Caveolin-1基因沉默对胰岛素刺激的内皮细胞表达PAI-1的影响
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摘要
目的利用RNA干扰技术,研究小凹蛋白(Caveolin-1)对胰岛素刺激的人脐静脉内皮细胞(ECV304细胞)纤维蛋白溶解酶原抑制物-1(Plasminogen Activator Inhibitor-1,PAI-1)表达的影响。
方法通过Ambion专用软件设计合成两条编码shRNA的互补DNA寡核苷酸单链,退火形成双链核苷酸。通过T4连接酶将双链克隆入含RNA PolIII聚合酶表达元件的pENTR~(TM)/U6入门载体后测序,利用Gateway技术构建Caveolin-1 RNAi慢病毒表达载体。运用实时定量RT-PCR、间接免疫荧光、Western-blot分析ECV304细胞Caveolin-1基因沉默效率。然后用生理浓度胰岛素(终浓度1×10~(-9)mol/L)和高浓度胰岛素(终浓度1×10~(-7)mol/L)处理ECV304细胞,检测Caveolin-1基因沉默前后不同浓度胰岛素处理的ECV304细胞中PAI-1mRNA和蛋白表达和不同浓度胰岛素处理后Caveolin-1mRNA和蛋白表达变化。
结果1.测序结果显示插入pENTR~(TM)/U6载体中的编码Caveolin-1基因shRNA序列的U6RNAi盒大小及阅读框架正确,Caveolin-1 pENTR~(TM)/U6入门载体构建成功。U6上游引物与V5下游引物PCR鉴定Caveolin-1 RNAi慢病毒表达载体,琼脂糖凝胶电泳片段与目的片段大小一致。
2、实时定量RT-PCR、间接免疫荧光、Western-blot分析入门载体、RNAi表达载体对Caveolin-1基因抑制效率达85%,且特异性较高,对无关基因如β-actin无影响,同时沉默无关基因S100A13不能干扰Caveolin-1表达。
3、高胰岛素可促使PAI-1mRNA和蛋白表达明显增加,在高胰岛素刺激PAI-1表达的同时,细胞中的Caveolin-1表达明显降低,在RNA干扰Caveolin-1基因后高胰岛素促PAI-1表达的作用更加明显;生理浓度胰岛素处理ECV304细胞后,PAI-1的表达增加,Caveolin-1表达无变化,shRNA诱导Caveolin-1沉默后生理浓度胰岛素并不明显增加PAI-1的表达。
结论1、成功构建了针对Caveolin-1基因的RNAi入门载体和表达载体,能有效而又特异地抑制Caveolin-1基因表达达85%。
2、Caveolin-1可能并不参与生理状态胰岛素促PAI-1表达的信号转导,高胰岛素则可能通过抑制Caveolin-1基因的表达来促使PAI-1的表达
【Objective】The aim of this study is to investigate how Caveolin-1 gene affects the expression of insulin-stimulated Plasminogen Activator Inhibitor-1 (PAI-1) in human vascular endothelial cells (ECV304) by RNA interfering technology.
【Method】Two complementary oligonucleotide strand coding small hair RNA for Caveolin-1 was designed by using siRNA Target Finder from Ambion. The Caveolin-1 shRNA double strand oligonucleotide was cloned into the pENTR~(TM)/U6 entry vector containing RNA Polymerase III express element by T4 ligase and then the Caveolin-1 RNAi lentiviral expression system was successfully constructed by utilizing gateway technology. To evaluate the efficiency of Caveolin-1 RNAi rate, Real-time RT-PCR, immunofluorescence staining and Western blot were used to analyze the Caveolin-1 mRNA and protein expression after ECV304 cells were transfected with Caveolin-1-shRNA. By utilizing Caveolin-1 RNAi expression system, we were able to compare the mRNA and protein levels of PAI-1 between ECV304 cells and Caveolin-1RNAi ECV304 cells after they cultured with physiological concentration of insulin (final concentration 1×10~(-9))and high concentration(final concentration 1×10~(-7)) as well as the mRNA and protein levels of Caveolin-1 stimulated by different concentration of insulin.
