Basic study for gene therapy by

基因治疗的基础研究

• 批准号: 13671222
• 负责人: OHTAKE Hiroshi
• 金额: $ 2.24万
• 依托单位: Kanazawa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671222/
• 关键词: Anastmotic stenosis; Patelet derived growth factor; Artificial graft; 人工血管吻合部; 遺伝子治療; 血小板由来増殖因子B鎖

[Purpose]As a factor of anastomotic stricture after bypass surgery using an artificial graft, involvement of platelet-derived growth factor beta : PDGF beta chain, has been reported. PDGF beta chain is a powerful cell growth factor for smooth muscle cells, and such cells at the anastomotic site have been reported to show high-level expression of PDGF beta chain receptor. The purpose of this study was to clarify the effect to avoid stricture, through gene introduction of the extracellular region of receptor EX, which inhibits intimal proliferation, into the anastomotic site.[Subjects and method]We grafted expanded polytetrafluoroethylene artificial blood vessels, three millimeters in diameter, to rat aorta.Experiment 1.We administered a replication-deficient recombinant adenovirus containing the E.coli LacZ gene(AxLacZ), at levels of 1x10^8, 1x10^9, 1x10^<10>, and 1x10^<11>pfu/ml, into the anastomotic blood vessels, (1)from the adventitial side, and (2)from the lumen. Two weeks after th … More e surgery, the introduction rates of E.coli LacZ gene were measured by X-Gal staining.Experiment 2.Using the method that showed the best introduction rate in Experiment 1, we administered a replication-deficient recombinant adenovirus containing the gene encoding EX(AxPDGFXR) into the anastomotic sites of the artificial vessels. Two weeks after the surgery, we determined the protein concentration of AxPDGFXR and mRNA expression. One month after the surgery, we conducted HE staining, and immunohistological staining for von Winebrand factor and Ki-67 antigen, to assess arterialization and the stricture rates of the anastomotic sites. We performed DNA fragmentation using the TUNEL method, and sought the rate of apoptosis.[Result]Experiment 1.Administration from the adventitial side of the anastomotic vessel at 1x10^<11> pfu/ml showed the best introduction efficiency.Experiment 2.EX protein expression and mRNA were identified at anastomotic artery. In the AxPDGFXR group, arterialization and cell growth of vessels immunohistologically stained for von Winebrand factor and Ki-67 antigen, were restricted, and the stricture rate was also significantly lower, compared with the control group. No apoptosis was found.[Conclusion]An experiment revealed that local expression of the extracellular region of platelet-derived growth factor receptor restricts intimal thickening of the artificial vessel anastomotic site. If effectiveness and safety of this method are established, clinical application to micro blood vessel grafting will become possible, and remarkable improvement of the patency rate can be expected. Less

[目的]作为人工血管搭桥术后吻合口狭窄的一个因素，已有报道涉及血小板衍生生长因子β：PDGFβ链。PDGFβ链是一种强大的细胞生长因子，在吻合口的这类细胞中高水平表达PDGFβ链受体。本研究的目的是通过将抑制内膜增殖的受体EX的胞外区基因导入吻合口，以避免狭窄。[对象和方法]我们将直径3毫米的扩张型聚四氟乙烯人工血管移植到大鼠主动脉。实验1.将含有E.coliLacZ基因的复制缺陷重组腺病毒(AxLacZ)以1x10^8、1x10^9、1x10^&lt；10-gt；和1x10^-lt；11&gt；pfu/ml的水平分别从外膜和管腔注入吻合口血管。TH…两周后用X-Gal染色检测大肠杆菌LacZ基因的导入速率。实验2采用实验1中最佳导入速率的方法，将含有ex基因的复制缺陷型重组腺病毒(AxPDGFXR)导入人工血管的吻合口。术后2周测定AxPDGFXR的蛋白浓度和mRNA的表达。术后1个月行HE染色、von Winebrand因子和Ki-67抗原免疫组织化学染色，观察动脉化程度和吻合口狭窄率。[结果]实验1.从吻合口血管外膜注入浓度为1×10~(-1)~lt；11~gt；pfu/ml的DNA片段效果最佳。实验2.在吻合口动脉检测到EX蛋白和mRNA的表达。与对照组相比，AxPDGFXR组血管动脉化和细胞生长受到抑制，血管狭窄率明显降低。[结论]血小板衍生生长因子受体胞外区的局部表达抑制了人工血管吻合口内膜的增厚。如果该方法的有效性和安全性得到证实，将有可能在临床上应用于微血管移植，并有望显著提高通畅率。较少

项目成果

Kaito K, Urayama H, Watanabe G.: "Doxycycline treatment in a model of early abdominal aortic aneurysm"Surg Today.. 36(6). 426-433 (2003)

Kaito K、Urayama H、Watanabe G.：“早期腹主动脉瘤模型中的多西环素治疗”Surg Today.. 36(6)。

Kosugi I, Urayama H, Kasashima F, Ohtake H, Watanabe Y: "Matrix metalloproteinase-9 and urokinase-type plasminogen activator in varicose veins."Ann Vase Surg.. 17(3). 234-238 (2003)

Kosugi I、Urayama H、Kasashima F、Ohtake H、Watanabe Y：“静脉曲张中的基质金属蛋白酶 9 和尿激酶型纤溶酶原激活剂。”Ann Vase Surg.. 17(3)。

Kaito K, Urayama H, Watanabe G.: "Doxycycline treatment in a model of early abdominal aortic aneurysm"Surg Today. 33(6). 426-433 (2003)

Kaito K、Urayama H、Watanabe G.：“早期腹主动脉瘤模型中的多西环素治疗”Surg Today。

Kosugi I, Urayama H, Kasashima F, Ohtake H, Watanabe Y: "Matrix metalloproteinase-9 and urokinase-type plasminogen activator in varicose veins"Ann Vase Surg.. 17(3). 234-238 (2003)

Kosugi I、Urayama H、Kasashima F、Ohtake H、Watanabe Y：“静脉曲张中的基质金属蛋白酶 9 和尿激酶型纤溶酶原激活剂”Ann Vase Surg.. 17(3)。