Basic study for gene therapy by

[目的]有报道称，血小板源生长因子β：血小板衍化生长因子β链参与了使用人工移植物的旁路手术后吻合口狭窄的因素。PDGF β链是平滑肌细胞的一种强有力的细胞生长因子，据报道，吻合部位的此类细胞显示出PDGF β链受体的高水平表达。本研究的目的是阐明通过将抑制内膜增殖的受体EX的细胞外区域基因导入吻合部位以避免狭窄的效果。【对象与方法】将直径为3 mm的聚四氟乙烯人工血管移植到大鼠主动脉上。实验1.将含有大肠杆菌LacZ基因的复制缺陷型重组腺病毒（AxLacZ）以1 × 10 ~（-8）、1 × 10 ~（-9）、1 × 10 ~（-9）<10>和1 × 10 ~（-9）pfu<11>/ml的浓度分别从外膜侧和管腔侧注入吻合血管。两周后， ...更多信息 实验2.采用实验1中最佳导入率的方法，将携带EX（AxPDGFXR）基因的复制缺陷型重组腺病毒导入人工血管吻合口，观察其对人工血管吻合口的影响。术后2周检测AxPDGFXR蛋白浓度及mRNA表达。术后1个月行HE染色、免疫组织化学vonWinebrand因子和Ki-67抗原染色，观察吻合口动脉化和狭窄率。我们用TUNEL法进行DNA片段化，并寻找凋亡率。[结果]实验1.从吻合血管外膜侧以1 × 10 ~（13）pfu<11>/ml的浓度给药，导入效率最高，实验2.在吻合血管处检测到EX蛋白和mRNA的表达。与对照组相比，AxPDGFXR组血管的动脉化和细胞生长受到抑制，血管内Winebrand因子和Ki-67抗原免疫组织化学染色，狭窄率也显著降低。未发现细胞凋亡。[结论]血小板源性生长因子受体胞外区的局部表达可抑制人工血管吻合口内膜增厚。如果这种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。少

[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