【Results】1.The size and sequence of Caveolin-1 U6 RNAi cassette insert in the pENTR?/U6 vector was confirmed by sequencing analysis.PCR were used to identifying the BLOCK-iT~(TM) Lentiviral RNAi Expression vector by U6 sense primer and V5 antisense. The PCR product analysed by electrophoresis was correct.
2.The mRNA and protein expression of Caveolin-1 decreased 85% measured by REAL-TIME RT-PCR, immunofluorescence staining and Western blot. Other genes, such as S100A13 andβ-actin did not affect the interfering course.
3.High insulin increase the mRNA and protein expression of PAI-1 whereas high insulin treatment suppressed Caveolin-1 gene expression in ECV304 cells. In contrast, PAI-1 mRNA and protein level was augmented dramatically in the Caveolin-1 RNAi ECV304 cells cultured in the high insulin;while cells were cultured in the physiology insulin culture,Caveolin-1 gene expression had no change, but PAI-1 did not increase when Caveolin-1 RNAi ECV304 cells were treated with physiology dose of insulin.
【Conclusions】1. The Caveolin-1 RNAi pENTR~(TM)/U6 vector and Lentiviral RNAi Expression vector were constructed successfully.The shRNA can inhabit the expression of Caveolin-1 gene efficiently and specially ,the inhabit rate reach 85%.
2. Perhaps,PAI-1 expression is a Caveolin-1 dependent in the pathophyiological conditions such as hyperinsulinism but not in the physiological ones.
方法通过Ambion专用软件设计合成两条编码shRNA的互补DNA寡核苷酸单链,退火形成双链核苷酸。通过T4连接酶将双链克隆入含RNA PolIII聚合酶表达元件的pENTR~(TM)/U6入门载体后测序,利用Gateway技术构建Caveolin-1 RNAi慢病毒表达载体。运用实时定量RT-PCR、间接免疫荧光、Western-blot分析ECV304细胞Caveolin-1基因沉默效率。然后用生理浓度胰岛素(终浓度1×10~(-9)mol/L)和高浓度胰岛素(终浓度1×10~(-7)mol/L)处理ECV304细胞,检测Caveolin-1基因沉默前后不同浓度胰岛素处理的ECV304细胞中PAI-1mRNA和蛋白表达和不同浓度胰岛素处理后Caveolin-1mRNA和蛋白表达变化。
结果1.测序结果显示插入pENTR~(TM)/U6载体中的编码Caveolin-1基因shRNA序列的U6RNAi盒大小及阅读框架正确,Caveolin-1 pENTR~(TM)/U6入门载体构建成功。U6上游引物与V5下游引物PCR鉴定Caveolin-1 RNAi慢病毒表达载体,琼脂糖凝胶电泳片段与目的片段大小一致。
2、实时定量RT-PCR、间接免疫荧光、Western-blot分析入门载体、RNAi表达载体对Caveolin-1基因抑制效率达85%,且特异性较高,对无关基因如β-actin无影响,同时沉默无关基因S100A13不能干扰Caveolin-1表达。
3、高胰岛素可促使PAI-1mRNA和蛋白表达明显增加,在高胰岛素刺激PAI-1表达的同时,细胞中的Caveolin-1表达明显降低,在RNA干扰Caveolin-1基因后高胰岛素促PAI-1表达的作用更加明显;生理浓度胰岛素处理ECV304细胞后,PAI-1的表达增加,Caveolin-1表达无变化,shRNA诱导Caveolin-1沉默后生理浓度胰岛素并不明显增加PAI-1的表达。
结论1、成功构建了针对Caveolin-1基因的RNAi入门载体和表达载体,能有效而又特异地抑制Caveolin-1基因表达达85%。
2、Caveolin-1可能并不参与生理状态胰岛素促PAI-1表达的信号转导,高胰岛素则可能通过抑制Caveolin-1基因的表达来促使PAI-1的表达
【Objective】The aim of this study is to investigate how Caveolin-1 gene affects the expression of insulin-stimulated Plasminogen Activator Inhibitor-1 (PAI-1) in human vascular endothelial cells (ECV304) by RNA interfering technology.
