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かぐらおか 第175号
Get PDF平成30年度学位記授与式 学長挨拶 2/ 定年退職にあたって～P型ATPase研究の発展と生化学～ 17/ 定年退職にあたって「學而不厭、誨人不倦」 19/ 卒業にあたって 20/ 卒業にあたって 21/ 卒業にあたって 22/ 卒業にあたって 23/ 一歩、前へ 24/ 卒業にあたって 25/ 大学生活4年間を振り返って 26/ 卒業にあたって 27/ 卒業にあたって 28/ 卒業にあたって 29/ 平成30年度 学位記授与式 30/ 平成30年度定年退職教授による最終講義が行われました 32/ 海外留学助成制度を利用して 33/ 学部学生海外留学助成制度報告書 34/ 平成30年度保健師卒業セミナーを開催 35/ 平成30年度助産師セミナーを開催 36/ 授業評価(平成30年度後期) 37/ 卒業製の動向(医学科) 57/ 卒業生の動向(看護学科) 58/ 飲酒事故の防止について 59/ 教員の異動 59/ 今後のスケジュール 60/ 第175号表紙 60
Development of the Gene Therapy with a CRE Decoy ODN to Prevent Vascular Intimal Hyperplasia
Get PDFOBJECTIVE:
Intimal hyperplasia (IH) is the main cause of therapeutic failure after vascular and endovascular surgery. However, there is currently no targeted therapy for the treatment of IH. We recently reported that the inhibition of cyclic adenosine monophosphate response element (CRE) binding protein (CREB) activation is important in vein graft IH. We focused on a decoy oligodeoxynucleotide (ODN) therapeutic strategy for suppressing IH as a clinical application. The objective of this study was to confirm the therapeutic effect of a CRE decoy ODN in an animal model as a novel therapy for preventing intimal hyperplasia as the first step of the preclinical study of our strategy.
METHODS:
We designed two phosphorothioate CREs and two scramble decoy ODNs and screened them using a CREB transcription assay to check their ability to bind to a CRE sequence. We chose a CRE decoy ODN with high first-binding ability and transfected it into vascular smooth muscle cells (VSMCs) in vitro. Proliferation and migration were assessed using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assays and modified Boyden chamber assays. We examined CRE activity using a luciferase reporter gene assay. We assessed the expression of messenger RNAs by quantitative real-time polymerase chain reaction. In a wire-injury mouse model (C57BL6, n = 6), CRE decoy ODN was transfected into the injured vessel wall using an ultrasound-sonoporation method in vivo. Mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3) and four and a half LIM domains 5 (FHL5) expression of pregrafting vein remnants were assessed by immunohistologic analyses.
RESULTS:
Compared with scramble decoy ODN, the selected CRE decoy ODN could significantly decrease CRE activity (mean ± standard error of the mean: 0.20 ± 0.03 vs 1.00 ± 0.16, n = 6; P < .05) as shown by a luciferase reporter gene assay, VSMC proliferation (0.73 ± 0.04 vs 0.89 ± 0.02, n = 6; P < .05) and migration (96.4 ± 6.1 vs 311.4 ± 19.1 migrated VSMCs/well, n = 6; P < .05) after 24-hour transfection. The CRE decoy ODN significantly suppressed the formation of IH at injured vessel walls in an animal model, as analyzed by pathologic staining (0.20 ± 0.02 vs 0.56 ± 0.08, area of the intima/area of the artery vs the control after 21 days' transfection, n = 6; P < .05). Furthermore, MAPKAPK3 and FHL5, which are CREB activators, were significantly expressed in pregrafting vein remnants in diabetes mellitus patients.
CONCLUSIONS:
CREB-CRE signaling is an important mechanism of IH formation, and CRE decoy therapy can help preventing IH. This study is the first part of the preclinical study of our strategy.博士（医学）旭川医科大