表达
15例肾癌组织中VEGF表达阳性者12例，同侧癌旁肾皮质组织则无VEGF阳性表达，经非常参数检验，两组间P＜0.05，统计学有显著性差异。
4.2肾癌和同侧癌旁肾皮质中CD34的表达
肾癌组织与同侧癌旁肾皮质MVD在200倍视野下测量值分别为：122.2±25.4条、28.2±14.7条。经独立样本t检验，两组间P＜0.05，统计学有显著性差异。
5 CT灌注成像参数值、VEGF及MVD的相关性
5.1 VEGF的表达与MVD之间的相关性
VEGF表达阳性的。肾癌病人的MVD值明显高于VEGF表达阴性者，经Spearman相关分析VEGF表达和MVD之间呈正相关(r=0.870，P=0.000)。
5.2肾癌CT灌注参数值BF、MTT与MVD的相关性
对肾癌组中MVD和BF、MTT的采用Pearson相关分析，发现肿瘤实质的BF值与MVD呈正相关(r值为r=0.565，P=0.028)，MTT与MVD呈负相关(r值为r=-0.878，P=0.000)。
5.3肾癌组CT灌注参数值BF与VEGF的相关性
对肾癌组中的VEGF和BF经Spearman相关分析，(P=0.06＞0.05)，统计结果表明VEGF表达和灌注参数值BF之间尚不能认为有相关性。
结论
1肾细胞癌CT灌注参数与癌旁肾皮质比较BF减低，而BV增高，MTT延长。CT灌注参数可以反映出肾癌组织血流动力学与正常肾皮质明显不同。
2肾癌CT灌注参数BF值与MVD存在正相关，MTT与MVD呈负相关；而VEGF在肾癌组织中表达较高，在癌旁肾皮质中表达较低。因此CT灌注成像可以在一定程度上反映肾癌的血管生成状况。
64-slice spiral CT was applied to perform CT perfusion imaging (CTPI) in rabbit VX2 renal carcinoma model and renal cell carcinoma (RCC), measuring the CT perfusion (CTP) value respectively, comparing with that in the normal group and making the statistical analyses, and studying the hemodynamic changes of RCC. The quantitative indexes of tumor angiogenesis including microvessel density (MVD) and vascular endothelial growth factor (VEGF) of RCC and peritumoral renal cortex by immunohistochemical techniques were compared to explore the correlations between CTP parameters of RCC and the quantitative indexes of tumor angiogenesis, and consequently, to evaluate the clinical value of CTPI in RCC angiogenesis evaluation.
PartⅠThe study of CTPI with 64-slice spiral CT on rabbit VX2 renal carcinoma model
Objective
To investigate the rule in hemodynamic changes of rabbit VX2 renal carcinoma model and provide theoretical bases for research in human renal tumor, through the establishment of rabbit VX2 renal carcinoma model and CTPI with 64-slice spiral CT on rabbit VX2 renal carcinoma model.
Materials and Methods
1 Major equipment and reagents
Brilliance 64-slice spiral CT scanner (Holland PHILIPS), with Mxview (with renal perfusion software) workstation and double-syringe power injector (US MEDRAD); non-ionic contrast agent, iopamidol (370mgl/ml).
2 Experimental data
2.1 Experimental groups
A total of 20 New Zealand white rabbits, weight ranging from 2-2.5kg, male or female, were randomly divided into the experimental group and the normal control group.
2.2 The establishment of rabbit VX2 renal carcinoma model
A tumor-transplanted VX2 New Zealand stud rabbit was prepared, tumor cells were inoculated in its hind leg subcutaneously to produce tumors and then the rabbit was sub-cultured to give birth to stud rabbits. Stud rabbits about two weeks were chosen and their tumors were removed; the vigorously-growing tissues surrounding the tumor masses were made into pieces about 1-2mm~3. Tumor masses transplantation by direct embedding was employed through the incisions in the posterior abdominal walls where kidneys were exposed, and the rabbit VX2 renal carcinomar model were established. Then these rabbits were fed for 16-20 days for subsequent experiments.
2.3 CTPI techniques and methods
First the conventional full renal axial plain scan was performed, CT slice thickness and spacing both being 2.5mm, and then dynamic enhanced CT scan in a wide range and at a single level was performed, the maximal range of scan being 40mm and the injection rate of double-syringe power injector being 1.5ml/s; 4ml of iopamidol (370mgl/ml), the non-ionic contrast agent was injected, then 2ml saline was injected and CT scan was performed is later after the injection, for 50 times, 50s in total. Parameters of perfusion scan were primarily made up of 64x0.625mm of the detector array, and in non-interval continuous scan, 2.5mm of CT slice thickness, 120KV of voltage, 150mAs of electric current and 0.5s of 360-degree rotation time.