【Method】Two complementary oligonucleotide strand coding small hair RNA for Caveolin-1 was designed by using siRNA Target Finder from Ambion. The Caveolin-1 shRNA double strand oligonucleotide was cloned into the pENTR~(TM)/U6 entry vector containing RNA Polymerase III express element by T4 ligase and then the Caveolin-1 RNAi lentiviral expression system was successfully constructed by utilizing gateway technology. To evaluate the efficiency of Caveolin-1 RNAi rate, Real-time RT-PCR, immunofluorescence staining and Western blot were used to analyze the Caveolin-1 mRNA and protein expression after ECV304 cells were transfected with Caveolin-1-shRNA. By utilizing Caveolin-1 RNAi expression system, we were able to compare the mRNA and protein levels of PAI-1 between ECV304 cells and Caveolin-1RNAi ECV304 cells after they cultured with physiological concentration of insulin (final concentration 1×10~(-9))and high concentration(final concentration 1×10~(-7)) as well as the mRNA and protein levels of Caveolin-1 stimulated by different concentration of insulin.
【Results】1.The size and sequence of Caveolin-1 U6 RNAi cassette insert in the pENTR?/U6 vector was confirmed by sequencing analysis.PCR were used to identifying the BLOCK-iT~(TM) Lentiviral RNAi Expression vector by U6 sense primer and V5 antisense. The PCR product analysed by electrophoresis was correct.
2.The mRNA and protein expression of Caveolin-1 decreased 85% measured by REAL-TIME RT-PCR, immunofluorescence staining and Western blot. Other genes, such as S100A13 andβ-actin did not affect the interfering course.
3.High insulin increase the mRNA and protein expression of PAI-1 whereas high insulin treatment suppressed Caveolin-1 gene expression in ECV304 cells. In contrast, PAI-1 mRNA and protein level was augmented dramatically in the Caveolin-1 RNAi ECV304 cells cultured in the high insulin;while cells were cultured in the physiology insulin culture,Caveolin-1 gene expression had no change, but PAI-1 did not increase when Caveolin-1 RNAi ECV304 cells were treated with physiology dose of insulin.
【Conclusions】1. The Caveolin-1 RNAi pENTR~(TM)/U6 vector and Lentiviral RNAi Expression vector were constructed successfully.The shRNA can inhabit the expression of Caveolin-1 gene efficiently and specially ,the inhabit rate reach 85%.
2. Perhaps,PAI-1 expression is a Caveolin-1 dependent in the pathophyiological conditions such as hyperinsulinism but not in the physiological ones.
引文
[1] Giltay EJ ,Elber JM. Visceral fat accumulation is an important determinate of PAI – 1 levels in young ,nonbese men and woman modulation bycross - sex hormone administration. Thromb Vasc Biol ,2004 , 18 (11) :1716 – 1722
[2]Frank PG, Lisanti MP.Caveolin-1 and caveolae in atherosclerosis: differential roles in fatty streak formation and neointimal hyperplasia. Curr Opin Lipidol. 2004 Oct;15(5):523-9
[3] Alessi MC, Juhan-Vague I.PAI-1 and the Metabolic Syndrome Links, Causes, and Consequences .Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26(10):2200-7.
[4] Rajjayabun PH , Garg S ,Durkan GC , et al. Caveolin-1 expression is associated with high-grade bladder cancer .Urology ,2001 ,58(5) :811-814
[5]Fra AM ,Pasqualetto E ,Mancini M ,et al. Genomic organization and transcriptional analysis of the human genes coding for caveolin-1 and caveolin-2 .Gene ,2000 ,243 :75-83.
[6] Hatanaka M ,Maeda T , Ikemoto T ,et al. Expression of caveolin-1 in human T cell leukemia cell lines[J ] . Biochem Biophys Res Comm ,1998 ,253 :382-387
[7] Harris J ,Werling D ,Hope JC ,et al. Caveolae and caveolin in immune cells : distribution and functions . Trends Immunol ,2002 ,23(3) :158-164.
[8]Fujimoto T. Cell biology of caveolae and its implication for clinical medicine . Nagoya J Med Sci ,2000 ,63 :9-18.
