shRNA慢病毒载体介导的SD大鼠心肌细胞Cx43基因的沉默作用
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摘要
目的构建缝隙连接蛋白(Cx)43基因shRNA'慢病毒表达载体,并检测其对SD大鼠心肌细胞Cx43基因的沉默效果。
方法针对Cx43基因序列,设计RNA干扰靶点序列,合成靶序列的双链DNA,接入pGCL-GFP载体,PCR筛选阳性克隆,测序鉴定。用pHelper1.0和pHelper2.0质粒共转染293T细胞,包装产生具备感染能力的慢病毒。以293T细胞中绿色荧光蛋白的表达水平测定病毒滴度,以最适感染复数感染SD大鼠心肌细胞,通过荧光显微镜检测感染效率,应用Real-Time PCR和Western blot检测Cx43mRNA及Cx43蛋白表达,评价其抑制效果。
结果经PCR和测序证实,成功构建Cx43慢病毒载体,病毒滴度为8×108TU/mL,慢病毒载体对SD大鼠心肌细胞感染效率为80%,荧光定量PCR检测提示慢病毒载体对心肌细胞Cx43mRNA的抑制率为92.4%,Westernblot检测提示慢病毒载体对心肌细胞Cx43蛋白的抑制率为86.5%。
结论成功构建Cx43shRNA慢病毒载体,有效感染SD大鼠心肌细胞后,显著抑制了心肌细胞Cx43的表达。
Objective:To construct a lentiviral RNAi vector that is capable of knocking down connexin43(Cx43) gene in rat cardiac myocytes.
Methords:Short hairpin RNA (shRNA) sequence targeting rat cardiac myocytes Cx43gene was designed. After synthesis and annealing, the double-stranded oligo nucleotides (ds oligo) were connect to pGC-LV vectors. Then, The viral particles were generated by cotransfection of293T cells with the pGC-lv-Cx43and two packaging vector(pHelper1.0,pHelper2.0)and the virus titer was determined by counting the percentage of GFP positive cell. After transfection of lentiviral into rat cardiac myocytes with multiplicity of infection (MOI),the level of Cx43mRNA was determined by RT-PCR, the level of Cx43protein was determined by western blot assay.
Results:Target Cx43lentiviral vector was confirmed by PCR, the final titer obtained was8×108TU/mL. the infection rate was80%,the Real-Time PCR show the inhibition rate of C×43mRNA was92.4%and the western blot show the inhibition rate of Cx43protein was86.5%the expression of Cx43in knock down group is significantly lower than in control group.
Conclusion:The lentiviral RNAi vector with the capability of knocking down Cx43gene in rat cardiac myocytes has been successfully constructed.
方法针对Cx43基因序列,设计RNA干扰靶点序列,合成靶序列的双链DNA,接入pGCL-GFP载体,PCR筛选阳性克隆,测序鉴定。用pHelper1.0和pHelper2.0质粒共转染293T细胞,包装产生具备感染能力的慢病毒。以293T细胞中绿色荧光蛋白的表达水平测定病毒滴度,以最适感染复数感染SD大鼠心肌细胞,通过荧光显微镜检测感染效率,应用Real-Time PCR和Western blot检测Cx43mRNA及Cx43蛋白表达,评价其抑制效果。
结果经PCR和测序证实,成功构建Cx43慢病毒载体,病毒滴度为8×108TU/mL,慢病毒载体对SD大鼠心肌细胞感染效率为80%,荧光定量PCR检测提示慢病毒载体对心肌细胞Cx43mRNA的抑制率为92.4%,Westernblot检测提示慢病毒载体对心肌细胞Cx43蛋白的抑制率为86.5%。
结论成功构建Cx43shRNA慢病毒载体,有效感染SD大鼠心肌细胞后,显著抑制了心肌细胞Cx43的表达。
Objective:To construct a lentiviral RNAi vector that is capable of knocking down connexin43(Cx43) gene in rat cardiac myocytes.
Methords:Short hairpin RNA (shRNA) sequence targeting rat cardiac myocytes Cx43gene was designed. After synthesis and annealing, the double-stranded oligo nucleotides (ds oligo) were connect to pGC-LV vectors. Then, The viral particles were generated by cotransfection of293T cells with the pGC-lv-Cx43and two packaging vector(pHelper1.0,pHelper2.0)and the virus titer was determined by counting the percentage of GFP positive cell. After transfection of lentiviral into rat cardiac myocytes with multiplicity of infection (MOI),the level of Cx43mRNA was determined by RT-PCR, the level of Cx43protein was determined by western blot assay.