Selection of ROI (region of interest), including the aorta, renal vein, tumor parenchyma and the renal cortex. The aorta and renal vein could be circular or elliptic, and the tumor parenchyma and renal cortex could be of the irregular shape. ROI should be as wide as possible to reduce the quantum noise, no fewer than 50 pixels; however, ROI could not reach the edge of the organ so as to avoid the impact of the partial volume effect (PVE); moreover, ROI of the parenchymatous area should not contain renal sinus fat and the lesion should avoid the necrotic area.
3 Image post-processing and data collection and observation
In Mxview workstation, renal perfusion software was employed for image post-processing. The abdominal aorta, taking the place of renal artery, was regarded as the afferent artery and the renal vein was regarded as the efferent artery. Regions of interest for the abdominal aorta, renal vein and renal cortex identified, the software would measure the CT value for each ROI automatically and the time-density curve (TDC) and the perfusion map were plotted; according to TDC, the software would generate and display perfusion indexes, including the renal blood flow (BF), renal blood volume (BV) and mean transit time (MTT) of the contrast agent and so on. All measurements should be executed twice during the period of study and their mean value was thus obtained.
The perfusion parameters of BF, BV and MTT etc in the experimental and control groups were collected and recorded, and TDC shapes were observed.
4 Pathological observations
Routine hematoxylin-eosin (HE) staining having been performed, the normal kidney and tumor tissues were observed under the microscope.
5 Statistical analyses
SPSS13.0 software package was applied for statistical analyses in this study. Each parameter index of peffusion was indicated by mean±standard deviation ((?)±s), data were indicated by two independent samples t-test and P＜0.05, was regarded as the significant difference.
Results
1 General shapes and pathological observation of tumor-transplanted models
The ten New Zealand white rabbits all gave birth to tumors, sizes ranging from 0.6-1.4cm; the diameter of the smallest tumor was 0.6cm, borders smooth and density uniform and the diameter of the largest was 1.4cm, most borders smooth, but a small part not smooth, due to the small flaky necrotic region observed in the center and some part of the tumor body protruding from the renal capsule.
HE staining for the VX2 renal carcinomar model group found that cell nucleus of tumors was large, abnormal or deep dyed, ranking in cluttered, clustered or mamillary state; by the HE staining of renal tissues, the comparatively normal glomerulus and renal tubule observed were regular in structure and relatively well in cell shape, and inflammatory cell infiltration could be observed in some part.
2 TDC features
Aorta TDC was divided into four segments, baseline segment, rising segment, declining segment and horizontal segment, based on the sequence. The baseline segment was relatively straight; then the curve suddenly rose to become wave crest, and the rising and declining segments showed sudden rise and sudden decline; in the end, the curve smoothly became the horizontal segment, declining gradually.
Renal vein TDC: the baseline segment was relatively straight, lasting for a long time; then the wave rose to form wave crest shortly after the formation of aorta wave crest, and declined fast, giving the impression of fast rise and fast decline; the crest value of renal vein was obviously lower than that of the aorta, but its time span was longer than that of the aorta; later, the curve declined gradually, when the small recycle crest could be seen.
Renal TDC in the normal control group: the baseline segment was longer than that in aortic TDC; the rising segment was comparatively steep, but gradient lower than that of the aorta; when the curve reached its crest value, it gradually rose for some period, later gradually declined, impressing us with fast rise and fast decline, and then it became the gradually declining horizontal segment, which was relatively smooth and straight.
TDC of the tumor-transplanted group: similar with that in the normal group, but the start point of rising was postponed; the rising speed slowed, so the rising segment was evidently smooth and the extent lowered; in addition, the crest value appeared later than that in the normal group.
3 Comparisons between the perfusion parameters of BF, BV and MTT in the normal control group and tumor-transplanted group
BF, BV and MTT in the normal control group was respectively (114.2±58.6)ml/ (min.100mg), (107.9±99.1)ml/100mg and (22.4±19.1)s, and in the tumor-transplanted group, (24.6±14.3)ml/(min.100mg), (106.7±64.5)ml/100mg and (40.9±13.1)s, respectively. P＜0.05, the reference value derived from the BF and MTT comparisons between the two groups suggested the significant difference; P＞0.05, the reference value obtained by comparing BV of the two groups suggested no evident significance.