[9] Babak R, Terry PC, Xiaobo W, et al.Caveolin-1-deficient mice are Lean, resistant to diet-induced obesity, and show hypertriglyceridemia with adipocyte abnormalitiesJBC2002,277(10)8635–8647.
[10] Rajakrishnan V,Irina Ch, Marie T et al Essential role for membrane lipid rafts in interleukin-1 induced nitric oxide release from insulin-secreting cells potential regulation by Caveolin-1. Diabetes,2005,54:2576–2585
[11] Muller G, Hanekop N, Wied S, Frick W. et al. Cholesterol depletion blocks redistribution of lipid raft components and insulin-mimetic signaling by glimepiride and phosphoinositolglycans in rat adipocytes. Mol Med 8: 120–136, 2002.
[12]Burant CF , Buse MG. Induced structural and functional changes in insulin receptors from rat skelatal muscle.Diabetes ,1990(48) ,236 :254.
[13]Changgui L , Guang N , Jialun C, et al.Expression of plasminogen activator inhibitor-1 gene by insulin resistant HepG2 cell line.Chin J Endocrinol Metab , 2000 , 16 (4),239-42.
[14]Couet J, Sargiacomo M, and Lisanti MP. Interaction of a receptor tyrosine kinase, EGF-R, with caveolins. Caveolin binding negatively regulates tyrosine and serine/threonine kinaseactivities. J Biol Chem ,2004,272: 30429–30438.
[15]Corely-Mastick C and Saltiel AR. Insulin-stimulated tyrosine phosphorylation of caveolin is specific for the differentiated adipocyte phenotype in 3T3-L1 cells. J Biol Chem 2005,272:20706–20714.
[16]Sergey VB, Kazimierz M, Marc G,et.al. Plasminogen Activator Inhibitor-1 Promotes Formation of Endothelial Microparticles With Procoagulant Potential. Circulation 2006;106;2372-2378.
[17] Ma GM, Halayko AJ, Stelmack GL, et al.Effects of oxidized and glycated low-density lipoproteins on transcription and secretion of plasminogen activator inhibitor-1 in vascular endothelial cells. Cardiovasc Pathol. 2006 Jan-Feb;15(1):3-10.
[18]Michael W,Mansfield J . Environment a land genetic factors in relation toelevated circulating l - evels of PAI - 1 in caucasian patients with type two diabetes. Thromb Haemost ,1997 ,79(7) :842 - 847.
[19] Hatanaka M ,Maeda T , Ikemoto T ,et al. Expression of caveolin-1 in human T cell leukemia cell lines[J ] . Biochem Biophys Res Comm ,1998 ,253 :382-387.
[20]Repetto S, Salani B, Maggi D, et al.Insulin and IGF-I phosphorylate eNOS in HUVECs by a caveolin-1 dependent mechanism. Biochem Biophys Res Commun. 2005 Nov 25;337(3):849-52.
[21]Alex WC,Babak B,Xiao BW, et al.Caveolin-1-deficient mice show insulin resistance and defective insulin receptor protein expression in adipose tissue. Am JPhysiol Cell Physiol 2003,285: C222–C235.
[22]Couet J, Sargiacomo M, Lisanti MP, et al. Interaction of a receptor tyrosine kinase, EGF-R, with caveolins. Caveolin binding negatively regulates tyrosine and serine/threonine kinaseactivities. J Biol Chem ,1997,272: 30429–30438.
[23]Corely-Mastick C ,Saltiel AR. et al. Insulin-stimulated tyrosine phosphorylation of caveolin is specific for the differentiated adipocyte phenotype in 3T3-L1 cells. J Biol Chem 1997,272:20706–20714.
[24]Eva G, Aaron N,Alison JL , et al.Small Interfering RNA-mediated Down-regulation of Caveolin-1 Differentially Modulates Signaling Pathways in Endothelial Cells.Biology Chemistry , 2004, 279, 40659–40669
[25]Cristina B, Eriksson P,Giovanna G,et al.Transcriptional Regulation of Plasminogen Activator Inhibitor Type 1 Gene by Insulin ,Insights Into the Signaling Pathway. Diabetes,2001,50:1522–1530.
