• 1.佳木斯大學(xué)附屬第一醫(yī)院普外一科(黑龍江佳木斯154003);;
  • 2.中國(guó)醫(yī)科大學(xué)附屬第一醫(yī)院血管外科(遼寧沈陽(yáng)110001);

目的探討早期生長(zhǎng)反應(yīng)基因-1(Egr-1)DNA酶(Egr-1 DNA enzyme，EDRz)對(duì)血管平滑肌細(xì)胞(VSMC)增殖和內(nèi)膜增生的抑制作用，從而證實(shí)基因治療靜脈移植后狹窄、閉塞的可行性。方法構(gòu)建EDRz，建立自體靜脈移植模型，將大鼠右頸總靜脈端-端吻合于腎下腹主動(dòng)脈，EDRz轉(zhuǎn)染移植靜脈，分別于移植后1、2、6、24 h及3、7、14、28、42 d切取移植靜脈標(biāo)本，每個(gè)時(shí)相按隨機(jī)數(shù)字表法隨機(jī)抽取10只大鼠。熒光顯微鏡下觀察EDRz轉(zhuǎn)染移植靜脈情況; 應(yīng)用原位雜交和RT-PCR方法檢測(cè)Egr-1 mRNA的表達(dá); 應(yīng)用Western蛋白印跡和免疫組織化學(xué)方法檢測(cè)Egr-1蛋白表達(dá)情況; HE染色光鏡下觀察組織學(xué)形態(tài)。結(jié)果①EDRz轉(zhuǎn)染移植靜脈情況: 移植后1 h時(shí)，EDRz主要位于移植靜脈的外膜、中膜和部分內(nèi)皮細(xì)胞; 2、6及24 h時(shí)，EDRz則主要位于移植靜脈的中膜; 7 d時(shí)，EDRz主要位于移植靜脈的內(nèi)膜; 14、28及42 d時(shí)未檢測(cè)到EDRz的表達(dá)。②Egr-1 mRNA表達(dá)結(jié)果。RT-PCR檢測(cè)結(jié)果: 轉(zhuǎn)染EDRz后1 h時(shí)，Egr-1 mRNA表達(dá)出現(xiàn)高峰，2、6及24 h表達(dá)下降，3 d時(shí)表達(dá)微弱，移植后7、14、28及42 d未見(jiàn)Egr-1 mRNA的表達(dá)，轉(zhuǎn)染EDRz后1 h時(shí)Egr-1 mRNA表達(dá)明顯高于其余各時(shí)相(P lt;0.01)。原位雜交檢測(cè)Egr-1 mRNA表達(dá)的變化趨勢(shì)與RT-PCR結(jié)果基本一致。③Egr-1蛋白表達(dá)結(jié)果。Western蛋白印跡結(jié)果: 正常靜脈中未檢測(cè)到Egr-1蛋白陽(yáng)性表達(dá)。轉(zhuǎn)染EDRz后2 h時(shí)，出現(xiàn)Egr-1蛋白陽(yáng)性表達(dá)，6 h、24 h及3 d時(shí)其表達(dá)逐漸降低，移植后1 h時(shí)和移植后7、14、28及42 d時(shí)未見(jiàn)Egr-1蛋白陽(yáng)性表達(dá)。移植后2 h時(shí)的Egr-1蛋白表達(dá)的吸光度值高于其他時(shí)相(P lt;0.01)。免疫組織化學(xué)方法檢測(cè)的Egr-1蛋白陽(yáng)性表達(dá)的變化趨勢(shì)與Western蛋白印跡結(jié)果基本一致。④EDRz轉(zhuǎn)染移植靜脈與未轉(zhuǎn)染同期相比VSMC的增殖程度和內(nèi)膜厚度明顯減輕。結(jié)論EDRz通過(guò)減少Egr-1在自體移植靜脈中的表達(dá)，可有效地抑制自體移植靜脈中VSMC增殖和內(nèi)膜增生，可用來(lái)防治自體靜脈移植后所導(dǎo)致的血管狹窄、閉塞。

引用本文： 劉程偉,張雪松,王石,田浩,程明勛,王樹(shù)卿,胡新華,張強(qiáng),辛世杰,段志泉. Egr-1 DNA酶抑制自體移植靜脈內(nèi)膜增生的實(shí)驗(yàn)研究. 中國(guó)普外基礎(chǔ)與臨床雜志, 2011, 18(10): 1043-1048. doi: 復(fù)制

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1. Paterson BM, Roberts BE, Kuff EL. Structural gene identification and mapping by DNAmRNA hybridarrested cellfree translation [J]. Proc Natl Acad Sci USA, 1977, 74(10): 43704374.
