抑制程序性坏死对缺氧/复氧H9c2心肌细胞损伤的影响
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摘要
本研究目的是观察程序性坏死中关键激酶基因ripk1和ripk3的表达被对应的shRNA质粒沉默后,对缺氧/复氧(H/R)导致的心肌细胞损伤的保护作用。研究采用DNA重组技术,将设计合成的shRNA序列插入pSUPER质粒中,构建出RIPK1-shRNA及RIPK3-shRNA表达质粒。用上述质粒转染H9c2心肌细胞,然后进行H/R处理,再用RT-PCR和Western blot方法研究相应基因的表达情况,检测程序性坏死及心肌损伤的相关指标。实验结果表明,基因ripk1和ripk3的表达被特异的shRNA抑制后,对H/R诱导的H9c2心肌细胞有明显的保护作用。
The aim of this study is to construct specific shRNA expressing plasmids,and to observe their effects on H9c2 cardiomyocytes injury induced by hypoxia/reoxygenation(H/R).RIPK1 and RIPK3are the key kinases mediating the process of necroptosis.Using recombinant DNA technology,we inserted the synthetic shRNA into pSUPER vector to construct RIPK1-shRNA or RIPK3-shRNA plasmid respectively.We transfected H9c2 cardiomyocytes with the two shRNA plasmids respectively,before we treated them with H/R stimulation.Then we measured the relevant genes and proteins by real-time PCR and Western blot.Meanwhile,we detected the markers of necroptosis and cardiomyocytes injury.The results showed that inhibition of ripk1 or ripk3gene expression by its specific shRNA might protect the cardiomyocytes injury induced by H/R stimulation.
The aim of this study is to construct specific shRNA expressing plasmids,and to observe their effects on H9c2 cardiomyocytes injury induced by hypoxia/reoxygenation(H/R).RIPK1 and RIPK3are the key kinases mediating the process of necroptosis.Using recombinant DNA technology,we inserted the synthetic shRNA into pSUPER vector to construct RIPK1-shRNA or RIPK3-shRNA plasmid respectively.We transfected H9c2 cardiomyocytes with the two shRNA plasmids respectively,before we treated them with H/R stimulation.Then we measured the relevant genes and proteins by real-time PCR and Western blot.Meanwhile,we detected the markers of necroptosis and cardiomyocytes injury.The results showed that inhibition of ripk1 or ripk3gene expression by its specific shRNA might protect the cardiomyocytes injury induced by H/R stimulation.
引文
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[14]HAN W,XIE J,LI L,et al.Necrostatin-1reverts shikonininduced necroptosis to apoptosis[J].Apoptosis,2009,14(5):674-686.
[15]CHIUSA M,HOOL S L,TRUETSCH P,et al.Cancer therapy modulates VEGF signaling and viability in adult rat cardiac microvascular endothelial cells and cardiomyocytes[J].J Mol Cell Cardiol,2012,52(5):1164-1175.
[2]LINKERMANN A,GREEN D R.Necroptosis[J].N Engl J Med,2014,370(5):455-465.
[3]LINKERMANN A,HACKL M J,KUNZENDORF U,et al.Necroptosis in immunity and ischemia-reperfusion injury[J].Am J Transplant,2013,13(11):2797-2804.
[4]OERLEMANS M I,LIU J,ARSLAN F,et al.Inhibition of RIP1-dependent necrosis prevents adverse cardiac remodeling after myocardial ischemia-reperfusion in vivo[J].Basic Res Cardiol,2012,107(4):270.
[5]KOSHINUMA S,MIYAMAE M,KANEDA K,et al.Combination of necroptosis and apoptosis inhibition enhances cardioprotection against myocardial ischemia-reperfusion injury[J].J Anesth,2014,28(2):235-241.
[6]SONG R,ZHANG J,ZHANG L,et al.H2O2Induces myocardial hypertrophy in H9c2cells:apotential role of Ube3a[J].Cardiovasc Toxicol,2014.
[7]LEE J H,PARK J W.Attenuated mitochondrial NADP+-dependent isocitrate dehydrogenase activity induces apoptosis and hypertrophy of H9c2 cardiomyocytes[J].Biochimie,2014,99:110-118.
[8]ZHAO M,SUN L,YU X J,et al.Acetylcholine mediates AMPK-dependent autophagic cytoprotection in H9c2 cells during hypoxia/reoxygenation injury[J].Cell Physiol Biochem,2013,32(3):601-613.
[9]TAFER H.Bioinformatics of siRNA design[J].Methods Mol Biol,2014,1097:477-490.
[10]文茂瑶,门若庭,但雪莲,等.NogoB-shRNA表达质粒的构建及其在大鼠原代肝星状细胞收缩中的作用[J].四川大学学报(医学版),2014,45(3):362-366.
[11]MAO Z,LIU Z,CHEN L,et al.Predictive value of the surface-enhanced resonance Raman scattering-based MTT assay:a rapid and ultrasensitive method for cell viability in situ[J].Anal Chem,2013,85(15):7361-7368.
[12]李君丽,廖延标,陆立慧,等.Visfatin诱导心肌细胞肥大的机制初探[J].生物医学工程学杂志,2014,31(2):379-384.
[13]SINGH M V,ANDERSON M E.Is CaMKII a link between inflammation and hypertrophy in heart?[J].J Mol Med(Berl),2011,89(6):537-543.
[14]HAN W,XIE J,LI L,et al.Necrostatin-1reverts shikonininduced necroptosis to apoptosis[J].Apoptosis,2009,14(5):674-686.
[15]CHIUSA M,HOOL S L,TRUETSCH P,et al.Cancer therapy modulates VEGF signaling and viability in adult rat cardiac microvascular endothelial cells and cardiomyocytes[J].J Mol Cell Cardiol,2012,52(5):1164-1175.