短发夹RNA敲低补体因子B可抑制大鼠激光诱导的脉络膜新生血管形成。
Complement factor B knockdown by short hairpin RNA inhibits laser-induced choroidal neovascularization in rats.
作者信息
Wang Xin, Shang Qing-Li, Ma Jing-Xue, Liu Shu-Xia, Wang Cai-Xia, Ma Cheng
机构信息
Department of Ophthalmology, the Second Hospital of Hebei Medical University, Shijiazhuang 050000, Hebei Province, China.
Department of Pathology, Hebei Medical University, Shijiazhuang 050017, Hebei Province, China.
出版信息
Int J Ophthalmol. 2020 Mar 18;13(3):382-389. doi: 10.18240/ijo.2020.03.03. eCollection 2020.
AIM
To evaluate whether recombinant complement factor B (CFB) short hairpin RNA (shRNA) reduces laser-induced choroidal neovascularization (CNV) in rats.
METHODS
Laser-induced rat CNV model was established, and then the animals underwent fundus fluorescence angiography (FFA) and hematoxylin and eosin (HE) staining. On day 3 and 7 after photocoagulation, the expression of CFB and membrane attack complex (MAC) was detected by immunhischemistry. A recombinant CFB-shRNA plasmid was constructed. CFB and scrambled shRNA plasmids were intravenous injected into rats the tail vein on the day of laser treatment, respectively. On day 7, the incidence of CNV was determined by FFA, and the expression of CFB and vascular endothelial growth factor (VEGF) in retinal pigment epithelium (RPE)/choroidal tissues was detected by immunhischemistry, Western blot and/or semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) in CFB and scrambled shRNA groups. The possible adverse effects of CFB-shRNA injection were assessed by transmission electron microscopy and electroretinography.
RESULTS
FFA and HE results indicated that a laser-induced rat CNV model was successfully established on day 7 after photocoagulation. The expression of CFB and MAC was extremely weak in normal retina and choroid, and increased on day 3 after photocoagulation. However, it started to reduce on day 7. CFB shRNA plasmid was successfully constructed and induced CFB knockdown in the retinal and choroidal tissues. FFA showed CFB knockdown significantly inhibited incidence of CNV in rats. Moreover, CFB knockdown significantly inhibited the expression of VEGF in RPE/choroidal tissues. CFB shRNA caused no obvious side effects in eyes.
CONCLUSION
CFB knockdown significantly inhibits the formation and development of CNV through reducing the expression of VEGF, which is a potential therapy target. The alternative pathway of complement activation plays an important role in CNV formation.
目的
评估重组补体因子B（CFB）短发夹RNA（shRNA）是否能减少大鼠激光诱导的脉络膜新生血管形成（CNV）。
方法
建立激光诱导的大鼠CNV模型，然后对动物进行眼底荧光血管造影（FFA）和苏木精-伊红（HE）染色。在光凝后第3天和第7天，通过免疫组织化学检测CFB和膜攻击复合物（MAC）的表达。构建重组CFB-shRNA质粒。在激光治疗当天，分别将CFB和乱序shRNA质粒经大鼠尾静脉注射。在第7天，通过FFA确定CNV的发生率，并通过免疫组织化学、蛋白质免疫印迹法和/或半定量逆转录-聚合酶链反应（RT-PCR）检测CFB和乱序shRNA组视网膜色素上皮（RPE）/脉络膜组织中CFB和血管内皮生长因子（VEGF）的表达。通过透射电子显微镜和视网膜电图评估注射CFB-shRNA可能产生的不良反应。
结果
FFA和HE结果表明，光凝后第7天成功建立了激光诱导的大鼠CNV模型。正常视网膜和脉络膜中CFB和MAC的表达极弱，光凝后第3天增加。然而，在第7天开始下降。成功构建了CFB shRNA质粒，并在视网膜和脉络膜组织中诱导CFB表达下调。FFA显示，CFB表达下调显著抑制了大鼠CNV的发生率。此外，CFB表达下调显著抑制了RPE/脉络膜组织中VEGF的表达。CFB shRNA对眼睛未造成明显副作用。
结论
CFB表达下调通过降低VEGF的表达显著抑制CNV的形成和发展，VEGF是一个潜在的治疗靶点。补体激活的替代途径在CNV形成中起重要作用。
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