糖尿病小鼠视网膜脉管系统分离铺片技术的改进与注意事项

Improvement and consideration of separation and spreading technique of retinal vessel system in diabetic mice

目的建立稳定的、能获得清晰小鼠视网膜脉管系统、血管内皮细胞及周细胞的实验条件。方法将C57BL/6J小鼠随机分为正常对照组和糖尿病模型组。正常组小鼠按55 mg/kg的剂量腹腔注射柠檬酸-柠檬酸钠缓冲液,模型组小鼠腹腔注射同等剂量的2%链脲佐菌素(STZ)溶液,连续注射5 d。待糖尿病模型组成模后3个月,2组同时取材,采用不同方法进行视网膜脉管系统消化分离及铺片。结果最佳方法为整只眼球在4%多聚甲醛中固定12 h,使用8%胰蛋白酶37℃单独消化2~2.5 h,每隔1 h配合1 ml移液枪在蒸馏水中反复吹打直至视网膜变得薄而清透。此时获得的视网膜脉管系统完整清晰,其他组织消化彻底,血管内皮细胞和周细胞清晰可见。且与正常组相比,模型组非细胞性毛细血管增多(t=2.668,P=0.024)。结论选择合适的固定和消化方法,重点关注消化过程中的时间、温度差异和使用移液器反复吹打使残余组织脱离是糖尿病小鼠视网膜脉管系统分离铺片技术成功与否的关键。

OBJECTIVE To establish stable test conditions for obtaining clear images of mouse retinal vasculature,vascular endothelial cells and pericytes.METHODS The C57BL/6J mice were divided into normal group and diabetic group randomly.Mice in the normal group were intraperitoneally injected with citrate-sodium citrate buffer in dose of 55 mg/kg.Correspondly,mice in the model group were injected with 2%streptozotocin(STZ)solution in the same dose for 5 consecutive days.On three months after the diabetes model was established,the eyeballs of all subjects were taken at the same time.Different methods were used to digest and separate the retinal vasculature.RESULTS The best method was to fix the whole eyeball in 4%paraformaldehyde for 12 hours,digest it with 8%trypsin at 37°C for 2 to 2.5 hours,and use of a pipette(1 ml)to repeatedly blow in the distilled water at one hour intervals until the retina became thin and clear.The retinal vasculature obtained by this method was complete and clear,other tissues were thoroughly digested,and vascular endothelial cells and pericytes were also clearly visible.Compared with the normal group,acellular capillaries increased in the model group(t=2.668,P=0.024).CONCLUSIONS Choosing the appropriate fixing and digestion methods,focusing on the time and temperature differences in the digestion process,and using pipette to repeatedly detach the residual tissue out of the retinal vasculature system in diabetic mice was the key of the technique.