Poloxamer 407-胰蛋白酶混合凝胶提高腺病毒转染移植静脉组织效率的研究
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摘要
安全高效地将治疗基因导入移植静脉组织是移植静脉再狭窄基因治疗研究的重要内容。该研究探讨应用Poloxamer 407-胰蛋白酶混合凝胶局部转染提高移植静脉的腺病毒转染效率的可行性。构建大鼠移植静脉再狭窄模型,应用Poloxamer 407-胰蛋白酶混合凝胶涂染法,通过增加腺病毒载体与血管组织的接触时间以及改善血管壁的渗透性,提高移植静脉的腺病毒转染效率。分别在术后7、14、28天采集标本,应用冰冻切片、免疫组化染色和qRT-PCR检测移植静脉中EGFP的表达以评估转染效率。Poloxamer 407-胰蛋白酶混合凝胶局部转染能够显著提高移植静脉的腺病毒转染效率,其中0.25%的胰蛋白酶转染效率最佳,并且不影响移植静脉血管的组织抗拉性。Poloxamer 407-胰蛋白酶混合凝胶涂染是一种简单、安全和高效的局部基因转染方法,可用于移植静脉再狭窄的防治研究。
Safe and effective vein grafts gene transfer remains elusive in vascular gene therapy. The aim of the present study was to investigate whether poloxamer 407-trypsin gel enhances the efficiency of adenovirus-mediated locally vein grafts gene transfection in vivo. Rat model of vein graft restenosis was established interposition bypass grafting from the autologous jugular vein to the carotid artery. We applied recombinant adenovirus encoding the reporter gene EGFP directly onto vein grafts with poloxamer 407 gel to increase virus contact time, and mild trypsinization to increase virus penetration. The expression of EGFP in vein grafts walls was determined by frozen section, qRT-PCR and immunohistochemistry staining staining at 7, 14 and 28 days after vein graft surgery. Structural integrity of the tissue was evaluated by measurement of tissue tensile strength. Transfection efficiency was significantly higher in vein grafts in poloxamer 407 gel contained varying concentrations of trypsin group versus control group. Trypsin at a concentration of 0.25% allowed marginally better penetration, and tissue tensile strength was not affected. These findings suggest that poloxamer 407-trypsin gel may be a safe and effective method to improve the efficiency of adenovirusmediated vein grafts gene transfer.
Safe and effective vein grafts gene transfer remains elusive in vascular gene therapy. The aim of the present study was to investigate whether poloxamer 407-trypsin gel enhances the efficiency of adenovirus-mediated locally vein grafts gene transfection in vivo. Rat model of vein graft restenosis was established interposition bypass grafting from the autologous jugular vein to the carotid artery. We applied recombinant adenovirus encoding the reporter gene EGFP directly onto vein grafts with poloxamer 407 gel to increase virus contact time, and mild trypsinization to increase virus penetration. The expression of EGFP in vein grafts walls was determined by frozen section, qRT-PCR and immunohistochemistry staining staining at 7, 14 and 28 days after vein graft surgery. Structural integrity of the tissue was evaluated by measurement of tissue tensile strength. Transfection efficiency was significantly higher in vein grafts in poloxamer 407 gel contained varying concentrations of trypsin group versus control group. Trypsin at a concentration of 0.25% allowed marginally better penetration, and tissue tensile strength was not affected. These findings suggest that poloxamer 407-trypsin gel may be a safe and effective method to improve the efficiency of adenovirusmediated vein grafts gene transfer.
引文
1 Luo J,Deng ZL,Luo X,Tang N,Song WX,Chen J,et al.A protocol for rapid generation of recombinant adenoviruses using the AdEasy system.Nat Protoc 2007;2(5):1236-47.
2 Wang XW,He XJ,Lee KC,Huang C,Hu JB,Zhou R,et al.Micro RNA-221 sponge therapy attenuates neointimal hyperplasia and improves blood flows in vein grafts.Int J Cardiol 2016;208:79-86.
3 Feldman LJ,Pastore CJ,Aubailly N,Kearney M,Chen D,Perricaudet M,et al.Improved efficiency of arterial gene transfer by use of poloxamer 407 as a vehicle for adenoviral vectors.Gene Ther 1997;4(3):189-98.
4 Kabanov AV,Batrakova EV,Alakhov VY.Pluronic block copolymers as novel polymer therapeutics for drug and gene delivery.JControl Release 2002;82(2/3):189-212.
5 March KL,Madison JE,Trapnell BC.Pharmacokinetics of adenoviral vector-mediated gene delivery to vascular smooth muscle cells:modulation by poloxamer 407 and implications for cardiovascular gene therapy.Hum Gene Ther 1995;6(1):41-53.