Conclusions
1 Through transplantation by embedding method by incision in the posterior abdominal wall, steady rabbit VX2 renal carcinoma model can be obtained.
2 When the CT perfusion parameter of BF in rabbit VX2 renal carcinoma model, is lower in that of the normal control group, M'IT shall be higher than that in the normal group; CT peffusion parameters may reflect the hemodynamic changes of VX2 renal carcinoma model to some extent, providing theoretical bases for research in human RCC.
PartⅡThe study of correlations between CTPI with 64-slice spiral CT and MVD, VEGF in renal cell carcinoma
Objective
To investigate with CTP techniques the rule of hemodynamic changes for RCC, especially the correlations between CT perfusion parameters BF, BV and MTT etc, and MVD and VEGF.
Materials and Methods
1 Major equipment and reagents Ditto!
2 Subjects
Ten cases in the normal control group: divided into two subgroups by the left kidney and right kidney.
Fifteen cases in RCC group: all identified as RCC by surgical-pathological diagnoses, pathological types including eight cases of clear cell carcinoma, four cases of granulosa cell carcinoma and three cases of undifferentiated carcinoma; RCC was compared with ipsilateral peritumoral renal cortex (over 2.0cm away from the tumor border).
3 The scanning program and parameters
Routine scan: first the conventional middle abdominal axial plain scan was performed, then the full renal perfusion pattern scan was performed (by the set procedures, bed-board shifting in circulatory and reciprocating modes); the double-syringe power injector was employed for injection, rate being 5ml/s, first the injection of 40ml iopamidol (370mgI/ml), the non-ionic contrast agent and then the injection of 20ml saline, and 8s later after injection, scanning was started, 15 times in total. Parameters of scanning consisted of 64×0.625mm of the detector array, 5.0mm of CT slice thickness, 5.0mm of spacing, Pitchl.156, 120KV, 100mAs, 0.4s of 360-degree rotation time, 4.7-5.6s of scan interval (minimum being the default value) and 85-90s of total time.
4 Image post-processing and data collection
Ditto! Image drift would occur to individual subjects due to respiratory effects, so the images should be adjusted layer by layer manually through delicate adjustment, to ensure the correctness and integrity of TDC.
5 Pathological examination and immunohistochemical observation
5.1 Pathological preparations
The positions of tumor tissues derived were in accordance with that of CTPI ROI. Each tissue mass was sliced into 3 pieces and CT slice thickness was 4μm. One piece of HE staining was used for conventional pathological diagnosis, one for MVD rabbit immunohistochemical staining and the other for VEGF immunohistochemical staining.
5.2 MVD calculation methods
By the evaluation criteria of Weidner etc, first the whole tomogram was observed under 100xmicroscope, to detect the high-density regions of tumor blood vessels, then four highest-density regions of blood vessels were chosen for observation under the high power microscope (200x), enumerating within 0.25μm~2 of ocular mesh micrometer scale, averaging the number of blood vessels in the four fields of vision and obtaining the average value.
5.3 Standards for the preparation of specimens for VEGF examination and result analyzing
In detection of VEGFs, streptavidin-biotin-peroxidase (SP) method was applied for immunohistochemical staining, and the color development kit of diamino benzidine tertrahydrochloride (DAB) was applied for VEGF detection and quantitative analysis. All color development kits were purchased from Roche Company. DAB color development, hematoxylin re-staining, and routine dehydration, transparency and mounting were compared with existed positive specimens for positive contrast. In the control group, phosphate buffer solution (PBS) was applied to replace the first antibody.
The evaluation for results of VEGF expression applied the criterion of balancing the intensity of VEGF positive staining and the percentage of positive cells. First the staining intensity was graded, 0 point for colorless, 1 point for light yellow, 2 points for brown yellow and 3 points for brown and then the percentage of positive cells was graded, 0 point for negative, 1 point for (positive cells =10%), 2 points for 11%～50%, 3 points for 51%～75% and 4 points for (＞75%). The arithmetic product of staining intensity and positive percentage＞3 points, the immune reaction was positive. Results of VEGF expression were then classified into four degrees by arithmetic product scores: - (0, 1, 2 points), + (3, 4points), ++ (6, 8points) and +++ (9, 12points).