2. Stephenson ML, Zamecnik PC. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide [J]. Proc Natl Acad Sci USA, 1978, 75(1): 285288.
3. Pavri R, Gazumyan A, Jankovic M, et al. Activationinduced cytidine deaminase targets DNA at sites of RNA polymerase Ⅱ stalling by interaction with Spt5 [J]. Cell, 2010, 143(1): 122133.
4. 劉程偉, 胡新華, 張雪松, 等. Egr1在自體移植靜脈中的表達(dá)及意義 [J]. 中國(guó)普外基礎(chǔ)與臨床雜志, 2006, 13(6): 642646.
5. Breaker RR, Joyce GF. A DNA enzyme that cleaves RNA [J]. Chem Biol, 1994, 1(4): 223229.
6. Tell G, Wilson DM, Lee CH. Intrusion of a DNA repair protein in the rnome world: is this the beginning of a new era? [J]. Mol Cell Biol, 2010, 30(2): 366371.
7. Lam CH, Perrin DM. Introduction of guanidiniummodified deoxyuridine into the substrate binding regions of dnazyme 10－23 to enhance target affinity: implications for dnazyme design [J]. Bioorg Med Chem Lett, 2010, 20(17): 51195122.
8. Li J, Wang N, Luo Q, et al. The 10－23 DNA enzyme generated by a novel expression vector mediate inhibition of taco expression in macrophage [J]. Oligonucleotides, 2010, 20(2): 6168.
9. Maeda J, Nishida M, Takikawa H, et al. Inhibitory effects of sulfobacin B on DNA polymerase and inflammation [J]. Int J Mol Med, 2010, 26(5): 751758.
10. Patel N, Kalra VK. Placenta growth factorinduced early growth response 1 (Egr1) regulates hypoxiainducible factor1alpha (HIF1alpha) in endothelial cells [J]. J Biol Chem, 2010, 285(27): 2057020579.
11. Mckinney KA, AlRawi N, Maciag PC, et al. Effect of a novel DNA vaccine on angiogenesis and tumor growth in vivo [J]. Arch Otolaryngol Head Neck Surg, 2010, 136(9): 859864.
12. Lockney D, Franzen S, Lommel S. Viruses as nanomaterials for drug delivery [J]. Methods Mol Biol, 2011, 726: 207221.
13. Yu HW, Liu QF, Liu GN. Positive regulation of the Egr1/osteopontin positive feedback loop in rat vascular smooth muscle cells by TGFbeta, ERK, JNK, and p38 MAPK signaling [J]. Biochem Biophys Res Commun, 2010, 396(2): 451456.
14. Wang B, Cao L, Chiuman W, et al. Probing the function of nucleotides in the catalytic cores of the 8-17 and 10-23 DNAzymes by abasic nucleotide and C3 spacer substitutions [J]. Biochemistry, 2010, 49(35): 75537562.
15. Deindl E, Fischer S, Preissner KT. New directions in inflammation and immunity: the multifunctional role of the extracellular RNA/RNase system [J]. Indian J Biochem Biophys, 2009, 46(6): 461466.
16. Ni J, Waldman A, Khachigian LM. cJun regulates shear and injuryinducible Egr1 expression, vein graft stenosis after autologous endtoside transplantation in rabbits, and intimal hyperplasia in human saphenous veins [J]. J Biol Chem, 2010, 285(6): 40384048.
17. Robaldo L, Montserrat JM, Iribarren AM. 10-23 DNA zyme modified with (2′R)and (2′S)2′deoxy2′Cmethyluridine in the catalytic core [J]. Bioorg Med Chem Lett, 2010, 20(15): 43674370.
18. Dyson OF, Traylen CM, Akula SM. Cell membranebound Kaposi’s sarcomaassociated herpesvirusencoded glycoprotein B promotes virus latency by regulating expression of cellular Egr1 [J]. J Biol Chem, 2010, 285(48): 3749137502.
19. Liu GN, Teng YX, Yan W. Transfected synthetic DNA enzyme gene specifically inhibits Egr1 gene expression and reduces neointimal hyperplasia following balloon injury in rats [J]. Int J Cardiol, 2008, 129(1): 118124.
20. 楊軍, 胡新華, 劉程偉, 等. 反義寡脫氧核苷酸抑制survivin基因表達(dá)對(duì)移植靜脈內(nèi)膜增生的影響 [J]. 中國(guó)普外基礎(chǔ)與臨床雜志, 2006, 13(6): 647652.
  1. 1. Paterson BM, Roberts BE, Kuff EL. Structural gene identification and mapping by DNAmRNA hybridarrested cellfree translation [J]. Proc Natl Acad Sci USA, 1977, 74(10): 43704374.
