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生存素通过线粒体途径影响K5诱导的血管内皮细胞凋亡

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  • R543

摘要:

目的 探讨生存素(Survivin)在人纤溶酶原K5诱导血管内皮细胞凋亡中的作用。方法 分
离培养脐带血管内皮细胞,用K5诱导处理,应用qRT-PCR和Western blot测定细胞中生存素表达。在细
胞中转染生存素过表达载体,同时用K5处理细胞,流式细胞术测定细胞凋亡情况,JC-1法测定细胞线粒
体膜电位,比色法测定细胞中三磷酸腺苷(ATP)的合成水平和细胞中Caspase-3、Caspase-9的活性,
用H2DCFDA法测定细胞中活性氧(ROS)水平,Western blot测定细胞色素C(Cyt C)在线粒体和胞质中
的水平。结果 K5诱导处理后,血管内皮细胞中生存素的转录和表达水平均降低,而转染生存素过表达
载体后的细胞中生存素表达水平升高。生存素过表达的血管内皮细胞经K5诱导处理以后,细胞凋亡率降
低,细胞线粒体膜电位升高,细胞合成的ATP增加,Caspase-3、Caspase-9活性降低,细胞中ROS水平减
少,线粒体中Cyt C水平升高,而胞质中Cyt C水平降低,与只经过K5处理的细胞相比,差异有统计学意
义(P<0.05)。结论 K5可诱导血管内皮细胞中生存素表达下调,而上调生存素表达可抑制K5诱导的内
皮细胞凋亡。

Abstract:

Objective To discuss the effect of Survivin on apoptosis of vascular endothelial cells induced by
Kringle 5 (K5). Methods The human umbilical vascular endothelial cells (HUVEC) were isolated and cultured
and then treated with K5. The expression of Survivin was detected by using qRT-PCR and Western blotting
assay. The overexpression vectors of Survivin were transfected in HUVEC and meanwhile HUVEC were treated
with K5. The apoptosis was detected by using flow cytometry. The mitochondrial membrane potential (MMP) was
detected by using JC-1 method, synthesis level of adenosine triphosphate (ATP) and activities of Caspase-3 and
Caspase-9 were detected by using chromatoptometry. The level of reactive oxygen species (ROS) was detected by
using H2DCFDA method. The level of cytochrome C (Cyt C) in mitochondrion and cytoplasma was detected by
using Western blotting assay. Results After K5 treatment, the transfection and expression of Survivin decreased,
and increased after transfected overexpression vectors of Survivin in HUVEC. After K5 treatment in HUVEC
with overexpression, apoptosis decreased, MMP increased, ATP synthesis increased, activities of Caspase-3
and Caspase-9 decreased, ROS level decreased, and Cyt C level increased in mitochondrion and decreased in
cytoplasma (P<0.05). Conclusion K5 can induce the down-regulation of Survivin expression, and up-regulation of
Survivin can inhibit HUVEC apoptosis induced by K5.

基金项目:

国家卫计委课题资助(2014SQ00271)

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