慢病毒介导转染Adra1d基因shRNA对大鼠主动脉血管平滑肌细胞钙离子和钙调蛋白的影响
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摘要
目的:探讨慢病毒介导转染α1D-肾上腺素能受体(Adra1d)基因shRNA对大鼠主动脉血管平滑肌细胞(vascular smooth muscle cells,VSMCs)中Ca~(2+)浓度及钙调蛋白(Ca M)表达的影响。方法:Adra1d基因shRNA与GV248载体拼接得到重组载体,经包装质粒共转染293T细胞,包装病毒,收集病毒原液,超速离心浓缩,并测定滴度得到重组慢病毒载体。用得到的重组慢病毒载体转染大鼠VSMCs,通过RT-q PCR和Western blot鉴定干扰效果,然后激光共聚焦检测VSMCs内Ca~(2+)浓度变化,RT-q PCR和Western blot法检测VSMCs Ca M的mRNA和蛋白表达。结果:慢病毒载体构建成功;收集得到293T细胞分泌的病毒上清,测定病毒的滴度为3×1011TU/L。转染后大鼠主动脉VSMCs的Adra1d mRNA和蛋白表达量均显著下调。慢病毒Lv-shRNA4-Adr对目的基因Adra1d的干扰效率大于85%;Adra1d基因被靶向沉默后,与scrambled组相比,大鼠主动脉VSMCs的Ca~(2+)荧光强度显著升高,而且Ca M的mRNA和蛋白表达量也显著增加。结论:慢病毒介导转染Adra1d基因shRNA可显著增加大鼠主动脉VSMCs中Ca~(2+)浓度和Ca M表达。
AIM: To investigate the effects of lentivirus-mediated transfection of shRNA targeting α1 D-adrenergic receptor( Adra1 d) gene on calcium ion( Ca~(2+)) and calmodulin( Ca M) in vascular smooth muscle cells( VSMCs) of rat aorta. METHODS: Single oligonucleotide sequences of shRNA targeting rat Adra1 d gene were design and synthesized,and then the shRNA was constructed and cloned into GV248 vector. The U6-shRNA carrier and expression vector were transfected into 293 T cells together and packed with lentivirus,and the supernatant was collected and concentrated by overspeed centrifugation. The VSMCs of rat aorta were transfected with recombinant lentivirus vector. The interference effects were identified by RT-q PCR and Western blot. The concentration of Ca~(2+)in VSMCs was detected by laser confocal inspection,and the expression of Ca M at mRNA and protein levels in the VSMCs was determined by RT-q PCR and Western blot.RESULTS: The lentiviral shRNA expression vector was successfully constructed. The titer of the concentrated virus was 3× 1011 TU/L. The mRNA and protein expression levels of Adra1 d in the rat aortic VSMCs were significantly reduced after transfection. The interference efficiency of Lv-shRNA4-Adr to Adrald gene was greater than 85%. After target silencing of Adra1 d gene,compared with scrambled group,the Ca~(2+)fluorescence intensity of rat aortic VSMCs was significantly increased. Moreover,the mRNA and protein expression levels of Ca M were also increased significantly. CONCLUSION: A lentiviral shRNA expression vector targeting rat Adra1 d gene was successfully constructed,which significantly increased Ca~(2+)concentration and Ca M expression in rat aortic VSMCs.
AIM: To investigate the effects of lentivirus-mediated transfection of shRNA targeting α1 D-adrenergic receptor( Adra1 d) gene on calcium ion( Ca~(2+)) and calmodulin( Ca M) in vascular smooth muscle cells( VSMCs) of rat aorta. METHODS: Single oligonucleotide sequences of shRNA targeting rat Adra1 d gene were design and synthesized,and then the shRNA was constructed and cloned into GV248 vector. The U6-shRNA carrier and expression vector were transfected into 293 T cells together and packed with lentivirus,and the supernatant was collected and concentrated by overspeed centrifugation. The VSMCs of rat aorta were transfected with recombinant lentivirus vector. The interference effects were identified by RT-q PCR and Western blot. The concentration of Ca~(2+)in VSMCs was detected by laser confocal inspection,and the expression of Ca M at mRNA and protein levels in the VSMCs was determined by RT-q PCR and Western blot.RESULTS: The lentiviral shRNA expression vector was successfully constructed. The titer of the concentrated virus was 3× 1011 TU/L. The mRNA and protein expression levels of Adra1 d in the rat aortic VSMCs were significantly reduced after transfection. The interference efficiency of Lv-shRNA4-Adr to Adrald gene was greater than 85%. After target silencing of Adra1 d gene,compared with scrambled group,the Ca~(2+)fluorescence intensity of rat aortic VSMCs was significantly increased. Moreover,the mRNA and protein expression levels of Ca M were also increased significantly. CONCLUSION: A lentiviral shRNA expression vector targeting rat Adra1 d gene was successfully constructed,which significantly increased Ca~(2+)concentration and Ca M expression in rat aortic VSMCs.
引文
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[9]郝礼森,宋小杰,章广玲,等.腺病毒介导的shRNA下调PTEN表达对活化肝星状细胞骨架蛋白F-actin的影响[J].中国病理生理杂志,2017,33(3):557-561.
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[11]梁旭竞,陈小佳,金玲,等.人端粒酶逆转录酶慢病毒载体的构建及人肝细胞的初步筛选[J].中国病理生理杂志,2009,25(4):826-829.
