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Elucidation of the mechanisms regulating the vascular tone and proliferation : Development of a novel technique to introduce protein into the intact cells and its applications

阐明调节血管张力和增殖的机制：开发将蛋白质引入完整细胞的新技术及其应用

基本信息

批准号：
15590758
负责人：
HIRANO Katsuya
金额：
$ 2.24万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

By using a cell-penetrating peptide, which is found in the human immunodeficiency viral transcription factor Tat and is composed of 11 amino acids, I have developed a novel technique to rapidly, reversibly and quantitatively introduce proteins into the vascular tissues and cells. The cargo protein to be introduced into the cells was prepared as a recombinant protein fused to the cell-penetrating peptide using a bacterial expression system. I have developed the expression vectors. Using this new technique of the protein transduction, I have made the following contribution to the vascular biology.(1)I have for the first time found that the N-terminal region of the regulatory subunit of myosin phosphatase, MYPT1, plays a physiological role in regulating the vascular tone in an intact artery.(2)By introducing the inhibitor proteins of the small G proteins RhoA and Rac1 in a time-specific manner, I have for the first time found that the activity of Rho proteins is required at the late G1 ph … More ase for the cell cycle to progress from the G1 phase to the S phase in the vascular endothelial cells.(3)I found that the 24 h treatment of the arterial tissue with the RhoA inhibitor protein attenuated the arterial contractility in a manner dependent on the endothelial NO production. However, Rac1 inhibitor protein had no such effect. This finding thus for the first time present direct evidence that RhoA plays a physiological role in the production of NO in in situ endothelial cells.(4)I found that the 24 h treatment of the cultured smooth muscle cells with the Rac1 inhibitor protein reduced the cell surface expression of the thrombin receptor PAR1. However, the RhoA inhibitor protein had no such effect. This observation thus suggests that Rac1 plays a critical role in determining the expression of PAR1 in the vascular smooth musclecells.(5)Estrogen inhibited the TNF-α-induced apoptosis in the vascular endothelial cells. However, the introduction of the dominant negative mutant of Akt inhibited the anti-apoptotic effect of estrogen. This finding thus provide the first direct evidence that Akt plays a critical role in the anti-apoptotic signaling elicited by estrogen in the vascular endothelial cells. Less

通过使用在人类免疫缺陷病毒转录因子TAT中发现的由11个氨基酸组成的细胞穿透肽，我开发了一种快速、可逆和定量地将蛋白质引入血管组织和细胞的新技术。利用细菌表达系统将待导入细胞的货物蛋白制备为与穿透性多肽融合的重组蛋白。我已经开发了表达载体。利用这种新的蛋白质转导技术，我对血管生物学做出了以下贡献：(1)我首次发现肌球蛋白磷酸酶调节亚单位MYPT1的N端区域在调节完整动脉的血管张力方面起着生理作用。(2)通过以特定时间的方式引入小G蛋白RhoA和Rac1的抑制蛋白，我首次发现在G1末期ph…需要Rho蛋白的活性血管内皮细胞有更多的细胞周期从G1期进入S期。(3)我发现动脉组织经RhoA抑制蛋白处理24 h后，动脉收缩功能减弱，这种作用依赖于内皮NO的产生。而rac1抑制蛋白则没有这种作用。这一发现首次为RhoA在原位内皮细胞产生NO中起生理学作用提供了直接的证据。(4)我发现用rac1抑制蛋白处理培养的平滑肌细胞24小时后，细胞表面凝血酶受体PAR1的表达减少。然而，RhoA抑制蛋白没有这种作用。这提示RAC1在决定血管平滑肌细胞中PAR1的表达中起关键作用。(5)雌激素抑制肿瘤坏死因子-α诱导的血管内皮细胞的凋亡。然而，Akt基因的显性负性突变体的引入抑制了雌激素的抗凋亡作用。这一发现为Akt在雌激素诱导的血管内皮细胞抗凋亡信号中发挥关键作用提供了第一个直接证据。较少