构建线粒体钙离子摄入蛋白1慢病毒表达载体及在H9C2细胞中的应用

被引：2

作者：

荆哲 ^{[1
]}

刘峰舟 ^{[2
]}

刘燕 ^{[1
]}

陈永清 ^{[1
]}

机构：

[1] 解放军兰州军区兰州总医院心内科

[2] 西京医院心内科

来源：

中国组织工程研究 | 2016年 / 20卷 / 37期

关键词：

慢病毒感染; 线粒体蛋白质类; 肌细胞,心脏; 组织构建; 组织工程; H9C2细胞; 线粒体钙离子摄入蛋白1(MICU1); 慢病毒载体; 病毒包装; 钙;

D O I：

暂无

中图分类号：

R587.2 [糖尿病性昏迷及其他并发症]; R542.2 [心肌疾病];

学科分类号：

100201 [内科学];

摘要：

背景:线粒体钙离子摄入蛋白1(mitochondrial calcium uptake 1,MICU1)是维持细胞线粒体钙稳态的重要分子,MICU1对线粒体钙稳态的调节可能在糖尿病心肌病的发生及发展中起着重要作用,但目前机制尚不明确。目的:构建MICU1基因的慢病毒表达载体,产毒感染H9C2细胞,评价MICU1基因在H9C2细胞中的表达效果,为后续在细胞水平研究糖尿病心肌病的发生及发展建立平台。方法:PCR提取H9C2细胞MICU1基因,SpeⅠ、EcoRⅠ双酶切后,将MICU1基因片段插入慢病毒载体pR RLsin.CMV.e FP中,构建慢病毒表达质粒pR RLsin.CMV.MICU1-e FP。使用pC MVDR8.91、pC MV-VSVG共转染于293T细胞中包装产毒,用于感染H9C2细胞。通过RT-PCR及Western blot检测感染后H9C2细胞中MICU1 m RNA及蛋白的表达。共聚焦显微镜检测Rhod-2染色H9C2细胞后线粒体钙水平。结果与结论:(1)MICU1基因成功插入pR RLsin.CMV.e FP慢病毒表达质粒;(2)转染pR RLsin.CMV.MICU1-e FP慢病毒表达质粒后,可见293T细胞表达绿色荧光蛋白,并且MICU1的蛋白表达明显升高;(3)病毒液感染H9C2细胞后,MICU1的蛋白及m RNA水平较未感染组及空质粒包装组明显升高;(4)Rhod-2染色后观察发现,MICU1能够明显增强线粒体钙水平;(5)结果表明,pR RLsin.CMV.MICU1-e FP慢病毒能够高效感染H9C2细胞,为构建永生化细胞系奠定基础。

引用

页码：5560 / 5566

页数：7

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