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Gene therapy for anastomotic stenosis after arterial reconstruction

动脉重建后吻合口狭窄的基因治疗

基本信息

批准号：
10671101
负责人：
OSHIRO Hidemi
金额：
$ 2.05万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10671101/
关键词：
Adenovirus Gene therapy Rat carotid balloon injury neointimal hyperplasia Cyclin D Rb protein p27 p21

项目摘要

Since neointimal hyperplasia is known to be critical in formation of anastomotic stenosis after arterial bypass operation, the purposes of the present study were to establish the method of gene transfer into neointima and to determine the important factor(s) regulating cell cycle in neointimal formation. For the first purpose, we applied adenovirus vectors containing LacZ gene to neointima of rat carotid artery, which was induced by balloon injury, and found that more than 1x10ィイD19ィエD1 PFU/mL of virus vector promoted high efficiency transfer of LacZ gene to neointimal cells. Miyata et al administrated the gene of mutant platelet-derived growth factor receptor to the rat carotid neointimal cells by the above method, and showed a significant suppression of the neointimal proliferation. To study the role of cell cycle in neointimal formation, expression of retinoblastoma protein (Rb) in balloon-injured rat carotid artery was assessed by western blot analysis, since phosphorylation of Rb releases the E2F transcription factor from its binding to Rb, which is a critical event in cell cycle. At 1 day after balloon injury, band shift of Rb, which indicates phosphorylation of Rb, was observed. The phosphorylation of Rb, became to be maximal at 2 days after injury, when maximal cell replication was detected in arterial media. The expression of cyclin D in arterial wall was also observed after balloon injury, though the expression increased gradually by 14 days after injury, indicating a discrepancy between Rb phosphorylation and cyclin D expression. Meanwhile, p27, cell cycle inhibitor, was expressed in uninjured arterial wall. The expression of p27 was down-regulated once immediately after injury but increased again from 7 days after injury These results suggested that p27 suppressed cyclin D after- day 7 and regulated phosphorylation of Rb of injured arterial cells.

鉴于动脉搭桥术后吻合口狭窄的形成与新生内膜的增殖密切相关，本研究旨在建立血管内膜基因转移的方法，并探讨细胞周期调控因子S在新生内膜形成中的作用。首先，我们将含有LacZ基因的重组腺病毒载体应用于球囊损伤诱导的大鼠颈动脉新生内膜，发现1×10ィイD19ィエD1pfu/mL腺病毒载体可促进LacZ基因高效转移至新生内膜细胞。Miyata等人通过上述方法将突变的血小板衍生生长因子受体基因导入大鼠颈动脉新生内膜细胞，并显示出明显的抑制新生内膜增殖的作用。为了研究细胞周期在新生内膜形成中的作用，用免疫印迹法检测了大鼠颈动脉球囊损伤后视网膜母细胞瘤蛋白(Rb)的表达，因为Rb的磷酸化使E2F转录因子从与Rb的结合中释放出来，这是细胞周期中的一个关键事件。球囊损伤后1d，Rb的条带发生移动，表明Rb的磷酸化。Rb的磷酸化在伤后2天达到最大，此时动脉中膜细胞复制最强。球囊损伤后动脉壁也有细胞周期蛋白D的表达，但14d后表达逐渐增加，提示Rb磷酸化与细胞周期蛋白D的表达存在差异。同时，细胞周期抑制因子p27在未损伤的动脉壁有表达。伤后即刻，p27的表达一度下调，7d后又开始升高，提示p27在伤后7d抑制细胞周期蛋白D的表达，并调节Rb的磷酸化。