  2. 2. Stephenson ML, Zamecnik PC. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide [J]. Proc Natl Acad Sci USA, 1978, 75(1): 285288.
  3. 3. Pavri R, Gazumyan A, Jankovic M, et al. Activationinduced cytidine deaminase targets DNA at sites of RNA polymerase Ⅱ stalling by interaction with Spt5 [J]. Cell, 2010, 143(1): 122133.
  4. 4. 劉程偉, 胡新華, 張雪松, 等. Egr1在自體移植靜脈中的表達(dá)及意義 [J]. 中國(guó)普外基礎(chǔ)與臨床雜志, 2006, 13(6): 642646.
  5. 5. Breaker RR, Joyce GF. A DNA enzyme that cleaves RNA [J]. Chem Biol, 1994, 1(4): 223229.
  6. 6. Tell G, Wilson DM, Lee CH. Intrusion of a DNA repair protein in the rnome world: is this the beginning of a new era? [J]. Mol Cell Biol, 2010, 30(2): 366371.
  7. 7. Lam CH, Perrin DM. Introduction of guanidiniummodified deoxyuridine into the substrate binding regions of dnazyme 10－23 to enhance target affinity: implications for dnazyme design [J]. Bioorg Med Chem Lett, 2010, 20(17): 51195122.
  8. 8. Li J, Wang N, Luo Q, et al. The 10－23 DNA enzyme generated by a novel expression vector mediate inhibition of taco expression in macrophage [J]. Oligonucleotides, 2010, 20(2): 6168.
  9. 9. Maeda J, Nishida M, Takikawa H, et al. Inhibitory effects of sulfobacin B on DNA polymerase and inflammation [J]. Int J Mol Med, 2010, 26(5): 751758.
  10. 10. Patel N, Kalra VK. Placenta growth factorinduced early growth response 1 (Egr1) regulates hypoxiainducible factor1alpha (HIF1alpha) in endothelial cells [J]. J Biol Chem, 2010, 285(27): 2057020579.
  11. 11. Mckinney KA, AlRawi N, Maciag PC, et al. Effect of a novel DNA vaccine on angiogenesis and tumor growth in vivo [J]. Arch Otolaryngol Head Neck Surg, 2010, 136(9): 859864.
  12. 12. Lockney D, Franzen S, Lommel S. Viruses as nanomaterials for drug delivery [J]. Methods Mol Biol, 2011, 726: 207221.
  13. 13. Yu HW, Liu QF, Liu GN. Positive regulation of the Egr1/osteopontin positive feedback loop in rat vascular smooth muscle cells by TGFbeta, ERK, JNK, and p38 MAPK signaling [J]. Biochem Biophys Res Commun, 2010, 396(2): 451456.
  14. 14. Wang B, Cao L, Chiuman W, et al. Probing the function of nucleotides in the catalytic cores of the 8-17 and 10-23 DNAzymes by abasic nucleotide and C3 spacer substitutions [J]. Biochemistry, 2010, 49(35): 75537562.
  15. 15. Deindl E, Fischer S, Preissner KT. New directions in inflammation and immunity: the multifunctional role of the extracellular RNA/RNase system [J]. Indian J Biochem Biophys, 2009, 46(6): 461466.
  16. 16. Ni J, Waldman A, Khachigian LM. cJun regulates shear and injuryinducible Egr1 expression, vein graft stenosis after autologous endtoside transplantation in rabbits, and intimal hyperplasia in human saphenous veins [J]. J Biol Chem, 2010, 285(6): 40384048.
  17. 17. Robaldo L, Montserrat JM, Iribarren AM. 10-23 DNA zyme modified with (2′R)and (2′S)2′deoxy2′Cmethyluridine in the catalytic core [J]. Bioorg Med Chem Lett, 2010, 20(15): 43674370.
  18. 18. Dyson OF, Traylen CM, Akula SM. Cell membranebound Kaposi’s sarcomaassociated herpesvirusencoded glycoprotein B promotes virus latency by regulating expression of cellular Egr1 [J]. J Biol Chem, 2010, 285(48): 3749137502.
  19. 19. Liu GN, Teng YX, Yan W. Transfected synthetic DNA enzyme gene specifically inhibits Egr1 gene expression and reduces neointimal hyperplasia following balloon injury in rats [J]. Int J Cardiol, 2008, 129(1): 118124.
  20. 20. 楊軍, 胡新華, 劉程偉, 等. 反義寡脫氧核苷酸抑制survivin基因表達(dá)對(duì)移植靜脈內(nèi)膜增生的影響 [J]. 中國(guó)普外基礎(chǔ)與臨床雜志, 2006, 13(6): 647652.