[26] Parpal S, Karlsson M, Thorn H, and Stralfors P. Cholesterol depletion disrupts caveolae and insulin receptor signaling for metabolic control via insulin receptor substrate-1, but not for mitogen-activated protein kinase control. J Biol Chem 2001,276: 9670–9678.
[27] Joshi V, Kazuhiko H, Zhong-Zhou Y, et al. Identification and modulation of a caveolae dependent signal pathway that regulates plasminogen activator inhibitor-1 in insulin-resistant adipocytes. PNAS, Dec 2004; 101: 17120 – 17125.
[1] Benjamin Lewin. <>[M].北京:科学教育出版社, 2005
[2] Yukihide Tomari and Phillip D. Zamore .Perspective: machines for RNAi Genes & Dev. Mar 2005; 19: 517 - 529.
[3] 康慧聪 唐洲平(综述) 朱遂强(审校). RNAi 及其基因治疗研究进展[J]. 卒中与神经疾病,2005,第12 卷第3期:191-193,
[4] Michelle A. C, Zhenyu X, Michael Q. Zhang, and Gregory J. The Argonaute family: tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis Genes & Dev.Nov 2002; 16: 2733
[5] George L. Sen and Helen M. Blau A brief history of RNAi: the silence of the genes,FASEB J, Jul 2006; 20: 1293 - 1299
[6] Steven Bartz and Aimee L. Jackson how will RNAi facilitate drug development?Sci. STKE, Aug 2005; 2005: pe39
[7] 刘宝玉,孟昭力. RNAi技术在药物研究中的应用[J]. Food and Drug,第8卷第04A期, Jun.2006
[8] Steven D. Buckingham, Behrooz Esmaeili, Matthew Wood, and David B. Sattelle RNA interference: from model organisms towards therapy for neural and neuromuscular disorders Hum. Mol. Genet. Oct 2004; 13: R275 - R288.
[9] 汪光蓉 ,朱道银 ,唐恩洁. RNAi抗病毒感染的研究进展[J]. Jounal of North Sichuan Medical College, Vol 120,No 14 ,Dec. 2005.
[10] Bradley SP,Kowalik TF, Rastellini C, Successful incorporation of short-interfering RNA into islet cells by in situ perfusion.Transplant Proc. 2005 Jan-Feb;37(1):233-6
[11] Hagerkvist R, Mokhtari DMyers JW.siRNA produced by recombinant dicer mediates efficient gene silencing in islet cells.Ann N Y Acad Sci. 2005 Apr;1040:114-22.
[12] Tang X,Gulherme Aetl.An RNA interference-based screen identifies MAP4K4/NIK as a negative regulator of PPARgamma, adipogenesis, and insulin-responsive hexose transport.Proc Natl Acad Sci U S A. 2006 Feb 14;103(7):2087-92.
[13] Xu J, Li L, Qian Z High glucose upregulates connective tissue growth factor expression in human vascular smooth muscle cells.Biochem Biophys Res Commun. 2005 Apr 8;329(2):538-43.
[14] Mussmann R, Geese M Harder F Inhibition of glycogen synthase kinase (GSK) 3 promotes replication and survival of pancreatic beta cells.J Biol Chem. 2007 Jan 22
[15] Goruppi S, Bonventre JV, Kyriakis JM. Signaling pathways and late-onset gene induction associated with renal mesangial cell hypertrophy.EMBO J. 2002 Oct15;21(20):5427-36
[16] Noh HJ, J Korean Med Sci. Kim HC, Lee SS, The inhibitory effect of siRNAs on the high glucose-induced overexpression of TGF-beta1 in mesangial cells.J Korean Med Sci. 2006 Jun;21(3):430-5
[17] Oshitari T, Brown D, Roy S. SiRNA strategy against overexpression of extracellular matrix in diabetic retinopathy. Exp Eye Res. 2005 Jul;81(1):32-7
[18] Zhang SX, Wang JJ. Pigment epithelium-derived factor (PEDF) is an endogenous antiinflammatory factor.FASEB J. 2006 Feb;20(2):323-5.
