Basic study for gene therapy by

【目的】血小板源性生长因子β: PDGF β链的影响是人工搭桥术后吻合口狭窄的一个因素，已有报道。PDGF β链是平滑肌细胞的一种强大的细胞生长因子，在吻合部位的平滑肌细胞中有报道显示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的细胞外区基因引入吻合口来避免狭窄的作用。【对象与方法】将直径为3毫米的聚四氟乙烯扩张人工血管移植至大鼠主动脉。实验1。我们将含有大肠杆菌LacZ基因的复制缺陷重组腺病毒（AxLacZ）以1x10^8、1x10^9、1x10^<10>和1x10^<11>pfu/ml的水平注入吻合血管，(1)从外膜侧，(2)从管腔。术后2周，采用X-Gal染色法测定大肠杆菌LacZ基因的引入率。实验2。采用实验1中引入率最高的方法，将含有EX基因编码的复制缺陷重组腺病毒（AxPDGFXR）注入人工血管吻合口。术后2周，我们检测AxPDGFXR蛋白浓度及mRNA表达。术后1个月行HE染色、von Winebrand因子、Ki-67抗原免疫组织染色，评估吻合口动脉化及狭窄率。我们使用TUNEL方法进行DNA片段化，并寻求细胞凋亡率。[结果]实验1。吻合血管外侧1 × 10^<11> pfu/ml给药效果最佳。实验2。在吻合动脉处检测EX蛋白和mRNA的表达。在AxPDGFXR组中，von Winebrand因子和Ki-67抗原免疫组织染色血管的动脉化和细胞生长受到限制，狭窄率也明显低于对照组。未见细胞凋亡。【结论】血小板源性生长因子受体细胞外区域的局部表达限制了人工血管吻合口内膜增厚。如果确立了该方法的有效性和安全性，将有可能在临床上应用于微血管移植，并有望显著提高血管通畅率。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