雷珠单抗竞争结合血管内皮生长因子A抑制裸鼠皮下血管瘤细胞生长的实验研究

Inhibition of Vascular Endothelial Growth Factor A by Ranibizumab in Subcutaneous Hemangiomas in Nude Mice

目的探索观察雷珠单抗(Ranibizumab)竞争结合血管内皮生长因子A(VEGF-A)抑制裸鼠皮下血管瘤的相对疗效及副作用。方法 BALB/c裸鼠30只,采用密度为1×10~7个/mL血管瘤内皮细胞(EOMA)200μL单侧腋下皮下注射法建立血管瘤裸鼠模型,在接种EOMA细胞9天后给予相应药物治疗。具体分组为:对照组只接种EOMA细胞,无药物治疗;雷珠单抗低、中、高剂量组接种EOMA细胞并给予0.25、0.5和0.75mg/cm^3雷珠单抗治疗;平阳霉素组接种EOMA细胞并给予1mg/cm^3平阳霉素治疗,每组6只。观察各组裸鼠成瘤情况并测量肿瘤体积,免疫组织化学(IHC)法检测各组裸鼠肿瘤组织CD34、Ki67表达,HE染色后观察各组裸鼠肺、心、肝、肾组织病理变化。结果第9~12天各组裸鼠血管瘤体积增长最快,第12~24天雷珠单抗低、中、高剂量组、平阳霉素组裸鼠血管瘤体积增长减缓,与对照组比较,差异有统计学意义[第12天:(44.44±13.11)mm^3、(45.71±7.69)mm^3、(35.37±11.96)mm^3、(41.44±3.75)mm^3比(61.91±17.09)mm^3,P均<0.05;第15天:(52.16±13.28)mm^3、(48.18±14.46)mm^3、(33.52±9.97)mm^3、(47.22±10.86)mm^3比(72.98±14.85)mm^3,P均<0.05;第18天:(56.50±6.96)mm^3、(51.56±11.10)mm^3、(36.57±18.79)mm^3、(51.36±5.21)mm^3比(95.58±19.10)mm^3,P均<0.01;第21天:(62.88±9.76)mm^3、(60.34±12.80)mm^3、(39.45±18.47)mm^3、(54.95±4.37)mm^3比(123.46±18.03)mm^3,P均<0.01;第24天:(70.85±8.73)mm^3、(69.84±16.33)mm^3、(41.83±18.65)mm^3、(61.52±8.12)mm^3比(171.76±52.46)mm^3,P均<0.01);与平阳霉素组比较,雷珠单抗高剂量组裸鼠血管瘤体积显著缩小[第12天:(35.37±11.96)mm^3比(41.44±3.75)mm^3,P<0.05;第15天:(33.52±9.97)mm^3比(47.22±10.86)mm^3,P<0.05;第18天:(36.57±18.79)mm^3比(51.36±5.21)mm^3,P<0.05;第21天:(39.45±18.47)mm^3比(54.95±4.37)mm^3,P<0.05;第24天:(41.83±18.65)mm^3比(61.52±8.12)mm^3,P<0.05);雷珠单抗低、中、高剂量组和平阳霉素组裸鼠血管瘤组织CD34和Ki67蛋白表达明显降低(P<0.01),且雷珠单抗浓度越高,降低越明显。各组裸鼠肺组织受到不同程度的损伤,但雷珠单抗高剂量组裸鼠肺组织损伤程度较平阳霉素轻;各组裸鼠心、肝、肾组织无明显变化。结论雷珠单抗抑制裸鼠皮下血管瘤细胞生长作用显著,其作用可能与下调血管瘤组织CD34、Ki67表达有关,对裸鼠肺组织损伤要较平阳霉素轻。

Objective To investigate the relative efficacy and side effects of ranibizumab competitive combined with vascular endothelial growth factor A（VEGF-A） in inhibiting subcutaneous hemangioma in nude mice. Methods Thirty animal model was established by injecting hemagioma cells（EOMA, 1×10^7/mL, 200μL） to subcutaneous tissue of nude mice. The nude mice were divided into 5 groups, with 6 mice in each. Drugs were given after 9 days incubation of EOMA. Group A： control group, without drug therapy. Group B： group of injection with 0.25mg/cm^3 ranibizumab. Group C： group of injection with 0.5mg/cm^3 ranibizumab. Group D： injection of 0.75mg/cm^3 ranibizumab. Group E： drug control group, injection of 1mg/cm^3 pingyangmycin. Tumor growth was observed and the body weight of mice was measured after treatment. Immunohistochemistry was used to detect the expression of CD34 and Ki67 in tumor tissue and HE staining was applied to observe the pathology of Lung, heart, liver and renal tissues of mice. Results Tumor grew the fastest during 9 to 12 days. On day 12, 15, 18, 21 and 24, the tumor volume was reduced in Group B, C, D, E compared with that in Group A（day 12： 44.44 ±13.11mm^3, 45.71 ±7.69mm^3,35.37 ±11.96mm^3, 41.44 ±3.75mm^3 vs 61.91 ±17.09mm^3, all P 0.05; day 15： 52.16 ±13.28mm^3, 48.18 ±14.46mm^3,33.52±9.97mm^3; 47.22±10.86mm^3 vs 72.98±14.85mm^3, all P〈0.05; day 18： 56.50±6.96mm^3, 51.56±11.10mm^3, 36.57±18.79mm^3, 51.36 ±5.21mm^3 vs 95.58 ±19.10mm^3, all P 0.01; day 21： 62.88 ±9.76mm^3, 60.34 ±12.80mm^3, 39.45 ±18.47mm^3, 54.95 ±4.37mm^3 vs 123.46 ±18.03mm^3, all P 0.01; day 24： 70.85 ±8.73mm^3, 69.84 ±16.33mm^3, 41.83 ±18.65mm^3, 61.52 ±8.12mm^3 vs 171.76 ±52.46mm^3, all P 0.01）; compared with Group E, the anti-tumor effect in Group D was more obvious at each time point（all P〈0.05）. The expression of CD34 and Ki67 protein in tumor tissue of nude mice in Group B-E was significantly decreased as compared to Group A; the decrease appeared to be in a dose-dependent manner in Group B-D. After treatment, the lung tissue of nude mice was damaged in different degrees in every group; however, the damage in Group D was the less; no pathological changes were observed in other tissues in every group. Conclusion Ranibizumab can obviously inhibit the growth of hemangioma,which may be related to its downregulation of CD34 and Ki67 in tumor tissue and ranibizumab has less damage to the lung tissue than Pingyangmycin does.