Results:Target Cx43lentiviral vector was confirmed by PCR, the final titer obtained was8×108TU/mL. the infection rate was80%,the Real-Time PCR show the inhibition rate of C×43mRNA was92.4%and the western blot show the inhibition rate of Cx43protein was86.5%the expression of Cx43in knock down group is significantly lower than in control group.
Conclusion:The lentiviral RNAi vector with the capability of knocking down Cx43gene in rat cardiac myocytes has been successfully constructed.
引文
1. Sekikawa A, Horiuchi BY, Edmundowicz D, et al. A "natural experiment" in cardiovascular epidemiology in the early 21st century [J]. Heart,2003,89 (3):255-7
2. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia:a delay of lethal cell injury in ischemic myocardium. Circulation,1986,74: 1124-1136
3. Miura T, Miki T, Yano T. Role of the gap preconditioning [J]. Am J Physiol Heart Circ Physiol,2009,296(2):H396-H403.
4.何燕,曾志羽,钟国强,等.线粒体连接蛋白43参与缺血后处理对兔急性心肌缺血再灌注损伤的保护作用[J].中华心血管病杂志,2010,38(4)：357-62.
5. De MA, Vega VL, Contreras JE. Gap junctions, homeostasis, and injury. J Cell Physiol,2002,191(3):269-282.
6. Evans W H, Boitano S. Connexin mimetic peptides:specific inhibitors of gap-junctional intercellular communication [J]. Biochem Soc Trans,2001, 29(Pt4):606-612.
7. Nakase T, Fushiki S,Naus CC. Astrocytic gap junctions composed of connexin 43 reduce apoptotic neuronal damage in cerebral ischemia [J]. Stroke,2003,34(9):e163-166.
8. Miura T, Miki T, Yano T. Role of the gap junction in ischemic preconditioning in the heart. [J]. Am J Physiol Heart Circ Physiol,2010,298: H1115-25.
9. Fire A, Xu S, Montgomery M K, et al. Potent and specific genetic interference bydoble-stranded RNA in caenorhabditis elegans. Nature, 1998,391 (6669):806-811.
10. Tabara H, Grishok A, Mello CC. RNAi in C. elegans:soaking in the genome sequence. Science,1998,282(5388):430-431.
11. Amado, R.G., and Chen, I.S.Y. The promise of gene therapy within reach? Science,1999,285:67-76.
12. Elbashir SM, HarborthJ, WeberK, et al. Analysis of gene function in somatic mammalian cells using small interfering RNAs[J]. Methods 2002, 26(2):199-213
13. Webster KA, Discher DJ, Bishopcic NH. Induction and muclear accumulation of fos and jun proto-oncogenes in hypoxic cardiac myocytes [J]. J Bio Chem,1993,268(22):16852-16853
14. Hannon G J. RNA interference[J]. Nature,2002,418:244-251.
15. Brown S J., Mahaffey J P., Lorenzen M D, et al. Using RNAi to investigate orthologous homeotic gene function during development of distantly related insects. Evol Dev,1999.1(1)11-15.
16. Elbashir S M., Harborth J., Lendeckel W., et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature,2001, 411(6836):494-498.
17. Martinez J., Tuschl T., RISC is a 5'phosphomonoester producing RNA endonuclease. Genes Dev,2004,18(9):975-980.
18. Tusterman M, Plasterk R H. Dicers at RISC; the mechanism of RNAi. Cell, 2004,117(1):1-3.
19. Zeng Y, Cullen B R.RNA interference in human cells is restricted to the cytoplasm. RNA,2002,8(7):855-860.
20. Kawaski H, Taira K. Short hairpin type of dsRNAs that are controlled by tRNA(Val)promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells. Nucleic Acids Res,2003,31(2):700-707.
21. Irie N, Sakai N, Ueyama T, et al. Subtype and species specific knockdown of PKC using short interfering RNA. Biochem Biophys Res Commun,2002, 298(5):738-743.