[19] Lorenz C,Hans-Peter Vornlocher.Steroid and lipid conjugates of siRNAs to enhance cellular uptake and gene silencing in liver cells
[20] Krutzfeldt J,Rajewsky N.Silencing of microRNAs in vivo with 'antagomirs'.Nature, Dec 2005; 438(7068): 685-9.
[2]Frank PG, Lisanti MP.Caveolin-1 and caveolae in atherosclerosis: differential roles in fatty streak formation and neointimal hyperplasia. Curr Opin Lipidol. 2004 Oct;15(5):523-9
[3] Alessi MC, Juhan-Vague I.PAI-1 and the Metabolic Syndrome Links, Causes, and Consequences .Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26(10):2200-7.
[4] Rajjayabun PH , Garg S ,Durkan GC , et al. Caveolin-1 expression is associated with high-grade bladder cancer .Urology ,2001 ,58(5) :811-814
[5]Fra AM ,Pasqualetto E ,Mancini M ,et al. Genomic organization and transcriptional analysis of the human genes coding for caveolin-1 and caveolin-2 .Gene ,2000 ,243 :75-83.
[6] Hatanaka M ,Maeda T , Ikemoto T ,et al. Expression of caveolin-1 in human T cell leukemia cell lines[J ] . Biochem Biophys Res Comm ,1998 ,253 :382-387
[7] Harris J ,Werling D ,Hope JC ,et al. Caveolae and caveolin in immune cells : distribution and functions . Trends Immunol ,2002 ,23(3) :158-164.
[8]Fujimoto T. Cell biology of caveolae and its implication for clinical medicine . Nagoya J Med Sci ,2000 ,63 :9-18.
[9] Babak R, Terry PC, Xiaobo W, et al.Caveolin-1-deficient mice are Lean, resistant to diet-induced obesity, and show hypertriglyceridemia with adipocyte abnormalitiesJBC2002,277(10)8635–8647.
[10] Rajakrishnan V,Irina Ch, Marie T et al Essential role for membrane lipid rafts in interleukin-1 induced nitric oxide release from insulin-secreting cells potential regulation by Caveolin-1. Diabetes,2005,54:2576–2585
[11] Muller G, Hanekop N, Wied S, Frick W. et al. Cholesterol depletion blocks redistribution of lipid raft components and insulin-mimetic signaling by glimepiride and phosphoinositolglycans in rat adipocytes. Mol Med 8: 120–136, 2002.
[12]Burant CF , Buse MG. Induced structural and functional changes in insulin receptors from rat skelatal muscle.Diabetes ,1990(48) ,236 :254.
[13]Changgui L , Guang N , Jialun C, et al.Expression of plasminogen activator inhibitor-1 gene by insulin resistant HepG2 cell line.Chin J Endocrinol Metab , 2000 , 16 (4),239-42.
[14]Couet J, Sargiacomo M, and Lisanti MP. Interaction of a receptor tyrosine kinase, EGF-R, with caveolins. Caveolin binding negatively regulates tyrosine and serine/threonine kinaseactivities. J Biol Chem ,2004,272: 30429–30438.
[15]Corely-Mastick C and Saltiel AR. Insulin-stimulated tyrosine phosphorylation of caveolin is specific for the differentiated adipocyte phenotype in 3T3-L1 cells. J Biol Chem 2005,272:20706–20714.
[16]Sergey VB, Kazimierz M, Marc G,et.al. Plasminogen Activator Inhibitor-1 Promotes Formation of Endothelial Microparticles With Procoagulant Potential. Circulation 2006;106;2372-2378.
[17] Ma GM, Halayko AJ, Stelmack GL, et al.Effects of oxidized and glycated low-density lipoproteins on transcription and secretion of plasminogen activator inhibitor-1 in vascular endothelial cells. Cardiovasc Pathol. 2006 Jan-Feb;15(1):3-10.
[18]Michael W,Mansfield J . Environment a land genetic factors in relation toelevated circulating l - evels of PAI - 1 in caucasian patients with type two diabetes. Thromb Haemost ,1997 ,79(7) :842 - 847.
