小鼠主动脉环血管新生实验模型的优化

目的: 建立优化体外培养小鼠主动脉环血管新生实验模型的条件和方法。方法: 分离C57BL/6小鼠的胸主动脉，剔除主动脉周围的脂肪及结缔组织，使用Ⅱ型胶原酶消化动脉，剥离血管外膜，将血管剪成0.5 mm长的动脉环，置于预先包被有鼠尾Ⅰ型胶原的96孔板中培养，给予DMEM+2.5%胎牛血清，DMEM+10%胎牛血清或DMEM+2.5%胎牛血清+血管内皮生长因子(VEGF，终浓度为50 ng/mL)100 μL/孔，覆盖胶原表面。倒置显微镜下观察动脉环新生微血管。免疫荧光染色鉴定内皮细胞。结果： 鼠尾Ⅰ型胶原包被的96孔板（DMEM+2.5%胎牛血清）内新生微血管数量明显多于24孔板（t=－6.000，P<0.05）。DMEM+10%胎牛血清组新生微血管数量明显高于DMEM+2.5%胎牛血清组和DMEM+2.5%胎牛血清+VEGF组。DMEM+10%胎牛血清组培养2~4 d后小鼠主动脉环开始出芽， 6~8 d后动脉环新生微血管分支、数量及长度达到高峰。结论： 优化后的方法可以得到生长状态良好，出芽率高，纯度高的离体小鼠动脉环血管新生实验模型。

A modification of the mouse aortic ring assay for angiogenesis

Objective: To optimize the method of the mouse aortic ring angiogenesis assay. Methods: The thoracic aorta of C57BL/6 mice were separated and all extraneous fat and connective tissues were removed. To remove the vascular adventitia, the aorta was digested with collagenase Ⅱ. The aorta were cut into rings～0.5 mm in width and the rings were embeded in the wells of a 96 well plate coated with type Ⅰ collagen. The embedded rings were fed with 100 μL of DMEM culture medium supplemented with 10% FBS or 2.5% FBS and VEGF to attain a final concentration of 50 ng/mL. Inverted microscope was used to observe microvessel outgrowth of the aortic rings. The endothelial cells were assayed by BS1 lectin FITC immunofluorescence. Results: The number of microvessels of the aortic rings in the 96 well plate was more than those in 24 well plate（t=-6.000，P<0.05）. The number of microvessels of the aortic rings cultured with DMEM+10% fatal bovine serum was more than those cultured with DMEM+2.5% fatal bovine serum or DMEM+2.5% fatal bovine serum+VEGF. Microvessel outgrowth of the aortic rings started at 2 to 4 days and reached the peak at day 6 to 8 using the modified method. Conclusion: A great quantity, purity and high survival rate of the mouse aortic ring model of angiogenesis can be effectively obtained by coating plates with type Ⅰ collagen and enhancing the concentration of FBS.