22. M ah C, Byme BJ, Floote TR. Virus-based gene deliver systems. Clin Phamacokinet 2002,41:901-911.
23. Naldini L, BlomerU, Gallay P, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science,1996, 272(5259):263-267.
24. Kafri T, Van Praag H, Ouyang L, et al. A packaging cell line for Lentivirus vectors. J Virol,1999,73:576.
25. Buchschacher GL Jr, Wong-Staal F. Development of lentiviral vectors for human diease. Blood,2000,95 (8):2499-2504.
1. Kowalt owski AJ, Castilh o RF, Vercesi AE. Mitochondrial permeability transit ion and oxidative tress. FEBS letters [J].Circ Res,2001,495(1/2) 12-5
2. Sjaast ad I, Bentzen JG, Semb SO, et al. Reduced calciumtolerance in rat cardiomyocytes after myocardial infarction [J]. Act a Physiol Scand,2002, 175 (4):261-269.
3. Yellon DM, Hausenloy DJ. Myocardial reperfusion injury [J]. N Engl J Med, 2007,357(11):112-135.
4. BohloolyY M, Bollano E, Mobini R, et al. Selective cerebral over expression of grow thhormon e alters cardiac function,morphology, energy metabolism and catecholamines intransgenic mice [J]. Growth Horm IGF Res,2005,15 (2):148-155.
5. Garcia Gonzalez MJ, Dominguez Rodriguez A, Abreu Gonzalez P. New pharmacologic opt ions in the treatment of acute coronary syndromes and myocardial ischemia reperfusion injury:potential role of levosimendan [J]. Minerva Cardioangiol,2007,55(5):625-635.
6. Nordlie MA, Wold LE, Simkhovich BZ, et al. Molecular aspects of ischemic heart disease:ischemia/reperfusion induced genetic changes and pot entail applications of gene and RNA interference therapy [J]. J Cardiovasc Pharmacol T her,2006,11 (1):17-30.
7. Engler RL, Gottlieb AR, Burlesin KO, et al. Myocytecell death by apoptosis during reperfusion [J]. Circulation,1993,88(Supply):1-5.
8. Qin F, Liang MC, Liang CS. Progressive left ventricular remodeling, myocyte apoptosis, and protein signaling cascades after myocardial infarction in rabbit s [J]. Bioch im Biophys Acta,2005,1740(3):499-513.
9. 杜健,王晨净,景志敏.心肌缺血再灌注损伤中钙超载和氧自由基作用机制研究[J].卫生职业教育,2009,27(11)：121-123.
10.孙海燕,薛富善,李成文,等.缺血再灌注对大鼠心肌内源性一氧化氮含量的影响[J].临床麻醉学杂志,2008,24(1)：49-51.
11.石高举,李玉新,世慧娜,等.通络定痛胶囊对心肌缺血再灌注损伤血浆中TNF、ICAM-1影响的临床观察[J].中国民康医学,2008,20(9)：894-896.
12.马国强,李跃荣.心肌缺血/再灌注损伤与细胞凋亡的机制及其防治研究进展[J].滨州医学院学报,2007,30(1)：50-52.
13. Adamek A, Jung S, Dienesch C, et al. Role of 5-lipoxygenase in myocardial ischemiareperf usion injury in mice[J]. Eur J Pharmacol,2007,571 (1): 51-54.
14. Venardos K M, Zatta A J, Marshall T, et al. Reduced L-arginine transport contributes to the pathogenesis of myocardial ischemiareperfusion injury [J]. J Cell Biochem,2009,108(1):156-168.
15. Wang X L, Liang F, Jiao X Y, et al. Diverse effects of L-arginine on cardiacfunction of rats subjected to myocardial ischemia and reperfunsion in vivo [J]. Acta Biochim Biophys Sin,2007,39(3):201-207.
16. Xie J R, Yu L N. Cardioprotective effects of cyclosporine Ainan in vivo model of myocardial ischemia and reperfusion [J]. Acta Anaesthesiol Scand, 2007,51(7):909-913.
17. Caplen N J, Parrish S, Imani F, et al. Specifc inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems [J]. Proc Natl Acad Sci,2001,98(17):9752-9747.