[19] Hatanaka M ,Maeda T , Ikemoto T ,et al. Expression of caveolin-1 in human T cell leukemia cell lines[J ] . Biochem Biophys Res Comm ,1998 ,253 :382-387.
[20]Repetto S, Salani B, Maggi D, et al.Insulin and IGF-I phosphorylate eNOS in HUVECs by a caveolin-1 dependent mechanism. Biochem Biophys Res Commun. 2005 Nov 25;337(3):849-52.
[21]Alex WC,Babak B,Xiao BW, et al.Caveolin-1-deficient mice show insulin resistance and defective insulin receptor protein expression in adipose tissue. Am JPhysiol Cell Physiol 2003,285: C222–C235.
[22]Couet J, Sargiacomo M, Lisanti MP, et al. Interaction of a receptor tyrosine kinase, EGF-R, with caveolins. Caveolin binding negatively regulates tyrosine and serine/threonine kinaseactivities. J Biol Chem ,1997,272: 30429–30438.
[23]Corely-Mastick C ,Saltiel AR. et al. Insulin-stimulated tyrosine phosphorylation of caveolin is specific for the differentiated adipocyte phenotype in 3T3-L1 cells. J Biol Chem 1997,272:20706–20714.
[24]Eva G, Aaron N,Alison JL , et al.Small Interfering RNA-mediated Down-regulation of Caveolin-1 Differentially Modulates Signaling Pathways in Endothelial Cells.Biology Chemistry , 2004, 279, 40659–40669
[25]Cristina B, Eriksson P,Giovanna G,et al.Transcriptional Regulation of Plasminogen Activator Inhibitor Type 1 Gene by Insulin ,Insights Into the Signaling Pathway. Diabetes,2001,50:1522–1530.
[26] Parpal S, Karlsson M, Thorn H, and Stralfors P. Cholesterol depletion disrupts caveolae and insulin receptor signaling for metabolic control via insulin receptor substrate-1, but not for mitogen-activated protein kinase control. J Biol Chem 2001,276: 9670–9678.
[27] Joshi V, Kazuhiko H, Zhong-Zhou Y, et al. Identification and modulation of a caveolae dependent signal pathway that regulates plasminogen activator inhibitor-1 in insulin-resistant adipocytes. PNAS, Dec 2004; 101: 17120 – 17125.
[1] Benjamin Lewin. <
[2] Yukihide Tomari and Phillip D. Zamore .Perspective: machines for RNAi Genes & Dev. Mar 2005; 19: 517 - 529.
[3] 康慧聪 唐洲平(综述) 朱遂强(审校). RNAi 及其基因治疗研究进展[J]. 卒中与神经疾病,2005,第12 卷第3期:191-193,
[4] Michelle A. C, Zhenyu X, Michael Q. Zhang, and Gregory J. The Argonaute family: tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis Genes & Dev.Nov 2002; 16: 2733
[5] George L. Sen and Helen M. Blau A brief history of RNAi: the silence of the genes,FASEB J, Jul 2006; 20: 1293 - 1299
[6] Steven Bartz and Aimee L. Jackson how will RNAi facilitate drug development?Sci. STKE, Aug 2005; 2005: pe39
[7] 刘宝玉,孟昭力. RNAi技术在药物研究中的应用[J]. Food and Drug,第8卷第04A期, Jun.2006
[8] Steven D. Buckingham, Behrooz Esmaeili, Matthew Wood, and David B. Sattelle RNA interference: from model organisms towards therapy for neural and neuromuscular disorders Hum. Mol. Genet. Oct 2004; 13: R275 - R288.
[9] 汪光蓉 ,朱道银 ,唐恩洁. RNAi抗病毒感染的研究进展[J]. Jounal of North Sichuan Medical College, Vol 120,No 14 ,Dec. 2005.
[10] Bradley SP,Kowalik TF, Rastellini C, Successful incorporation of short-interfering RNA into islet cells by in situ perfusion.Transplant Proc. 2005 Jan-Feb;37(1):233-6
[11] Hagerkvist R, Mokhtari DMyers JW.siRNA produced by recombinant dicer mediates efficient gene silencing in islet cells.Ann N Y Acad Sci. 2005 Apr;1040:114-22.
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