【作者】 华文敏；

【导师】 梁中琴；

【作者基本信息】 苏州大学 ， 药理学， 2009， 硕士

【摘要】 目的:研究自噬/cathepsins激活机制在姜黄素(curcumin, Cur)抗人脐静脉内皮细胞(HUVEC)在缺血再灌注(ischemia /reperfusion ,I/R)过程中的作用。方法:建立缺氧复氧(hypoxia/reoxygenation,H/R)损伤模型,用MTT法观察不同的缺氧和复氧时间对HUVEC的损伤作用以及Cur对HUVEC在H/R模型中的保护作用;应用免疫荧光和Western Blot法观察Cur预给药后H/R对HUVEC的微管相关蛋白LC3、溶酶体酶cathepsin B、cathepsin L、促凋亡蛋白Bax、抗凋亡蛋白Bcl-2表达的影响。Western Blot法检测在H/R过程加入自噬特异性抑制剂3-MA后Bax、Bcl-2的表达。结果:单纯缺氧后LC3、Beclin 1、cathepsin B、Bax/Bcl-2表达增强,cathepsin L被激活并发生核转位,H/R后LC3、Beclin 1、cathepsin B、Bax/Bcl-2表达进一步增强,cathepsin L核转位现象更为明显,给与3-MA后,HUVEC单纯缺氧和H/R诱导表达的Bax/Bcl-2比值升高;Cur(1.25～5μmol·L﹣1)可剂量依赖性的保护H/R的HUVEC;Cur预处理后在增强LC3表达的同时,明显抑制cathepsin B和Bax/Bcl-2的表达、并抑制cathepsin L的核转位。结论缺氧诱导发生自噬,复氧后自噬表达进一步增强,用3-MA抑制自噬后, Bax/Bcl-2表达升高,说明自噬在单纯缺氧阶段和复氧阶段都是起保护作用的,H/R后cathepsin B、L表达上调,启动凋亡程序。Cur能有效提高HUVEC在H/R损伤中的存活率,并通过诱导其自噬活性、下调cathepsin B、L来抑制Bax/Bcl-2的表达保护内皮细胞。更多还原

【Abstract】 Aim To investigate the effect of autophagy/cathepsins activation mechanisms on protecting HUVEC against ischemia /reperfusion (I/R) injury directed by curcumin.Methods Establish hypoxia/reoxgenation(H/R) model on HUVEC. Using MTT colorimetric assay to observe the injury degree of hypoxia and reoxygenation at the diferent time. Preconditioning with different concentration of Cur, the survival rate of HUVEC subjected to H/R was assessed by MTT colorimetric assay. Pretreated with Cur, the expression of LC3、cathepsin B、cathepsin L、Bax and Bcl-2 was observed by fluorescent staining and Western Blot in HUVEC during H/R process. The level of Bax and Bcl-2 was tested by Western Blot during H/R when 3-methyladenine(3- MA), an inhibitor of autophagy was added.Results In the hypoxia phase , the expression of LC3、Beclin 1、cathepsin B and the ratio of Bax /Bcl-2 increased, and the nuclear translocation of cathepsin L was induced; and in the H/R phase ,these proteins just refered further enhanced;Inhibition of autophagy by 3-MA significantly increased the ratio of Bax/Bcl-2 which was induced by H/R. Cur(1.25～5μmol·L﹣1) played a protective role during H/R in HUVEC in a dose-dependent manner . When Cur (5μM) pretreated , LC3 further strengthened , at the same time , the up-regulation of cathepsin B、the ratio of Bax /Bcl-2 and the nuclei-location of cathepsin L was inhibited partly by Cur.Conclusions Autophagy was induced by hypoxia and further enhanced by reoxgenation in HUVEC, in vitro. Apoptosis was enhanced by 3-MA suggesting that autophagy is protective against hypoxia and H/R in HUVEC. Cathepsin B and cathepsin L protein up-ragulated and the apoptosis increased. Cur can raised the survival rate of HUVEC in the process of H/R. Cur increased the autophagy activity , down-regulated cathepsin B、L and inhibited the ratio of Bax /Bcl-2 to protect the endothelial cells.更多还原