18. Meister G, Tuschl T. Mechanisms of gene silencing by double-stranded RNA[J]. Nature,2004,431:343-349.
19. Dunn SR, Philips WS, Green DR, et al. Knockdown of actin and caspase gene by RNA interference in the symbio ticanemone Aiptasia pallid [J]. Biol Bull,2007,212(3):250-258.
20. James CC, Carolyn AW, Nancy SL, et al. Use of double-stranded RNA interference in Drosophila cell lines to dissect singal transduction pathway s [J]. RNAs,2006,97(5):6499-6503.
21. Li F, Wang XY, Chen L. Effects of RNAi targeting-L-survivin on proliferation and apoptosis of MDA-MB-231 cells [J]. Actad Acad IAE M ed Milit Tertian,2008,12 (30):2302-2320.
22. Shukla S, Sumaria CS, Pradeepkumar PI. Exploring chemical modifications for siRNA therapeutics:a structural and functional outlook [J]. Chem Med Chem,2010,5(3):328-349.
23. Lee AJ, Kolesnick R, Swanton C. RNAi mediated functional analysis o f pathways influencing cancer cell drug resistance [J]. Ex pert Rev Mole Med [electronic resource],2009,11(21):15-19
24. Subramanya S, Kim SS, Manjunath N, et al. RNA-interference based therapeutics for human immunodeficiency virus HIV-1 treatment:synthetic siRNA or vector-based shRNA [J]. Expert Opin Biol Ther,2010,10(2):201-213.
25.王丽,梅长林.缺氧诱导因子在大鼠肾缺血再灌注损伤中的表达和意义[J].中国中西医结合肾病杂志,2007,8(2)：77-80.
26.李虹,白小涓,刘强,等.siRNA靶向沉默p22phox表达对内皮细胞衰老抑制作用的研究[J].遗传,2008,30(9)：1175-118.
27.杨勇,王玮,黄从新,等.ACE siRNA对人脐静脉内皮细胞ET1表达的影响[J].郧阳医学院学报,2009,28(2)：109-112.
28.王岚,吕家高,张存泰,等.RNA干扰下调钙调蛋白激酶Ⅱα表达对血管紧张素Ⅱ诱导心肌细胞肥大的影响[J].临床心血管病杂志,2008,24(5)：378-382.
29.郭英竹,矫强,王世婷,等.Ryanodine受体2基因沉默对大鼠心肌细胞缺血再灌注损伤的影响[J].中华病理学杂志,2008,37(11)：760-764.
30. TobiasW, Hans PW, Liane T, et al. Suppression of ICAM-1 in human venous endothelial cells by small interfering RNAs [J]. Eur J Cardiothorac Surg, 2005,28(6):816-820.
31.周晓彤,沈振亚,于曙东,等.siRNA对小鼠血管内皮细胞NF-κBp65表达的抑制作用[J].现代免疫学,2005,25(3)：208-212.
32. Lisovyy OO, Dosenko VE, Nagibin VS, et al. Cardioprotective effect of 5-lipoxygenase gene (ALOX5) silencing in ischemia-reperfusion [J]. Acta Biochim Po 1,2009; 56(4):687-694.
2. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia:a delay of lethal cell injury in ischemic myocardium. Circulation,1986,74: 1124-1136
3. Miura T, Miki T, Yano T. Role of the gap preconditioning [J]. Am J Physiol Heart Circ Physiol,2009,296(2):H396-H403.
4.何燕,曾志羽,钟国强,等.线粒体连接蛋白43参与缺血后处理对兔急性心肌缺血再灌注损伤的保护作用[J].中华心血管病杂志,2010,38(4)：357-62.
5. De MA, Vega VL, Contreras JE. Gap junctions, homeostasis, and injury. J Cell Physiol,2002,191(3):269-282.
6. Evans W H, Boitano S. Connexin mimetic peptides:specific inhibitors of gap-junctional intercellular communication [J]. Biochem Soc Trans,2001, 29(Pt4):606-612.
7. Nakase T, Fushiki S,Naus CC. Astrocytic gap junctions composed of connexin 43 reduce apoptotic neuronal damage in cerebral ischemia [J]. Stroke,2003,34(9):e163-166.
8. Miura T, Miki T, Yano T. Role of the gap junction in ischemic preconditioning in the heart. [J]. Am J Physiol Heart Circ Physiol,2010,298: H1115-25.