6 Statistical analyses
All enumeration data were indicated by ((?)±s), analyzed by SPSS13.0 software, and the comparison between mean values of each two samples underwent t-test; comparisons between mean values of multiple samples underwent variance analysis. The comparison between classified data underwent non-parametric test, the relationships between BF of RCC parenchyma and MVD as well as VEGF underwent Pearson correlation analysis and Spearman's correlation analysis. P＜0.05 suggested the significant difference.
Results
1 TDC shapes
1.1 TDC shapes of the aorta and renal vein: in the same TDC segment, higher than that of experimental rabbits.
1.2 Normal renal TDC shapes
Renal cortex TDC rose fast and declined fast, its crest value occurred after that in the aorta; the shape of rising segment was similar with that of the aorta, but smoother and lower in amplitude; the declining segment first declined fast and slightly, and then it turned into plateau period, maintaining its steady declining.
1.3 RCC TDC shapes
According to the CTP TDC of RCC, the 15 cases with RCC were classified into two types with rich and poor blood supply, respectively, among which eight cases had rich blood supply RCC, criteria as below: the tumor body TDC shape was similar with the aorta TDC, and the amplitude and steep degree of the curve were lower than that of the abdominal aorta; seven cases had poor blood supply RCC, criteria as below: the tumor parenchyma TDC was evidently different from that of the abdominal aorta, and the amplitude and steep degree of the curve were lower than that of the abdominal aorta obviously, with slow rising segment and indistinct or even wavy declining segment.
2 Comparisons between perfusion parameters in normal left and right renal cortexes BF, BV and MTI" of the left renal cortex were respectively, (182.99±25.89)ml/ (min.100mg), (109.74±13.07)ml/100mg and (9.50±3.13)s, and in the right renal cortex, (190.68±33.68 (ml/(min.100mg), (122.84±19.75)ml/100mg and (8.70±3.30)s, respectively. Comparisons between BF, BV and MTT of the left and right renal cortexes indicated P＞0.05, no significant statistical difference.
3 Comparisons between perfusion parameters of the RCC group and ipsilateral peritumoral renal cortex group
In the RCC group, BF (133.0±29.9)ml/ (min.100mg), BV (107.2±23.1)ml/100mg and MTT (15.6±6.5)s were obtained, and in the ipsilateral peritumoral renal cortex group, BF (166.1±26.0)ml/ (min.100mg), BV (84.8±31.6)ml/100mg and MTT (11.3±3.3)s were obtained, P＜0.05, indicating significant statistical difference.
4 Comparisons between MVD as well as VEGF and perfusion parameters
4.1 VEGF expressions in RCC and ipsilateral peritumoral renal cortex
In the RCC group with 15 cases, 12 cases had positive VEGF expression, but in the ipsilateral peritumoral renal cortex group, no positive VEGF expression. After non-parametric test, we found that P＜0.05, suggesting significant statistical difference.
4.2 CD34 expressions in RCC and ipsilateral peritumoral renal cortex
The measurements of MVD for the RCC and ipsilateral peritumoral renal groups under 200×microscope were respectively, 122.2±25.4 stripes and 28.2±14.7 strips. After independent sample t-test, we found that P<0.05, indicating significant statistical difference.
5 The correlation between CTPI parameters and VEGF as well as MVD
5.1 The correlation between VEGF expression and MVD
MVD value of RCC patients with positive VEGF expression was evidently higher than that with negative VEGF expression; Spearman correlation analysis showed that VEGF expression was positively correlated with MVD (r=0.870, P=0.000).
5.2 Correlations between RCC CTP parameters, BF and MTT, and MVD
Pearson correlation analysis for MVD and BF as well as MTT in the RCC group found that BF value of tumor parenchyma was positively correlated with MVD (r=0.565, P=0. 028) and MTT was negatively correlated with MVD (r=-0.878, P=0.000).
5.3 The correlation between RCC CTP parameter BF and VEGF
Spearman correlation analysis for RCC VEGF and BF found that (P=0.06＞0.05), indicating that the correlation between VEGF expression and the perfusion parameter BF was unclear.
Conclusions
1 Among RCC CTP parameters, BF were lower than that of the normal renal cortex, but MTI" and BV increased. CTP parameters may reflect the evident differences lying between hemodynamics of RCC tissues and the normal renal cortex.
2 Among RCC CTP parameters, BF Was positively correlated with MVD and MTT was negatively correlated with MVD; in addition, VEGF had a high expression in RCC tissues, but had a low expression in the peritumoral renal cortex. Therefore, CTPI might reflect the angiogenesis situation of renal cell carcinoma.
引文
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