脂质体介导胶质细胞生长因子2基因治疗颅脑损伤的实验研究

Liposome-mediated glial growth factor 2 gene therapy in brain injury:an experimental study with rats

目的探讨胶质细胞生长因子2(GGF2)对大鼠液压打击颅脑损伤的神经保护作用。方法采用侧方液压打击装置制备大鼠颅脑损伤模型。将 pEGFP-N1-GGF2表达质粒或 pEGFP-N1载体质粒与阳离子脂质体混合后,脑内直接注射转染大鼠脑组织。将34大鼠随机分为4组,分别为治疗组(pEGFP-N1-GGF2+脂质体,n=10)、载体对照组(pEGFP-N1载体+脂质体,n=10)、脂质体对照组(脂质体,n=10)和假手术组(n=4)。伤后连续观察爬坡、平衡和行走试验,于伤后第10天处死大鼠取脑组织,进行 HE、尼氏及 MBP、NSE、GFAP 免疫组化染色。结果伤后第5d,治疗组大鼠行为学指标的恢复优于载体对照组和脂质体对照组[爬坡试验(角度):66.25±3.54 vs 58.31±3.72、57.21±3.93,P<0.05;平衡试验(评分):2.59±0.21 vs 3.41±0.25、3.24±0.22,P<0.05;行走试验(s):20.15±2.59 vs 27.00±3.47、27.80±3.00,P<0.05],以行走实验改善最为明显。伤后第10天,治疗组大鼠的皮层、海马神经元计数多于载体对照组和脂质体对照组(皮层外颗粒和外锥体层:98±10 vs 75±7、67±8,P<0.05;皮层内锥体层:37±4 vs 19±3、23±4,P<0.05;海马 CA1区:102±11 vs67±8、58±9,P<0.01),皮层、皮层下白质 MBP 染色信号强于对照组。结论阳离子脂质体介导GGF2基因治疗能有效地促进大鼠颅脑损伤后的恢复。

Objective To explore the protective effect of glial growth factor-2 （GGF2） on brain injury. Methods Thirty-four SD rats underwent lateral fluid percussion to establish brain injury models and then were randomly divided into 4 groups： treatment group （n = 10, the plasmid pEGFP-N1-GGF2 mixed with liposome was injected into the brain tissue directly）, vector control group （n = 10, the vector pEGFP- N1 mixed with liposome was injected into the brain tissue directly） , . liposome control group （n = 10, liposome was injected）, and sham operation group （ n = 4）. Three assessment tasks were performed for neurobehavioral evaluation： Clivas Test, Beam Balance Test and Beam Walking Test. 10 days after brain injury, the rats were sacrificed and their brains were embedded in paraffin for HE staining, Nissle staining and immunohistochemical examination of MBP, NSE, and GFAP. Results The Clivas test score of the treatment group was 66.25 ± 3.54, significantly higher than those of the vector control group and . liposome control group （58.31 ± 3.72 and 57.21 ± 3.93 respectively,both P 〈 0.05）. The beam test score of the treatment group was 2.59 ± 0.21, significantly lower than those the vector control group and . liposome control group （3.41 ±0.25 and 3.24 ±0.22 respectively, both P 〈0.05）. The walking test score of the treatment group was 20. 15 ± 2.59, significantly lower than those of control group and . liposome control group （27.00 ± 3.47 and 27.80 ± 3.00 respectively, both P 〈 0.05 ）. The improvement in beam walking test was the greatest. The neuron number in the external granular layer and external pyramidal layer in cortex of the treatment group was 98 ± 10, significantly more than those of the vector control group and liposome group （ 75 ±7 and 67±8,both P〈0.05）.The neuron number in the internal pyramidal layer in cortex of the treatment group was 37±4,significantly more than those of the vector control group and liposome group （19±3 and 23 ± 4 respectively,both P〈0.05）.The neuron number in the CAI region in hippocampus of the treatment group was 102 ± 11,significantly more than those of the vector control group and liposme group （67±8 and 58±9 respectively,both P〈0.01）.Higher level of immunoreactivity with MBP was also delected in the cortex in the rats of the treatment group.Conclusion Cationic liposme-mediated GGF2 gene therapy effectively promotes the recovery of brain injury.