我们的网站为什么显示成这样？

可能因为您的浏览器不支持样式，您可以更新您的浏览器到最新版本，以获取对此功能的支持，访问下面的网站，获取关于浏览器的信息：

|本期目录/Table of Contents|

人QKI6基因重组腺病毒载体的构建及其在心肌细胞中的表达鉴定

《心脏杂志》[ISSN:1009-7236/CN:61-1268/R]

期数:
2013年第4期
页码:
408-413
栏目:
基础研究
出版日期:
2013-07-25

文章信息/Info

Title:
Construction of human QKI6 recombinant adenovirus vector and identity of its expression in cardiomyocytes
作者:
胡杏林; 郑强荪; 郭万刚; 曾 迪; 李 雪; 苏菲菲; 欧东波
(第四军医大学唐都医院心内科，陕西 西安 710038)
Author(s):
HU Xinglin;  ZHENG Qiangsun;  GUO Wangang;  ZENG Di;  LI Xue;  SU Feifei;  OU Dongbo
(Department of Cardiology, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, Shaanxi, China)
关键词:
RNA结合蛋白6; 重组腺病毒; 载体构建
Keywords:
RNAbinding protein 6;  recombinant adenovirus vector;  vector construction
分类号:
Q343.1
DOI:
-
文献标识码:
A
摘要:
目的:构建人RNA结合蛋白（QKI6）基因的重组腺病毒载体，并在心肌细胞中表达。方法:利用PCR技术，从原始质粒中扩增出QKI6目的基因片段，酶切后连接到pShuttle-GFP-CMV重组穿梭质粒上。再将pShuttle-GFP-QKI6 转移到pAdxsi载体上，得到带有QKI6目的基因及GFP荧光指针的重组腺病毒载体pAdxsi-GFP-QKI6。酶切、PCR鉴定重组腺病毒载体pAdxsi-GFP-QKI6，并完成扩增与纯化。经Pacl线性化后，转染HEK 293细胞进行腺病毒包装，并测定病毒的滴度及目的基因的表达。以包装好的重组腺病毒感染大鼠新生心肌细胞，通过GFP表达观察病毒感染效率。用PCR检测目的基因QKI6在感染的心肌细胞中的表达。结果:含QKI6基因及GFP荧光指针的重组腺病毒载体pAdxsi-GFP-QKI6构建成功，酶切、PCR鉴定及DNA测序证实了其正确性。重组腺病毒可重复感染HEK 293细胞，荧光显微镜下可见GFP表达，且QKI6基因的表达明显升高。包装的重组腺病毒可有效感染新生心肌细胞，并表达目的基因QKI6。结论:成功地构建QKI6基因重组腺病毒载体，以其感染293细胞及新生心肌细胞后，可有效地表达QKI6基因，为进一步研究QKI6基因在糖尿病心肌细胞凋亡的机制以及在糖尿病性心肌病治疗中的应用奠定实验基础。
Abstract:
AIM:To construct recombinant adenovirus vector containing human RNAbinding protein (QKI6) gene and provide an experimental foundation for further research on the protective mechanisms of the QKI6 gene in cardiomyocyte apoptosis and applications of QKI6 in the treatment of diabetic cardiomyopathy. METHODS: Using PCR, QKI6 target gene fragment was amplified from the original plasmid and ligated into recombinant shuttle plasmid (pShuttleGFPCMV). pShuttleGFPQKI6 was then transferred into pAdxsi vector to gain adenovirus plasmids pAdxsiGFPQKI6 containing QKI6 target gene and GFP fluorescence reporter. PCR was performed to identify the recombinant adenovirus following amplification and purification of pAdxsiGFPQKI6. After linearization by Pacl, the recombinant adenovirus vector transfected HEK 293 cells for adenovirus packaging. Viral titer was determined and the expression of the target gene was measured. The wrapped adenovirus vector transfected rat neonatal cardiomyocytes. GFP expression was observed to determine the efficiency of viral infection and PCR was performed to detect QKI6 expression in infected cardiomyocytes. RESULTS: The recombinant adenovirus vector pAdxsiGFPQKI6 containing QKI6 gene and GFP fluorescence reporter was constructed successfully, which was confirmed by PCR and DNA sequencing. Recombinant adenovirus was reusable to infect HEK 293 cells. GFP and QKI6 expression was detected by fluorescence microscopy and PCR. The packaged recombinant adenovirus efficiently infected neonatal cardiomyocytes and QKI6 expression in cardiomyocytes increased. CONCLUSION: We successfully constructed the adenovirus containing QKI6 gene. QKI6 expression increases after infection of 293 cells and neonatal cardiomyocytes, which establishes the foundation for further research on effects and mechanisms of myocardial protection of QKI6 in diabetic rats.

参考文献/References

[1]Artzt K,Wu JI.STAR trek: An introduction to STAR family proteins and review of quaking (QKI)[J]. Adv Exp Med Biol,2010,693:1-24.

[2]Guo W,Shi X,Liu A,et al.RNA binding protein QKI inhibits the ischemia/reperfusioninduced apoptosis in neonatal cardiomyocytes[J].Cellular Physiol Biochem, 2011,28(4):593-602.

[3]Penna C,Tullio F,Perrelli MG,et al.Cardioprotection against ischemia/reperfusion injury and chromogranin Aderived peptides[J].Curr Med Chem,2012,19(24):4074-4085.

[4]Kannel WB,McGee DL.“Diabetes and cardiovascular risk factors:the framingham study”[J].Circulation,1979,59(1):8-13.

[5]Hausenloy DJ,Maddock HL,Baxter GF,et al.Inhibiting mitochondrial permeability transition pore opening:a new paradigm for myocardial preconditioning?[J].Cardiovasc Res,2002,55(3):534-543.

[6]Hausenloy DJ,Yellon DM.Reperfusion injury salvage kinase signalling:taking a RISK for cardioprotection[J].Heart Fail Rev,2007,12(3-4):217-234.

[7]Hausenloy DJ,Yellon DM.The mitochondrial permeability transition pore:its fundamental role in mediating cell death during ischaemia andreperfusion[J].Mol Cell Cardiol,2003,35(4):339-341.

[8]Hardy RJ.Molecular defects in the dysmyelinating mutant quaking[J].Neurosci Res,1998,51(4):417-422.

[9]Kondo T,Furuta T,Mitsunaga K,et al.Genomic organization and expression analysis of the mouse qkI locus[J].Mamm Genome,1999,10(7):662-669.

[10]Cattaneo I,Figliuzzi M,Azzollini N,et al.In vivo regeneration of elastic lamina on fibroin biodegradable vascular scaffold[J].Int J Artif Organs,2013,36(3):166-174.

[11]Larocque D,Pilotte J,Chen T,et al.Nuclear retention of MBP mRNAs in the quaking viable mice[J].Neuron,2002, 36(5):815-829.[12]Larocque D,Richard S.QUAKING KH domain proteins as regulators of glial cell fate and myelination[J].RNA Biol,2005,2(2):37-40.

[13]Ehrengruber MU,Hennou S,Büeler H,et al.Gene transfer into neurons from hippocampal slices:comparison of recombinant Semliki Forest Virus,adenovirus,adenoassociated virus, lentivirus, and measles virus[J].Mol Cell Neurosci,2001,17(5):855-871.

备注/Memo

备注/Memo:
收稿日期：2013-03-21.通讯作者：郑强荪，教授，主要从事心电生理及冠心病诊治研究 Email：qiangsunzheng@gmail.com 作者简介：胡杏林，硕士生 Email：xinglinhu@yeah.net
更新日期/Last Update: 2013-07-29