9. Fire A, Xu S, Montgomery M K, et al. Potent and specific genetic interference bydoble-stranded RNA in caenorhabditis elegans. Nature, 1998,391 (6669):806-811.
10. Tabara H, Grishok A, Mello CC. RNAi in C. elegans:soaking in the genome sequence. Science,1998,282(5388):430-431.
11. Amado, R.G., and Chen, I.S.Y. The promise of gene therapy within reach? Science,1999,285:67-76.
12. Elbashir SM, HarborthJ, WeberK, et al. Analysis of gene function in somatic mammalian cells using small interfering RNAs[J]. Methods 2002, 26(2):199-213
13. Webster KA, Discher DJ, Bishopcic NH. Induction and muclear accumulation of fos and jun proto-oncogenes in hypoxic cardiac myocytes [J]. J Bio Chem,1993,268(22):16852-16853
14. Hannon G J. RNA interference[J]. Nature,2002,418:244-251.
15. Brown S J., Mahaffey J P., Lorenzen M D, et al. Using RNAi to investigate orthologous homeotic gene function during development of distantly related insects. Evol Dev,1999.1(1)11-15.
16. Elbashir S M., Harborth J., Lendeckel W., et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature,2001, 411(6836):494-498.
17. Martinez J., Tuschl T., RISC is a 5'phosphomonoester producing RNA endonuclease. Genes Dev,2004,18(9):975-980.
18. Tusterman M, Plasterk R H. Dicers at RISC; the mechanism of RNAi. Cell, 2004,117(1):1-3.
19. Zeng Y, Cullen B R.RNA interference in human cells is restricted to the cytoplasm. RNA,2002,8(7):855-860.
20. Kawaski H, Taira K. Short hairpin type of dsRNAs that are controlled by tRNA(Val)promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells. Nucleic Acids Res,2003,31(2):700-707.
21. Irie N, Sakai N, Ueyama T, et al. Subtype and species specific knockdown of PKC using short interfering RNA. Biochem Biophys Res Commun,2002, 298(5):738-743.
22. M ah C, Byme BJ, Floote TR. Virus-based gene deliver systems. Clin Phamacokinet 2002,41:901-911.
23. Naldini L, BlomerU, Gallay P, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science,1996, 272(5259):263-267.
24. Kafri T, Van Praag H, Ouyang L, et al. A packaging cell line for Lentivirus vectors. J Virol,1999,73:576.
25. Buchschacher GL Jr, Wong-Staal F. Development of lentiviral vectors for human diease. Blood,2000,95 (8):2499-2504.
1. Kowalt owski AJ, Castilh o RF, Vercesi AE. Mitochondrial permeability transit ion and oxidative tress. FEBS letters [J].Circ Res,2001,495(1/2) 12-5
2. Sjaast ad I, Bentzen JG, Semb SO, et al. Reduced calciumtolerance in rat cardiomyocytes after myocardial infarction [J]. Act a Physiol Scand,2002, 175 (4):261-269.
3. Yellon DM, Hausenloy DJ. Myocardial reperfusion injury [J]. N Engl J Med, 2007,357(11):112-135.
4. BohloolyY M, Bollano E, Mobini R, et al. Selective cerebral over expression of grow thhormon e alters cardiac function,morphology, energy metabolism and catecholamines intransgenic mice [J]. Growth Horm IGF Res,2005,15 (2):148-155.
5. Garcia Gonzalez MJ, Dominguez Rodriguez A, Abreu Gonzalez P. New pharmacologic opt ions in the treatment of acute coronary syndromes and myocardial ischemia reperfusion injury:potential role of levosimendan [J]. Minerva Cardioangiol,2007,55(5):625-635.
6. Nordlie MA, Wold LE, Simkhovich BZ, et al. Molecular aspects of ischemic heart disease:ischemia/reperfusion induced genetic changes and pot entail applications of gene and RNA interference therapy [J]. J Cardiovasc Pharmacol T her,2006,11 (1):17-30.
7. Engler RL, Gottlieb AR, Burlesin KO, et al. Myocytecell death by apoptosis during reperfusion [J]. Circulation,1993,88(Supply):1-5.
