胶质细胞源性神经营养因子体内转基因对皮质脊髓束再生的影响

The experimental study of liposome-mediated glial cell line-derived neurotrophic factor gene transfer in vivo on cortical spinal tract regenera tion after spinal cord injury in adult rats

目的 通过非病毒载体阳离子脂质体介导 ,探讨胶质细胞源性神经营养因子(GDNF)基因体内转染对成年大鼠脊髓损伤后皮质脊髓束再生的影响。方法 采用NystrOm法制备大鼠胸髓后路压迫损伤模型 ,压迫重量为 5 0 g ,时间为 5min ,造成大鼠胸段脊髓急性重度损伤。用微量注射器将阳离子脂质体DC Chol 6μl和重组质粒pEGFP GDNFcDNA 2 μg混合后注入大鼠损伤脊髓。应用逆转录 聚合酶链反应 (RT PCR)技术和荧光显微镜检测GDNF体内转基因后在局部的表达 ,通过辣根过氧化物酶 (HRP)顺行追踪技术和神经微丝 (NF)免疫组织化学活性的变化来评价GDNF基因体内转染对大鼠皮质脊髓束再生的影响。结果 GDNF基因体内转染 1周后显示在基因注射局部有转录和蛋白水平表达。脊髓损伤后 4周在损伤区可见较多的皮质脊髓束再生 ,损伤区NF阳性轴突数 ( 5 2 4.33± 80 .5 5 ) /低倍视野较对照组 ( 30 9.84± 5 6.65 ) /低倍视野明显增多( P <0 .0 1)。结论 阳离子脂质体介导GDNF体内转基因能促进近端皮质脊髓束再生和神经骨架蛋白的修复。

Objective To study the effect of cation ic liposome-mediated glial cell line-derived neurotrophic factor (GDNF) gene tra nsfer in vivo on the regeneration of corticospinal tract in spinal cord injured adult rats.Methods The model of acute spinal cord compression injury in rats was established according to the method of Nystr m.The compressive loa d was 50g for 5min,causing severe spinal cord injury.6μl recombinant pla smid pEGFP-GDNF and 2μg DC-Chol liposome complexes were injected into the i njured spinal cord by microsyringe.RT-PCR and fluorescence observation confirme d the presence of expression of GDNF mRNA and GDNF protein for 1 week after inje ction.Using the technique of HRP anterograde tracing and the immunohistochemical reactivity of NF in the injured spinal cord,the effect of GDNF in vivo gene tr ansfer on corticospianl tract regeneration was observed.Results One week after GDNF gene transfer,the expression of GDNF in the level of transcription and protein was increased in the injected area.Long-distance regeneration of corticospinal tract was observed in the inj ured area 4 weeks after spinal cord injury.The number of NF-positive axons in t he GDNF group (524.33±80.55/low power field)was more than that in the contr ol group (309.84±56.65/low power field)(P<(0.01).)Conclusion Cationic liposome-mediated GDNF cDNA in vivo transfer can promote regeneration of corticospinal tract and facilitate repair o f the cytoskeleton in the injured neurons.