β-榄香烯抑制氧诱导小鼠视网膜新生血管形成的实验研究

Inhibitive effects of β-elemene on hyperoxia induced retinal neovascularization in mice

目的探讨β-榄香烯抑制氧诱导小鼠视网膜新生血管形成的作用。方法 64只鼠龄为7d的C57BL/6J小鼠随机分为4组:A组(空气对照组)、B组(高氧对照组)、C组(玻璃体内注射组)及D组(球后注射组)。A组在正常环境中饲养,其余3组建立氧诱导视网膜新生血管小鼠模型,造模后C组玻璃体内注射1μLβ-榄香烯、D组球后注射2μLβ-榄香烯。比较4组HE染色计数突破视网膜内界膜的血管内皮细胞核数,ADP酶法视网膜铺片测量视网膜无灌注区面积和视网膜新生血管数量。免疫组织化学染色检测VEGF蛋白的表达。结果 HE染色突破视网膜内界膜的血管内皮细胞核数4组分别为:A组(0.90±1.52)个、B组(27.15±9.83)个、C组(2.90±2.02)个、D组(13.33±4.83)个,4组比较差异有统计学意义(P<0.05);其中C组与D组相比差异有统计学意义(P<0.05)。视网膜铺片各组新生血管钟点数:A组未见新生血管、B组(8.00±1.26)个、C组(3.67±0.82)个、D组(6.12±1.17)个,4组比较差异有统计学意义(P<0.05);C组与D组相比差异有统计学意义(P<0.05)。4组视网膜铺片无灌注区面积百分比:A组未见无灌注区、B组(17.11±2.49)%、C组(9.98±2.44)%、D组(13.66±2.62)%,后3组比较差异有统计学意义(P<0.05);C组与D组相比差异有统计学意义(P<0.05)。免疫组织化学染色3组累积光密度值:A组32.17±20.98、B组144.04±65.72、C组55.56±36.69;3组间差异有统计学意义(P<0.05)。结论β-榄香烯注射乳可以抑制视网膜新生血管的发生发展,玻璃体内注射的给药途径治疗效果较好。

Objective To investigate the inhibitive effects of β-elemene on hy- peroxia induced retinal neovascularization in mice. Methods A total of 64 postnatal 7 days （p7） C57BL/6J mice born in the same day were divided into group A （normal control group）, group B （ model group）, group C （ intravitreal group） and group D （ ret- robulbar group）. Group A was fostered in normal condition, and the other three groups were fostered in hyperoxia condition for five days and then backed into-normal condi- tion. Group C treated with intravitreal injections of β-elemene （ I ixL） and group D trea- ted with retrobulbar injections of β-elemene （2 μL） at p12. At p17,all the mice were sacrificed and the eye balls were extracted. Adenosine diphosphate （ADPase） staining was adopted to assess the vascular pattern, HE staining methods was applied to count the nuclei of neovascular cells anterior to the inner layer membrane （ ILM）, and immu- nohistochemistry was used to detect the expression of VEGF protein. Results The nuclei of endothelial cells breaking through the ILM could be found in all groups：0.90 ± 1.52 in group A,27.15 ± 9.83 in group B,2.90 ± 2.02 in group C and 13.33 ±4.83 in group D, there was statistical difference among four groups （P 〈 0.05 ）, also between group C and group D （P 〈 0.05 ）. There was no new vessel in group A, but 8.00 ± 1.25 in group B,3.57 ＋0.82 in group C and 5.12± 1.17 in group D,there was statistical differ- ence among later 3 groups （P 〈 0.05 ）, also between group C and group D （ P 〈 0.05 ）. There was no avascular area in group A,but （ 17.11±2.49 ）96 in group B, （9.98 ± 2.44）% in group C and （ 13.55 ±2.52）% in group D,there was statistical difference compared among later 3 groups （ P 〈 0.05 ）, also between group C and group D （P 〈 0. 05 ）. The integrity olotical density of VEGF were 32. 17 ± 20.98 in group A. 144.04 ±65.72 in group B and 55.56± 36.69 in group C, there was statistical difference （P 〈 0.05 ）. Conclusion β-elemene can in- hibit the development of retinal neovascularization, and the intravitreal injection is more effective.