Alternative TitleBasic study for gene therapy by
Periodical title平成14(2002)年度 科学研究費補助金 基盤研究(C) 研究報告書概要 = 2002 Fiscal Year Final Research Report Summary
Note (General)金沢大学医学部附属病院
【目的】人工血管を用いたバイパス術後の吻合部狭窄の一因として血小板由来増殖因子B鎖(platele-derived growth factor beta : PDGF beta chain)の関与が報告されている.PDGF beta chainは平滑筋細胞に対する強力な細胞増殖因子であり,吻合部位の平滑筋細胞にはPDGF beta receptorに対する本受容体が高度に発現することが報告されている.本研究の目的,内膜増殖を阻害する本受容体の細胞外領域(extracellular region of the receptor : EX)を吻合部に遺伝子導入することによる狭窄回避に関する効果を明らかにすることである.【対象及び方法】ラットの大動脈に直径3mmのexpanded polytetrafluoroethylene製人工血管(artificial graft)を移植した.実験1.ラットに大腸菌LacZ遺伝子を組み込んだ非増殖性組み換えアデノウイルス(a replication-deficient recombinant adenovirus containing the E.coli LacZ gene)(AxLacZ)を1x10^8,1x10^9,1x10^<10>,1x10^<11>pfu/mlの濃度や吻合部血管に1.外膜側から,また2.内腔から投与した.術2週間後に大腸菌LacZ遺伝子の導入率をX-Gal染色を施行し測定した.実験2.実験1で得られた最も導入率の良い方法でEXの遺伝子を組み込んだ非増殖性組み換えアデノウイルス(a replication-deficient recombinant adenovirus containing the gene encoding the EX)(AxPDGFXR)を人工血管吻合部に投与した.術2週間後にAxPDGFXRの蛋白定量,mRNA発現の検索を行った.術1カ月後にHE染'色を行い,von Winebrand因子およびKi-67抗原に対する免疫組織染色を行い,吻合部の血管新生および狭窄率を求めた.TUNE法を用いてDNA断片化,アポトーシス細胞の割合を求めた.【結果】実験1.吻合部の血管の外膜側から1x10^<11>pfu/mlの濃度での投与が最も導入効率が良かった.実験2.吻合部動脈にEXの蛋白発現,mRNAが同定された.AxPDGFXR群はControl群に比しvon Winebrand 因子およびKi-67抗原に対する免疫組織染色血管新生・細胞増殖が抑制されて,狭窄率も有意に低かった.アポトーシス細胞はなかった.【結語】血小板由来増殖因子受容体細胞外領域の局所発現による人工血管吻合部内膜肥厚の抑制が実験的に明らかにされた.本法の有効性,安全性が確立されれば,細小人工血管移植への臨床応用が可能となり開存率の飛躍的向上が期待できる.
[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 the 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.
研究課題/領域番号:13671222, 研究期間(年度):2001 – 2002
出典：「細径人工血管吻合部狭窄に対する血小板由来増殖因子を標的とした遺伝子治療の研究」研究成果報告書 課題番号13671222（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-13671222/136712222002kenkyu_seika_hokoku_gaiyo/)を加工して作成
Sourcehttps://kanazawa-u.repo.nii.ac.jp/?action=repository_action_common_download&item_id=57493&item_no=1&attribute_id=26&file_no=1
Related Materialhttps://kaken.nii.ac.jp/ja/search/?kw=60283131
https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-13671222/
https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-13671222/136712222002kenkyu_seika_hokoku_gaiyo/
Data Provider (Database)国立情報学研究所 : 学術機関リポジトリデータベース（IRDB）（機関リポジトリ）