裸鼠肺癌移植瘤抗血管生成治疗后肿瘤灌注变化的定量分析

Quantitatively evaluating the evolution of the tumor perfusion in A549 lung adenocareinoma transplantation model induced by antiangiogenic treatment

目的对裸鼠肺癌移植瘤抗血管生成治疗后肿瘤灌注的变化进行定量分析。方法60只纯BALB／CA-nu品系裸小鼠腋下接种A549肺癌细胞建立裸鼠移植瘤模型，按随机数字表法分成贝伐单抗组和对照组各30只，前者以10rag／kg体质量尾静脉注射贝伐单抗，后者以等量生理盐水代替。两组于处理前、处理后第2、4、6、10天各随机选择6只小鼠行容积灌注CT（VPCT），获得血流量后处死，获取肿瘤组织病理切片，量子点荧光免疫双标法测定微血管密度（MVD）、CD31表达量、α-平滑肌肌动蛋白（α-SMA）表达量和血管成熟度指数（VMI）。比较两组肿瘤血流量、VMI和MVD的差异及变化。结果处理前，贝伐单抗组和对照组血流量、VMI、MVD分别为（13．5±1．5）ml·（100ml）-1·min-1、0．14±0．04、（45．7±16．5）条／高倍视野和（13．4±1．6）ml·（100ml）-1·min-1、0．14±0．05、（48．0±7．0）条／高倍视野（均P〉0．05）。处理后第2、4、6、10天，两组血流量分别为（17．9±7．3）、（32．2±6．9）、（18．5±2．4）、（13．8±1．8）和（10．5±0．6）、（9．6±0．8）、（5．7±1．2）、（1．9±1．0）ml·（100ml）-1·min-1，VMI分另0为1．17±0．22、3．25±0．23、2．94±0．31、1．07±0．18和0．12±0．03、0．13±0．03、0．15±0．03、0．13±0．03，MVD分别为（38．0±6．3）、（24．3±5．4）、（15．2±3．4）、（13．5±4．7）和（44．8±5．9）、（48．0±12．8）、（41．8±5．7）、（45．7±20．3）条／高倍视野。处理后贝伐单抗组血流量与VMI均显著高于对照组（均P〈0．01）；与处理前相比，血流量与VMI均于处理后第2天上升，第4天显著增高（P〈0．01）；第6天较第4天下降，但仍显著高于处理前水平（P〈0．01）；第10天血流量下降到处理前水平，VMI仍高于处理前水平（P〈0．01）；第4、6、10天MVD均显著低于对照组及处理前（均P〈0．05）。对照组血流量逐渐下降（均P〈0．01），MVD与VMI无明显变化。结论裸鼠肺癌移植瘤模型抗血管生成治疗后VMI及血流灌注短暂性增高，VPCT有助于通过量化抗血管生成后肿瘤血流灌注的变化来评价VMI的变化。

Objective To quantitatively evaluate the evolution of the tumor perfusion in A549 lung adenocarcinoma transplantation model induced by antiangiogenic treatment. Methods To establish the preclinical transplantation model of lung adenocarcinoma, 60 BALB/c nu/nu mice was inoculated with A549 cell lines via axilla. Sixty mice were randomly divided into 2 groups. The treatment group was treated with intravenous Bevacizumab （10 mg/kg weight, in a single injection）, and the control group received saline only in the same dose. Five times of volume perfusion CT （VPCT） scan was performed before treatment, and on the second, forth, sixth and tenth days of treatment, respectively. The values of blood flow （BF） in the A549 tumors were measured after scanning. The microvessel density （ MVD ） , vessel maturity index （VMI） in the tumors were determined using multiplexed QDs-based immunohistochemical staining. ResultsComparing the values of BF, VMI and MVD between the two groups on the same day before treatment, the values of BF, VMI and MVD of the treatment group were （ 13.5 ± 1.5） ml ·（ 100 ml） -1 . min-1, 0. 14± 0.04, （45.7±16.5）/HPF, respectively, and those in the control group were （13.4 ± 1.6） ml · （ 100 ml） -1 . min-1, 0. 14±0. 05, （48.0 ±7. 0） /HPF , respectively. There was no significant difference between the two groups （ all P 〉 0. 05）. And on the second, forth, sixth, tenth days of treatment, the values of BF of the treatment group were （ 17. 9± 7.3 ）, （ 32. 2 ± 6.9 ）, （ 18.5 ± 2. 4） and （ 13.8 ± 1.8 ） ml ·（100 ml）-1 . min-1, respectively, and those in the control group were （10. 5 ± 0. 6）, （9.6 ± 0. 8 ）, （5.7± 1.2） and （ 1.9 ±1.0） ml · （ 100 ml） -1 . min-1, respectively. The values of VMI of the treatment group were 1.17 ±0. 22, 3.25 ±0. 23, 2.94 ± 0. 31 and 1.07 ± 0. 18, respectively, and those in the control group were 0. 12 ±0. 03, 0. 13 ±0. 03, 0. 15 ±0. 03, and 0. 13 ±0. 03, respectively. The values of MVD of the treatment group were （38.0 ±6.3）, （24.3 ±5.4）, （15.2±3.4） and （13.5 ±4.7）/HPF, respectively, and those in the control group were （44. 8 ± 5.9 ）, （ 48.0 ±12. 8 ）, （ 41.8± 5.7 ） and （45.7 ± 20. 3 ）/HPF, respectively. In treated mice, BF and VMI were significantly higher than those in the control group （ all P 〈 0. 01 ）. BF and VMI increased from day2, and reached the peak at day4 （ P 〈 0. 01 ） , then decreased at day6, however the value of BF at day6 was still higher than that in the baseline （P 〈 0. 01 ） and decreased to the baseline level at day10; while the value of VMI was still higher than that in the baseline at day10 And on the forth, sixth, tenth days of treatment, in treated mice, the values of MVD were significantly lower than those in the control group and the baseline level before treatment （ all P 〈 0. 01 ）. In control mice, BF decreased （ all P 〈 0. 01 ） with the time, while MVD and VMI had no changes. Conclusions The tumor perfusion and vessel maturity are transiently increased in A549 lung adenocarcinoma transplantation model induced by antiangiogenie treatment. VPCT is helpful to quantify the evolution of the tumor perfusion and then evaluate the functional changes of tumor vessel maturity.