8. Qin F, Liang MC, Liang CS. Progressive left ventricular remodeling, myocyte apoptosis, and protein signaling cascades after myocardial infarction in rabbit s [J]. Bioch im Biophys Acta,2005,1740(3):499-513.
9. 杜健,王晨净,景志敏.心肌缺血再灌注损伤中钙超载和氧自由基作用机制研究[J].卫生职业教育,2009,27(11)：121-123.
10.孙海燕,薛富善,李成文,等.缺血再灌注对大鼠心肌内源性一氧化氮含量的影响[J].临床麻醉学杂志,2008,24(1)：49-51.
11.石高举,李玉新,世慧娜,等.通络定痛胶囊对心肌缺血再灌注损伤血浆中TNF、ICAM-1影响的临床观察[J].中国民康医学,2008,20(9)：894-896.
12.马国强,李跃荣.心肌缺血/再灌注损伤与细胞凋亡的机制及其防治研究进展[J].滨州医学院学报,2007,30(1)：50-52.
13. Adamek A, Jung S, Dienesch C, et al. Role of 5-lipoxygenase in myocardial ischemiareperf usion injury in mice[J]. Eur J Pharmacol,2007,571 (1): 51-54.
14. Venardos K M, Zatta A J, Marshall T, et al. Reduced L-arginine transport contributes to the pathogenesis of myocardial ischemiareperfusion injury [J]. J Cell Biochem,2009,108(1):156-168.
15. Wang X L, Liang F, Jiao X Y, et al. Diverse effects of L-arginine on cardiacfunction of rats subjected to myocardial ischemia and reperfunsion in vivo [J]. Acta Biochim Biophys Sin,2007,39(3):201-207.
16. Xie J R, Yu L N. Cardioprotective effects of cyclosporine Ainan in vivo model of myocardial ischemia and reperfusion [J]. Acta Anaesthesiol Scand, 2007,51(7):909-913.
17. Caplen N J, Parrish S, Imani F, et al. Specifc inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems [J]. Proc Natl Acad Sci,2001,98(17):9752-9747.
18. Meister G, Tuschl T. Mechanisms of gene silencing by double-stranded RNA[J]. Nature,2004,431:343-349.
19. Dunn SR, Philips WS, Green DR, et al. Knockdown of actin and caspase gene by RNA interference in the symbio ticanemone Aiptasia pallid [J]. Biol Bull,2007,212(3):250-258.
20. James CC, Carolyn AW, Nancy SL, et al. Use of double-stranded RNA interference in Drosophila cell lines to dissect singal transduction pathway s [J]. RNAs,2006,97(5):6499-6503.
21. Li F, Wang XY, Chen L. Effects of RNAi targeting-L-survivin on proliferation and apoptosis of MDA-MB-231 cells [J]. Actad Acad IAE M ed Milit Tertian,2008,12 (30):2302-2320.
22. Shukla S, Sumaria CS, Pradeepkumar PI. Exploring chemical modifications for siRNA therapeutics:a structural and functional outlook [J]. Chem Med Chem,2010,5(3):328-349.
23. Lee AJ, Kolesnick R, Swanton C. RNAi mediated functional analysis o f pathways influencing cancer cell drug resistance [J]. Ex pert Rev Mole Med [electronic resource],2009,11(21):15-19
24. Subramanya S, Kim SS, Manjunath N, et al. RNA-interference based therapeutics for human immunodeficiency virus HIV-1 treatment:synthetic siRNA or vector-based shRNA [J]. Expert Opin Biol Ther,2010,10(2):201-213.
25.王丽,梅长林.缺氧诱导因子在大鼠肾缺血再灌注损伤中的表达和意义[J].中国中西医结合肾病杂志,2007,8(2)：77-80.
26.李虹,白小涓,刘强,等.siRNA靶向沉默p22phox表达对内皮细胞衰老抑制作用的研究[J].遗传,2008,30(9)：1175-118.
27.杨勇,王玮,黄从新,等.ACE siRNA对人脐静脉内皮细胞ET1表达的影响[J].郧阳医学院学报,2009,28(2)：109-112.
28.王岚,吕家高,张存泰,等.RNA干扰下调钙调蛋白激酶Ⅱα表达对血管紧张素Ⅱ诱导心肌细胞肥大的影响[J].临床心血管病杂志,2008,24(5)：378-382.
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