短发夹RNA对人视网膜色素上皮细胞血管内皮生长因子表达的抑制

目的探讨针对人血管内皮生长因子（VEGF）的短发夹RNA（shRNA）在体外对视网膜色素上皮（RUE）细胞VEGF表达的影响。方法用酶辅助显微分离法分离人RPE细胞,并用细胞角蛋白和S-100进行免疫组化鉴定。设计针对人VEGF的短发夹RNA（shRNAs,P1,P2）,P3为阴性对照即不含特异性shRNA,P1、P2退火后双链DNA连接到质粒pSilencer 4．1-CMV的BamHⅠ和Hjnd Ⅲ双酶切位点。转化细菌后,提取的质粒用EcoRⅠ和SamⅠ进行酶切鉴定。实验分5组,第1组：RPE细胞中在含有100μmol／LCoCl2,10％胎牛血清（FBS）的DMEM培养基中培养30h;第2组：在常规含有10％FBS的DMEM培养基中培养30h;第3、4、5组：分别P1、P2、P3转染细胞24h后在含有100μmoL／LCoCl2的10％FBS的DMEM培养基中培养30h;用免疫印迹法检测各组细胞VEGF表达水平。结果培养的细胞用细胞角蛋白和S-100免疫组化染色阳性。提取的质粒经酶切后片断相对分子质量分别为3300和1600,说明质粒成功地提取和纯化。VEGF表达水平1组明显高于2、3、4组（均P〈0．001）。乏氧（2组与1组比）可以明显增加RPE细胞中VEGF的表达。3、4组（P1和P2）对VEGF表达抑制分别为65．9％和52．4％。5组与1组比差异无统计学意义（P=0．147）。各组间β肌动蛋白表达量差异无统计学意义。结论针对人VEGF的特异性shRNA可以明显降低人RPE细胞VEGF的表达,为RNA干扰治疗新生血管性眼病,尤其是脉络膜新生血管奠定了基础。

Short hairpin RNA targeting vascular endothelial growth factor effectively inhibits expression of vascular endothelial growth factor in human retinal pigment epithelium

OBJECTIVE: To design shRNA targeted to human vascular endothelial growth factor (VEGF) and to evaluate the effect of VEGF. shRNA on expression of VEGF in human retinal pigment epithelium (RPE) cells in vitro. METHODS: Human RPE cells were isolated with enzyme-assisted microdissection. The cells were identified by immunohistochemical method with antibody to cytokeratin and S-100. Plasma DNA was identified via restriction enzyme EcoRI and SamI. shRNAs (P1, P2) specific for human VEGF were designed. DNA expression vector is pSilencer 4.1-CMV of Ambion company. P3 is negative control nonspecific shRNA. There are 5 groups. Group 1: VEGF in cultured human RPE exposed to 100 micromol/L CoCl2 30 h; Group 2: VEGF in cultured human RPE in normal culture medium; Group 3, 4, 5: VEGF in cultured human RPE exposed to 100 micromol/L CoCl2 30 h after P1, P2, P3 transfection, respectively. VEGF level in conditioned media was measured by Western blot. RESULTS: The cells in culture could be stained with both cytokeratin and S-100 antibodies. The length of two fragment was 3.3 kb and 1.6 kb, respectively, which indicated that the extraction and purification were successful. The expression of VEGF in RPE was increased significantly (P < 0. 001) in group 1 as compared with group 2. Hypoxia-induced upregulation of human VEGF is halted by siRNA application in vitro (P < 0. 001 and P < 0. 001 in group 3 and 4 compared with group 1, respectively). shRNAs targeted hVEGF effectively and specifically inhibited hypoxia-induced VEGF levels in human RPE. The level of VEGF was reduced 65.9% and 52.4% in groups 3 and 4, respectively. There was no difference between group 5 and 1 (P = 0. 147). There was no difference of beta-actin production in RPE cells among groups. CONCLUSIONS: Delivery of shRNA can be used in vitro to target specific RNAs of VEGF and to reduce the level of the specific protein product (VEGF) in the targeted cells (human RPE). This work established the basis for the using of RNA interference in studies of retinal biology and for the treatment of a variety of retinal angiogenic diseases, especially the choroidal neovascularization.