动态增强MRI、扩散加权成像及光学成像联合监测抗血管生成治疗后肿瘤反应的动物实验研究

Monitoring tumor response to antiangiogenic treatment by integrating of dynamic contrast enhanced MRI, diffusion weighted imaging and optical imaging in animal model

目的利用多模态成像技术即小动物活体光学成像、动态增强MRI（DCE—MRI）及DWI，评价裸鼠肺癌皮下移植瘤经抗血管治疗后肿瘤解剖形态、血管功能及细胞分子水平的改变。方法将绿色荧光蛋白（GFP）标记的人NCI—H460肺癌细胞接种于裸鼠皮下，接种第10天选取肿瘤直径生长至0．5～1．0cm的裸鼠12只，按数字法随机等分为2组，并分别给予重组人血管内皮抑制素注射液（治疗组）和生理盐水（对照组）处理。治疗7d后利用小动物光学活体成像系统、DCE—MRI、DWI评价两组裸鼠肿瘤体积、荧光信号强度值、血管功能参数[对比剂容积转移常量（Ktrans）、速度常量（Kep）、血管外细胞外间隙容积比（Ve）、对比剂浓度下峰面积（iAUC）]以及ADC值；并与肿瘤组织透射电镜、HE染色的病理结果，及免疫组织化学染色检查的微血管密度（MVD）及血管内皮生长因子（VEGF）表达结果进行对照。对结果采用Kolmogorov—Smirnov方法进行正态性检验。Kep为非正态分布，数据以肘（范围）表示，两组间比较采用秩和检验；VEGF为等级资料，两组间表达水平的比较采用X2检验；其他两组间符合正态分布的定量资料的比较均采用t检验；符合正态分布的定量资料问的相关性分析采用Pearson相关分析，定性资料用Spearman相关分析。结果肿瘤细胞接种第7天两组裸鼠皮下均出现荧光团，随着时间的延长荧光团范围逐渐增大，强度也逐渐增加。治疗7d后，治疗组肿瘤平均光信号强度值为（2．51±2．43）×10^10（光子数／s），对照组为（5．77±3．25）×10^10（光子数／s），差异无统计学意义（t=1．964，P〉0．05）；治疗组肿瘤体积为（365±56）mm2，小于对照组的（987±265）mm2，差异有统计学意义（t=0．001，P〈0．01），抑瘤率为63．0％。两组肿瘤荧光信号强度值与体积之间存在正相关（r=0．673，P〈0．05）。治疗组的血管功能定量参数Ktrans、Kep,Ve及iAUC分别为（0．055±0．012）min-1、0．335（0．184—0．894）min～、0．297±0．041和7．334±3．930，均低于对照组[对照组分别为（0．117±0．027）rain～、0．417（0．324—1．736）min-1、0．326±0．062和13．280±4．245]，但两组Ktrans和iAUC间差异有统计学意义（t值分别为5．155、2．518，P值均〈0．05），Kep,Ve间差异无统计学意义。iAUC与MVD呈正相关（r=0．715，P〈0．01），与VEGF无相关性（r=0．484，P〉0．05）。治疗组裸鼠肿瘤的ADC值为（791±38）×10^-6mm2／s，高于对照组的（737±43）×10^-6mm2／s，差异有统计学意义（t=-2．299，P〈0．05）。免疫组织化学检查结果示：治疗组MVD[（11．9±4．8）条／高倍视野]低于对照组[（19．2±4．3）条／高倍视野]（t=2．774，P〈0．05），VEGF表达较对照组弱，差异无统计学意义（x2=4．000，P〉0．05）。透射电镜检查结果显示：治疗组部分肿瘤细胞发生变性，并出现凋亡现象。结论利用多模态成像技术能够提供肿瘤抗血管生成治疗后解剖形态、血管功能及细胞分子水平的动态信息，有利于及时评估治疗效果。

Objective To evaluate the response of the lung tumor xenografts in nude mice to antiangiogenic treatment from perspectives of anatomic, vessel function, cellular and molecular level using the multimodality imaging techniques including optical imaging, dynamic contrast enhanced MRI （DCE- MRI） and diffusion weighted imaging （DWI）. Methods The green fluorescent protein （GFP） was transplanted labeled using GFP-expressing NCI-H460 cells. After the transfection of GFP, NCI-H460 cells were implanted subcutaneously into nude mice. Ten days after implantion, 12 nude mice whose tumor xenografts grew to 0. 5--1.0 cm in the maximum diameter were randomly divided into 2 groups, and injected with phosphate-buffered saline and recombinant human endostatin respectively. Then the nude mice in the two groups underwent optical imaging, DCE-MRI and DWI. The volumes, photon counts, the quantitative MR vessel functional parameters including volume transfer constant （ Ktra ） , rate constant （ Kop ） , volume of extravascular extracellular space （ Vo ） and maximum area under the enhancement curve （ iAUC ）, and apparent diffusion coefficient （ADC）values of the tumors were recorded. Then tumors were collected and observed using the transmission electron microscopy and pathology examination, including HE staining, microvessel density（MVD） and the expressions of vascular endothelial cell growth factor （VEGF）. The Kop and VEGF expressions in experimental group and control group were compared with X2 text, and other values were compared with t test. The Pearson and Spearman test were used for analyzing the correlation of values in the two groups. Results Seven days after inoculation, the fluorescence signals were detected and grew with the growth of the tumors.