臺灣博碩士論文加值系統

異常的血管平滑肌細胞增生和爬行對於在經皮冠狀動脈血管成形術(PTCA)後形成的動脈再狹窄扮演很重要的角色。血小板衍生因子(platelet-derived growth factor) 在這過程中被認為是一個重要的生長因子。化合物5185是具有抗血栓形成作用的benzimidazole衍生物。在本研究中，我們調查在活體外5185對於血管平滑肌細胞增生和爬行的影響，以及在活體內大鼠氣球損傷後對於動脈新生內膜的作用。我們的結果證實5185能夠濃度相關的抑制血清、血小板衍生因子或U46619所引起的血管平滑肌細胞增生，以及血清或血小板衍生因子引起的血管平滑肌細胞爬行。我們也藉由乳酸脫氫酶釋放實驗(LDH release assay)確認5185的這些作用並非來自於它的細胞毒性。此外，我們利用PI染色來分析5185對細胞週期分布的影響，觀察到有輕微的sub-G1累積現象。而5185也會抑制血小板衍生因子刺激後和細胞週期相關的Cdk2和Cdk4。更進一步，我們的資料顯示5185會抑制血小板衍生因子引起的PDGFRβ、PI3K、 P38、 PLCγ1和c-Src在大鼠主動脈平滑肌細胞的磷酸化，但是ERK1/2、JNK和Akt的磷酸化則不受影響。我們認為抑制PDGFRβ下游的訊息傳導途徑，是造成5185能夠抑制血小板衍生因子引起的血管平滑肌細胞增生和爬行的原因。最後，我們的活體內實驗結果顯示5185每天每公斤2毫克的劑量，能夠顯著的抑制大鼠頸動脈的新生內膜形成，這顯示著5185在治療動脈損傷後血管再狹窄的療效潛力。

Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) play an important role in the development of restenosis after percutaneous transluminal angioplasty (PTCA). PDGF has been recognized as one of the most important growth factors involved in this process. Compound 5185 [1-benzyl-2-(5-methyl-2-furyl) benzimidazole] is a benzimidazole derivative with anti-thrombotic activities. In this study, we investigated the effects of 5185 on VSMC proliferation, migration in vitro and neointima formation in rat balloon injury model in vivo. Our results demonstrated that 5185 could inhibit serum, PDGF, and U46619-induced VSMC proliferation, and serum or PDGF-induced migration in a concentration-dependent manner. We also confirmed that these effects of 5185 on VSMCs were not due to its cytotoxicity by LDH release assay. In addition, we analyzed the effects of 5185 on cell cycle distribution by PI staining, and a slight accumulation of sub-G1 was observed. Cell cycle associated Cdk2 and Cdk4 activities were inhibited by 5185 in the presence of PDGF stimulation. Furthermore, our data showed that 5185 inhibited PDGF-induced phosphorylation of PDGFRβ, PI3K, P38, PLCγ1 and c-Src in RASMCs, but phosphorylations of ERK1/2, JNK and Akt were not obviously affected. We suggest that the suppression of PDGFRβ downstream signaling transduction pathway may be responsible for the inhibitory effect of 5185 of PDGF-induced VSMC migration and proliferation. Finally, our in vivo results showed that 5185 could significantly inhibit neointima formation of rat carotid artery at a dose of 2mg/kg daily, indicating the therapeutic potential for treating restenosis after arterial injury.

目錄 …………………………………………………………………… i
中文摘要…………………………………………………………… iii
英文摘要…………………………………………………………… iv
縮寫表………………………………………………………………… vi
第一章 Introduction………………………………………………… 1
1.1 Vascular smooth muscle cells ……………………………… 1
1.2 PDGF and receptor ………………………………………… 2
1.3 Signal transduction induced by PDGF……………………… 4
1.4 Migration and proliferation of VSMCs……………………… 6
1.5 Restenosis…………………………………………………… 8
1.6 Aims of this thesis………………………………………… 13
第二章 Materials and methods…………………………………… 25
第三章 Results…………………………………………………… 33
3.1 5185 inhibited VSMC viability…………………………… 33
3.2 5185 inhibited PDGF, serum, and U46619-induced DNA synthesis in VSMCs……………………………………………… 33
3.3 5185 does not cause LDH release in RASMCs……………34
3.4 Effect of 5185 on VSMC cell cycle progression………… 35
3.5 5185 reduced serum and PDGF-induced VSMC migration... 36
3.6 5185 inhibited PDGF-induced PDGFRβ phosphorylation and downstream PI3K and MAPKs activation…………………… 36
3.7 5185 attenuated the expression of Cdk2 and Cdk4……… 37
3.8 5185 inhibited PDGF-induced PLCγ1 and c-Src phosphorylation……………………………………………… 38
3.9 5185 suppressed neointima formation after balloon injury in rat carotid artery in vivo……………………………39
第四章 Discussion…………………………………………………… 66
第五章 Conclusion and perspectives………………………… 72
Reference……………………………………………………………… 76

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