贝伐单抗对人眼脉络膜黑色素瘤MUM-2B细胞血管生成拟态的影响

目的:通过体内外实验探讨贝伐单抗对人眼脉络膜黑色素瘤细胞（MUM-2B）血管生成拟态的影响及可能相关的分子通路。方法:通过不同浓度的贝伐单抗（0，0.1，1，2，3 mg／mL）处理MUM-2B细胞，观察其对MUM-2B细胞成管能力的影响。通过CCK-8法及Transwell 法检测其对MUM-2B细胞增殖及侵袭的影响。然后，建立裸鼠皮下移植瘤模型，通过不同剂量的贝伐单抗（5 mg／kg，7.5 mg／kg，10 mg／kg）干预，探究贝伐单抗对裸鼠皮下移植瘤VM形成的影响，并通过CD31/PAS免疫组化双重染色法检测VM的表达情况，免疫组化法检测HIF-1a、VE-cadherin、EphA2、PI3K蛋白表达水平变化。结果:体外实验中，不同浓度的贝伐单抗（0，0.1，1，2，3 mg／mL）处理MUM-2B细胞24小时后，对MUM-2B细胞的成管能力、增殖、侵袭能力并没有表现出明显的抑制或促进作用。实验组与对照组相比差异无统计学意义（P＞0.05）。在体内实验中，贝伐单抗实验组（5 mg／kg，7.5 mg／kg，10 mg／kg）与对照组相比较能显著的促进MUM-2B细胞形成VM的能力（P＜0.05），且形成管道的数量与贝伐单抗的浓度呈正相关（r＝0.942，P＜0.05）。实验组HIF-1a、VE-cadherin、EphA2和PI3K-Akt蛋白的表达水平均明显高于对照组（P＜0.05），且各分子的表达量随着贝伐单抗浓度的升高呈浓度依赖性的升高。结论:体外实验中，贝伐单抗对MUM-2B细胞的成管能力，增殖，侵袭能力并没有表现出明显的抑制或促进作用。体内实验中，贝伐单抗的使用能促进HIF-1a的表达、上调VE-cadherin／EphA2／PI3K-Akt后续的级连信号通路，从而加速VM的生成。

The effect of bevacizumab on the angiogenesis mimicry of human choroidal melanoma MUM-2B cells

Objective:To investigate the effect of bevacizumab on the angiogenic mimicry of human choroidal melanoma cell (MUM-2B) and the possible related molecular pathways in vitro and in vivo.Methods:MUM-2B cells were treated with different concentrations of bevacizumab (0,0.1,1,2,3 mg/mL) to observe its effect on the tube-forming ability of MUM-2B cells.The effects of the proliferation and invasion of MUM-2B cells were detected by CCK-8 and Transwell assay.Then,the subcutaneous transplantation tumor models were established in nude mice,and the effect of bevacizumab on VM formation of subcutaneous transplantation tumors in it was investigated by different doses of bevacizumab (5 mg/kg,7.5 mg/kg,10 mg/kg) intervention,and the expression of VM was detected by double staining with CD31/PAS immunohistochemistry.The changes in the expression levels of HIF-1a,VE-cadherin,EphA2,and PI3K proteins were detected by immunohistochemistry.Results:In vitro,different concentrations of bevacizumab (0,0.1,1,2,3 mg/mL) treated with MUM-2B cells for 24 hours did not show significant inhibitory or promotional effects on the tube-forming ability,proliferation,and invasion ability of MUM-2B cells.The difference between the experimental group and the control group was not statistically significant (P＞0.05).In vivo,compared with the control group,the bevacizumab experimental group (5 mg/kg,7.5 mg/kg,10 mg/kg) could significantly promote the ability of MUM-2B cells to form VM (P＜0.05),and the number of tubes was positively correlated with the concentration of bevacizumab (r=0.942,P＜0.05).The expression levels of HIF-1a,VE-cadherin,EphA2,and PI3K-Akt proteins were significantly higher in the experimental group than in the control group (P＜0.05),and the expression of each molecule increased in a concentration-dependently manner with the increase of bevacizumab concentration.Conclusion:In vitro,bevacizumab did not significantly inhibit or promote the tubulation,proliferation and invasion of MUM-2B cells.In vivo,Bevacizumab can promote the expression of HIF-1a and up-regulate the subsequent cascade signal pathway of VE-cadherin/EphA2/PI3K-Akt to accelerate the generation of VM.