重组人血管内皮抑制素注射液与多西紫杉醇不同顺序用药的抗肿瘤效应观察

Observation of the antitumor effect of endostar combined with docetaxel under different administration sequences

目的 观察重组人血管内皮抑制素注射液（商品名：恩度）与多西紫杉醇联合使用时不同给药顺序的抗肿瘤效应.方法 建立肺腺癌A549荷瘤裸鼠模型,随机分为3组,每组8只.（1）同时用药组：每只小鼠恩度400μg/d,第1～35天;多西紫杉醇10 mg/kg,第1～19天,每3 d给药1次.（2）先恩度组：每只小鼠恩度400 μg/d,第1～35天;多西紫杉醇10 mg/kg,第16～34天,每3 d给药1次.（3）模型组：每只小鼠生理盐水100 μl/d,第1～35天;注射用水200 μl/d,第1～35天,每3 d注射1次.同时设立空白对照组（未荷瘤的正常裸鼠,8只）,注射方法同模型组.实验过程中测量各组裸鼠体重、移植瘤体积,并计算抑瘤率.实验结束后,采用流式细胞术检测裸鼠外周血循环内皮细胞（CECs）数,采用免疫组化法检测移植瘤组织中基质金属蛋白酶2（MMP-2）、MMP-9、MMP-2的抑制剂（TIMP-2）、MMP-9的抑制剂（TIMP-1）、细胞外MMP诱导因子（EMMPRIN）、α-平滑肌肌动蛋白（SMA）的表达情况,并计数微血管密度（MVD）.结果 同时用药组肿瘤体积增长为39.94 mm3,先恩度组肿瘤体积增长为（99.57±74.48）mm3,二者均明显小于模型组[（217.67±95.44）mm3,均P＜0.05].同时用药组、先恩度组、模型组和空白对照组裸小鼠外周血中CECs的数量分别为（25.86±11.77）个/104个细胞、（77.25±24.02）个/104个细胞、（14.71±11.07）个/104个细胞和（12.90±11.20）个/104个细胞,同时用药组、模型组和空白对照组均明显低于先恩度组（均P＜0.01）.同时用药组和先恩度组移植瘤组织中MMP-2和MMP-9的表达均较模型组下调（均P＜0.05）,先恩度组移植瘤组织中TIMP-1和同时用药组TIMP-2的表达均较模型组上调（均P＜0.05）,同时用药组EMMPRIN的表达较模型组下调（P＜0.05）.同时用药组和先恩度组移植瘤组织的MVD及α-SMA的水平均低于模型组（均P＜0.05）.结论 同时用药组的抑瘤效果及小鼠生存质量更好.同时用药组和先恩度组均可通过下调MMPs的表达以限制肿瘤进展.CECs的变化可能是先上升后下降的动态过程,早期升高可能提示新生血管床面积减小,继而下降则反映联合治疗后的CECs凋亡和肿瘤消退,动态观察CECs的变化可能有助于了解肿瘤负荷及其血管床的变化并预测疗效.

Objective To observe and analyze the antitumor effect of endostar combined with docetaxel under different administration sequences. Methods Nude mice with xenograft tumor（ A549 cell line） were randomized into 3 groups, 8 mice/group： （ 1 ） Concurrent administration group （ each mouse：endostar400 μg/d, d1-d35, docetaxel 10 mg/kg, every 3 days, d1-d19）; （2）Endo-first group （each mouse： endostar 400 μg/d, d1-d35, docetaxel 10 mg/kg, every 3 days, d16-d34）; （3）Model group （positive control, tumor-bearing mice without treatment, each mouse： physiological saline, 100 μl/d, d1-d35, water for injection, 200 μl/d, d1-d35, every 3 days）, and blank control group （ negative control,normal mice without treatment, 8 mice）, the administration method was the same to the model group. The volume of tumor and the weight of mouse were measured during treatment. Circulating endothelial cells （CECs） were detected by flowcytometry, and the expression of matrix metalloproteinase （MMP-2, MMP-9）, the tissue inhibitor of MMP （TIMP-1, TIMP-2）, the extracellular MMP inducer （EMMPRIN）, CD34,α-smooth muscle actin （α-SMA） were determined by immunohistochemistry. Results The tumor growth of concurrent administration group（ 39.94 mm3） was lower than that of the endo-first group[（99.57 ± 74.48 ）mm3]during treatment, both of them were smaller than that of the model group[（217.67±95.44） mm3,P 〈0.05]. The amount of CECs in the endo-first group[（ 77.25 ± 24.02 ） cells/104cells]was more than that of the concurrent administration group[（25.86 ± 11.77） cells/104 cells], the model group[（ 14.71 ±11.07 ） cells/104 cells], and the blank control group[（ 12.90 ± 11.20 ） cells/104 cells, P 〈 0.01]. The expression of MMPs in the treatment groups was obviously downregulated. The expressions of TIMP-1 in the endo-first group and TIMP-2 in the concurrent administration group were upregulated （ P 〈 0. 05 ）. The expression of EMMPRIN was significantly down-regulated in the concurrent administration group （P 〈0.05）. The MVD and α-SMA expressions of the treatment groups were less than that of the model group （P 〈0.05）. Conclusion In comparison with the endo-first group, the anti-tumor effect and survival quality of the concurrent administration group are better. Both of the administration groups may have ＂vascular normalization effect＂ by down-regulating MMPs expression through different points, and inhibit the cancer-induced stromal reaction, restraining the cancer progress to a certain extent. The changes of CECs should be a dynamic process with an initial rise in the early-stage suggesting the decrease of vascular bed and subsequent decline ascribed to apoptosis of CECs and the tumor-regression after combined therapy.Investigation of its dynamic changes may be helpful to know the change of tumor burden and vascular bed and predict the antitumor effect.