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。
BACKGROUND： Mitochondrial calcium uptake 1（MICU1） is one of the important molecules to maintain the mitochondrial calcium homeostasis. The regulation of MICU1 to mitochondrial calcium homeostasis may play an important role in diabetic cardiomyopathy, but the underlying mechanism remains unclear. OBJECTIVE： To construct a lentiviral vector carrying MICU1 gene to transfect H9C2 cells, and then to assess MICU1 level in H9C2 cells thereby establishing a platform for researching the occurrence and development of diabetic cardiomyopathy at a cellular level.METHODS： DNA fragments of MICU1 were amplified by PCR, cleaved with Spe I, Eco R I and cloned into the lentiviral vector p RRLsin.CMV.eGFP to construct p RRLsin.CMV.MICU1-e FP vector. 293 T cells were co-transfected with recombined p CMVDR8.91 and p CMV-VSVG to produce p RRLsin.CMV.MICU1-eGFP lentiviral viruses, and then used to infect H9C2 cells. m RNA and protein expressions of MICU1 in the transfected H9C2 cells were evaluated by real-time PCR and western blot assay. Mitochondrial calcium level in Rhod-2-stained H9C2 cells was tested under confocal microscope. RESULTS AND CONCLUSION： The recombinant inducible lentiviral vector containing MICU1 gene was successfully constructed. 293 T could express green fluorescent protein with increased MICU1 level after p RRLsin.CMV.MICU1-eGFP transfection. The m RNA and protein expressions of MICU1 in the infected H9C2 group were obviously up-regulated compared with the other groups. MICU1 could remarkably improve the mitochondrial calcium level under Rhod-2 staining. These results show that p RRLsin.CMV.MICU1-eGFP lentiviral viruses are efficient to transfect H9C2 cells, which will be powered to lay a foundation for the immortalized cell line establishment.
出处
《中国组织工程研究》
CAS
北大核心
2016年第37期5560-5566,共7页
Chinese Journal of Tissue Engineering Research
