重组腺伴随病毒载体介导Kringle5基因对SD大鼠视网膜神经细胞及血.docVIP

重组腺伴随病毒载体介导Kringle5基因对SD大鼠视网膜神经细胞及血 　 作者：宋哲　黎晓新　于文贞　董建强　闫征 【摘要】 　　目的：研究腺伴随病毒载体介导Kringle5基因对人脐静脉内皮细胞(human umbilical vein endothelial cells, HUC)及视网膜神经细胞影响作用。方法：亚克隆构建pSNAV-Kringle5-gfp载体，腺伴随病毒包装形成rAAV-Kringle5-gfp；HUC培养及SD大鼠视网膜神经细胞培养；按设计分为试验组、阴性对照组和空白对照组；聚合酶链反应(PCR), MTT法对各组进行细胞活力测定；荧光显微镜照相。 结果：腺伴随病毒载体介导Kringle5基因产生Kringle5物质对HUC有抑制作用，实验组与对照组比较P lt;0.01，实验组与空白对照组比较P gt;0.05；腺伴随病毒载体介导Kringle5基因产生Kringle5物质对SD大鼠视网膜神经细胞实验组和空白对照组没有影响，实验组与对照组及实验组与空白对照组比较P gt;0.05。结论：腺伴随病毒载体介导Kringle5基因能够对新生血管有抑制作用而对视网膜神经细胞无不良影响作用。 【关键词】 腺伴随病毒载体　血管抑制素Kringle5　血管内皮细胞　SD大鼠视网膜神经细胞 　　Adeno-associated virus type-2 expression of Kringle5 effect on HUC and Retinal cell of SD rat AbstractAIM: To investigate the effect of recombinant adeno-associated virus vector expressing human Kringle5 on the HUC(Human Umbilical Vein Endothelial Cells) and the retina nerve cell of SD rats. METHODS: The human kringle5 cDNA was plugged into expression vector of pSNAV to construct the plasmid pSNAV-Kringle5-gfp. HUC and the retina nerve cell of SD rats were cultured. All the samples were devided into three groups: experimental group, negative control group and blank control group. Polymerase chain reaction (PCR), MTT assay and fluorescence microscopy were applied.RESULTS: rAAV-Kringle5 produced kringle5, which inhibits the HUC (P lt;0.01 between experimental group and controls; Pgt;0.05 between experimental group and blank control) but has no effect on the retinal nerve cell of rats (P lt;0.01 between experimental group and controls; P lt;0.05 between experimental group and blank control).· CONCLUSION: The method of producing kringle5 by recombinant adeno-associated virus can be used to inhibit retina angiogenesis with no effect on retinal nerve cells. · KEYWORDS: adeno-associated virus vector; Kringle5; HUC; retinal nerve cells 　　 0引言 纤维溶酶原Kringle5片断能够抑制肿瘤的生长，对新生血管形成具有极强抑制作用，是目前发现抑制新生血管作用最强的抑制剂[1]。在眼科方面，国内外文献曾报道用Kringle5制剂治疗新生血管如脉络膜新生血管，视网膜新生血管，角膜新生血管等报道[2-4]。Kringle5是纤维溶酶原片段,由80个氨基酸组成[1,5]，它的获得主要是通过提取纤维溶酶原裂解