重组pEGFP-N1-IGF-1体内转基因预处理对脊髓损伤后一氧化氮合酶活性的影响

Effects of pre-disposal treatment with pEGFP-N1-IGF-1 gene transfer on nitric oxide synthase activity after spinal cord injury in rats

目的:观察阳离子脂质体介导的重组pEGFP-N1-IGF-1体内转基因预处理对脊髓损伤后血清一氧化氮合酶及脊髓组织诱导型一氧化氮合酶影响,进一步探讨转基因治疗脊髓损伤的机制。方法:实验于2005-07/11在解放军第二军医大学动物实验中心完成。取54只雄性SD大鼠单纯随机分为3组:①生理盐水组(24只):采用脊髓内直接注射法,将2μL生理盐水+6μLTransfectamreagent共8μL注入T8～T10脊髓内,无下肢功能障碍者1周后按改良Nystr"m’s压迫法制作大鼠脊髓不完全损伤模型(压迫总质量35g,压迫时间5min)。②pEGFP-N1-IGF-1转基因组(24只):给药方法、部位、剂量以及造模方法与生理盐水组相同,但脊髓内注入6μLTransfectamreagent+2μg质粒pEGFP-N1-IGF-1。③正常组(6只):不损伤脊髓,不给药,作为正常对照。前2组损伤后1,4,8,24h、1,2周,取尾静脉血和损伤区域脊髓,测定血清一氧化氮合酶活性及局部脊髓组织诱导型一氧化氮合酶活性变化。结果:经补充后54只大鼠进入结果分析。①血清一氧化氮合酶活性:正常组为(305.4±52.0)μkat/L,其他2组在损伤后1～8h迅速升高,并于伤后24h达到高峰[pEGFP-N1-IGF-1转基因组:(522.6±60.3)μkat/L;生理盐水组:(757.0±83.7)μkat/L],其后逐渐降低,术后一两周仍维持在较高水平。pEGFP-N1-IGF-1转基因组各个时间点均低于生理盐水组(P<0.05,0.01)。②脊髓组织诱导型一氧化氮合酶活性:正常组为(72.5±17.3)μkat/g,其他2组在损伤后1～8h迅速升高,并于伤后24h达到高峰[pEGFP-N1-IGF-1转基因组:(140.2±24.2)μkat/g;生理盐水组:(207.2±36.8)μkat/g],其后逐渐降低,术后一两周仍维持在较高水平。pEGFP-N1-IGF-1转基因组各个时间点均低于生理盐水组(P<0.05,0.01)。结论:阳离子脂质体介导胰岛素样生长因子1基因转染预处理能够有效地下调大鼠一氧化氮合酶活性,特别是其可有效抑制诱导型一氧化氮合酶,从而减轻继发性损伤所致的神经组织的损害,侧面证明了胰岛素样生长因子1转基因治疗的可行性。

AIM： To study the effect of cationic liposome mediated pre-disposal treatment reconstructed pEGFP-N1-IGF-1 gene transfer and its potential mechanism for spinal cord injury （SCI） in vivo on nitric oxide synthase （NOS） and inducible NOS （iNOS） activity after acute SCI in rats. METHODS： The experiments were fulfilled on Shanghai animal experiment laboratory of the Second Military Medical University of Chinese PEA from July to November 2005. A total of 54 male SD rats were randomized into： （1）Contrel group （n =24）： A direct injection of 2 μL saline and 6 μL Transfectam reagent was given to T8-T10 spinal segments. One week later, those rats with no low limb disorder were adopted to establish the rat models of incomplete SCI by modified Nystroem＇s compression method （total weight of 35 g for 5 minutes）.（2）Experiment group （n =24）： A direct injection of 2 μg pEGFP-N1-IGF-1 and 6 μL Transfectam reagent was given identically with that in control group.（3）Normal group （n =6）： No SCI or administration was managed. In control group and experiment group, the activities of serum NOS and iNOS were detected at different time points （1, 4, 8, 24 hours, 1 week, 2 weeks after SCI） on the tail vein and injured spinal cord of rats. RESULTS： Totally 54 rats were involved in the result analysis.（1）The activity of NOS in serum raised quickly at 1-8 hours and peaked at 24 hours after SCI in control group and experiment group [normal group： （305.4±52.0） μkat/L; experiment group： （522.6±60.3） μkat/L; control group： （757.0±83.7） μkat/L] and then decreased to higher level at 1-2 weeks. There was significantly declined activities in experiment group compared with control group at different time points after SCI （P 〈 0.05, 0.01）. （2）The activity of iNOS in normal control spinal cord tissue raised quickly at 1-8 hours and peaked at 24 hours after SCI in experiment group and control group [normal group： （72.5±17.3） μkat/g; experiment group： （140.2±24.2） μkat/g; control group： （207.2±36.8） μkat/g] and then depressed to higher level at 1-2 weeks. There was significantly difference between experiment group and control group at different time points after SCI （P 〈 0.05, 0.01）. CONCLUSION： Pre-disposal treatment with PEGFP-N1-IGF-1 gene transfection medicated by cationic liposome can obviously down-regulate the activity of NOS, especially the expression of iNOS. The damage of nervous tissue caused by the secondary lesion can be reduced, proving the reliability of IGF-1 gene transfer therapy.