[12]陈舒,罗铭,蔡望青,等.靶向MAG基因shRNA慢病毒载体的构建及其对MAG基因表达的沉默[J].中国病理生理杂志,2013,29(9):1725-1728.
[13]Endoh M,Hiramoto T,Ishihata A,et al.Myocardialα1-adrenoceptors mediate positive inotropic effect and changes in phosphatidylinositol metabolism.Species differences in receptor distribution and the intracellular coupling process in mammalian ventricular myocardium[J].Circ Res,1991,68(5):1179-1190.
[14]Duka I,Gavras I,Johns C,et al.Role of the postsynapticα2-adrenergic receptor subtypes in catecholamine-induced vasoconstriction[J].Gen Pharmacol,2000,34(2):101-106.
[15]Tanoue A,Nasa Y,Koshimizu T,et al.Theα1D-adrenergic receptor directly regulates arterial blood pressure via vasoconstriction[J].J Clin Invest,2002,109(6):765-775.
[16]Li YF,Cao FF,Liu F,et al.Effects of Shexiangbaoxin pills on the expression of cardiacα1-andβ-adrenergic receptor subtypes in rat hearts with heart failure induced by myocardial infarction[J].Chin Med J(Engl),2012,125(9):1556-1562.
[17]Xin X,Yang N,Faber JE.Platelet-derived growth factorBB inhibits ratα1D-adrenergic receptor gene expression in vascular smooth muscle cells by inducing AP-2-like protein binding toα1D proximal promoter region[J].Mol Pharmacol,1999,56(6):1152-1161.
[2]Liu R,Leslie KL,Martin KA.Epigenetic regulation of smooth muscle cell plasticity[J].Biochim Biophys Acta,2015,1849(4):448-453.
[3]Docherty JR.Subtypes of functionalα1-adrenoceptor[J].Cell Mol Life Sci,2010,67(3):405-417.
[4]Gericke A,Martinka P,Nazarenko I,et al.Impact ofα1-adrenoceptor expression on contractile properties of vascular smooth muscle cells[J].Am J Physiol Regul Integr Comp Physiol,2007,293(3):R1215-R1221.
[5]Fan LL,Ren S,Zhou H,et al.α1D-Adrenergic receptor insensitivity is associated with alterations in its expression and distribution in cultured vascular myocytes[J].Acta Pharmacol Sin,2009,30(12):1585-1593.
[6]Sun B,Kintsurashvili E,Ona D,et al.Inhibition of theα1D-adrenergic receptor gene by RNA interference(RNAi)in rat vascular smooth muscle cells and its effects on other adrenergic receptors[J].Vasc Pharmacol,2007,46(5):367-372.
[7]孙成林,段志泉,辛世杰,等.血管紧张素Ⅱ1型受体的短发夹环RNA抑制大鼠血管平滑肌细胞增殖的研究[J].中华外科杂志,2004,42(22):1357-1362.
[8]秦小江.杜鹃素舒张大鼠主动脉及对自发性高血压大鼠主动脉损伤的保护作用[D].太原:山西医科大学,2015.
[9]郝礼森,宋小杰,章广玲,等.腺病毒介导的shRNA下调PTEN表达对活化肝星状细胞骨架蛋白F-actin的影响[J].中国病理生理杂志,2017,33(3):557-561.
[10]Liu S,Yi F,Cheng W,et al.Molecular mechanisms in vascular injury induced by hypertension:expression and role of microRNA-34a[J].Exp Ther Med,2017,14(6):5497-5502.
[11]梁旭竞,陈小佳,金玲,等.人端粒酶逆转录酶慢病毒载体的构建及人肝细胞的初步筛选[J].中国病理生理杂志,2009,25(4):826-829.
[12]陈舒,罗铭,蔡望青,等.靶向MAG基因shRNA慢病毒载体的构建及其对MAG基因表达的沉默[J].中国病理生理杂志,2013,29(9):1725-1728.
[13]Endoh M,Hiramoto T,Ishihata A,et al.Myocardialα1-adrenoceptors mediate positive inotropic effect and changes in phosphatidylinositol metabolism.Species differences in receptor distribution and the intracellular coupling process in mammalian ventricular myocardium[J].Circ Res,1991,68(5):1179-1190.
[14]Duka I,Gavras I,Johns C,et al.Role of the postsynapticα2-adrenergic receptor subtypes in catecholamine-induced vasoconstriction[J].Gen Pharmacol,2000,34(2):101-106.
[15]Tanoue A,Nasa Y,Koshimizu T,et al.Theα1D-adrenergic receptor directly regulates arterial blood pressure via vasoconstriction[J].J Clin Invest,2002,109(6):765-775.
[16]Li YF,Cao FF,Liu F,et al.Effects of Shexiangbaoxin pills on the expression of cardiacα1-andβ-adrenergic receptor subtypes in rat hearts with heart failure induced by myocardial infarction[J].Chin Med J(Engl),2012,125(9):1556-1562.
[17]Xin X,Yang N,Faber JE.Platelet-derived growth factorBB inhibits ratα1D-adrenergic receptor gene expression in vascular smooth muscle cells by inducing AP-2-like protein binding toα1D proximal promoter region[J].Mol Pharmacol,1999,56(6):1152-1161.