6 Fromes Y,Salmon A,Wang X,Collin H,Rouche A,Hagege A,et al.Gene delivery to the myocardium by intrapericardial injection.Gene Ther 1999;6(4):683-8.
7 Wan S,George SJ,Nicklin SA,Yim AP,Baker AH.Overexpression of p53 increases lumen size and blocks neointima formation in porcine interposition vein grafts.Mol Ther 2004;9(5):689-98.
8 West NE,Qian H,Guzik TJ,Black E,Cai S,George SE,et al.Nitric oxide synthase(nNOS)gene transfer modifies venous bypass graft remodeling:effects on vascular smooth muscle cell differentiation and superoxide production.Circulation 2001;104(13):1526-32.
9 Cooney R,Hynes SO,Sharif F,Howard L,O’Brien T.Effect of gene delivery of NOS isoforms on intimal hyperplasia and endothelial regeneration after balloon injury.Gene Ther 2007;14(5):396-404.
10陶登顺,张仁福,王辉山,汪曾炜,朱洪玉,谭丽丽,等.内皮型一氧化氮合酶基因转染在兔颈静脉移植血管桥中的表达.第四军医大学学报(Tao Dengshun,Zhang Renfu,Wang Huishan,Wang Zengwei,Zhu Hongyu,Tan Lili,et al.Expression of transfected endothelialnitric oxide synthase transfer on carotid vein graft in rabbits.J Fourth Mil Univ)2005;26:591-3.
11 Zhou X,Xiao Y,Mao Z,Huang J,Geng Q,Wang W,et al.Soluble Jagged-1 inhibits restenosis of vein graft by attenuating Notch signaling.Microvasc Res 2015;100:9-16.
12 Xiang S,Liu J,Dong N,Shi J,Xiao Y,Wang Y,et al.Suppressor of cytokine signaling 3 is a negative regulator for neointimal hyperplasia of vein graft stenosis.J Vasc Res 2014;51(2):132-43.
2 Wang XW,He XJ,Lee KC,Huang C,Hu JB,Zhou R,et al.Micro RNA-221 sponge therapy attenuates neointimal hyperplasia and improves blood flows in vein grafts.Int J Cardiol 2016;208:79-86.
3 Feldman LJ,Pastore CJ,Aubailly N,Kearney M,Chen D,Perricaudet M,et al.Improved efficiency of arterial gene transfer by use of poloxamer 407 as a vehicle for adenoviral vectors.Gene Ther 1997;4(3):189-98.
4 Kabanov AV,Batrakova EV,Alakhov VY.Pluronic block copolymers as novel polymer therapeutics for drug and gene delivery.JControl Release 2002;82(2/3):189-212.
5 March KL,Madison JE,Trapnell BC.Pharmacokinetics of adenoviral vector-mediated gene delivery to vascular smooth muscle cells:modulation by poloxamer 407 and implications for cardiovascular gene therapy.Hum Gene Ther 1995;6(1):41-53.
6 Fromes Y,Salmon A,Wang X,Collin H,Rouche A,Hagege A,et al.Gene delivery to the myocardium by intrapericardial injection.Gene Ther 1999;6(4):683-8.
7 Wan S,George SJ,Nicklin SA,Yim AP,Baker AH.Overexpression of p53 increases lumen size and blocks neointima formation in porcine interposition vein grafts.Mol Ther 2004;9(5):689-98.
8 West NE,Qian H,Guzik TJ,Black E,Cai S,George SE,et al.Nitric oxide synthase(nNOS)gene transfer modifies venous bypass graft remodeling:effects on vascular smooth muscle cell differentiation and superoxide production.Circulation 2001;104(13):1526-32.
9 Cooney R,Hynes SO,Sharif F,Howard L,O’Brien T.Effect of gene delivery of NOS isoforms on intimal hyperplasia and endothelial regeneration after balloon injury.Gene Ther 2007;14(5):396-404.
10陶登顺,张仁福,王辉山,汪曾炜,朱洪玉,谭丽丽,等.内皮型一氧化氮合酶基因转染在兔颈静脉移植血管桥中的表达.第四军医大学学报(Tao Dengshun,Zhang Renfu,Wang Huishan,Wang Zengwei,Zhu Hongyu,Tan Lili,et al.Expression of transfected endothelialnitric oxide synthase transfer on carotid vein graft in rabbits.J Fourth Mil Univ)2005;26:591-3.
11 Zhou X,Xiao Y,Mao Z,Huang J,Geng Q,Wang W,et al.Soluble Jagged-1 inhibits restenosis of vein graft by attenuating Notch signaling.Microvasc Res 2015;100:9-16.
12 Xiang S,Liu J,Dong N,Shi J,Xiao Y,Wang Y,et al.Suppressor of cytokine signaling 3 is a negative regulator for neointimal hyperplasia of vein graft stenosis.J Vasc Res 2014;51(2):132-43.