Apelin-13对缺血再灌注损伤大鼠视网膜血管新生的作用

Effect of Apelin-13 on angiogenesis in retina of rats with ischemia-reperfusion injury

目的研究Apelin-13是否具有促进大鼠视网膜血管新生的作用。方法采用腹腔注射戊巴比妥钠全身麻醉于左眼和右眼均建立大鼠视网膜缺血再灌注损伤模型。利用玻璃体注射技术,大鼠右眼视网膜玻璃体注射250pmol/μl的Apelin-13多肽作为实验组,左眼注射磷酸盐缓冲溶液(PBS)作为对照组。采用免疫荧光染色法,利用荧光共聚焦显微镜的分层扫描功能分别对视网膜表层和深层血管进行拍照,分析视网膜血管新生情况;大鼠玻璃体注射Apelin-13多肽2周后处死大鼠,收取视网膜组织。采用逆转录聚合酶链反应(RT-PCR)法检测Apelin-13(Apln)及其受体(Aplnr)的mRNA水平;免疫印迹检测胶质纤维酸性蛋白(GFAP)的表达。对视网膜石蜡标本纵切片进行过碘酸雪夫苏木精(PASH)染色,在400倍的放大倍数下,同一个视网膜上分别在外周和中央各取两个视野拍照,计算视网膜节细胞层(GCL)中的细胞核总数和视野中视网膜的长度。结果玻璃体注射250 pmol/μl Apelin-13可引起视网膜深层血管新生,表现为视网膜深层血管分支数的增加,并可引起视网膜星形胶质细胞的激活,GFAP蛋白水平的上升以及神经节细胞的缺失。结论 Apelin-13多肽能引起视网膜深层血管一定程度的血管增生,这种促进血管生长的作用在视网膜表层血管未见明显表现。

Objective To investigate whether Apelin-13 can promote angiogenesis of retina in rats. Methods After general anesthesia by intraperitoneal injection of pentobarbital sodium,retinal ischemia-reperfusion injury model was established in the rat with right eye and left eye. With vitreous injection technology,injection of 250 pmol/μl of Apelin-13 peptides was administered in the right eye（ experiment group）,and injection of phosphate buffer saline（ PBS） was administered in the left eye（ control group）. Using mmunofluorescence staining method and the hierarchical scan function of confocal fluorescent microscopy to take a picture forblood vessels of retinal surface layer and deep layer,the retinal angiogenesis was detected and analyzed. The rats were killed two weeks after vitreous injection,and the retinal tissue was taken. Reversetranscription-polymerase chain reaction（ RT-PCR） was used to detect Apelin-13（ Apln） and Apelin-13 receptor（ Aplnr） mRNAs levels. Immunoblotting was used to detect glialfibrillaryacidicprotein（ GFAP） expression. Periodic acid-schiff hematoxylin stain（ PASH） was carried on in the retinal paraffin specimen longitudinal section. Under the magnification of 400 times,two vision fields at the outside and central areas on the same retina were taken,respectively to take pictures,and total number of cell nuclei in retinal ganglion cell layer（ GCL） and the length of the retina in vision field were calculated. Results Intravitrealinjection of 250 pmol/μl of Apelin-13 can cause deep retinal angiogenesiswhich showed an increase of the number of branches of the deep retinal vessels and can cause the activation of retinal astrocytes,the rise of GFAP protein level and the loss of ganglion cells. Conclusion Apelin-13 peptides can cause deep retinal vascular hyperplasia to a certain degree,while this role in promoting vascular growth has no obvious performance in the retinal superficial blood vessels.