慢病毒介导RNA干扰Nogo受体基因治疗脊髓损伤的实验研究  

Lentiviral vector-mediated RNA interfere gene Nogo receptor to repair spinal cord injury

作　　者：吕碧涛[1] 袁文[1] 徐盛明[1] 

机构地区：[1]第二军医大学附属长征医院骨科,上海200003

出　　处：《中华外科杂志》2010年第20期1573-1576,共4页Chinese Journal of Surgery

基　　金：国家自然科学基金资助项目（30471757）

摘　　要：目的观察慢病毒介导RNA干扰大鼠神经元Nogo受体（NgR）基因表达对脊髓损伤的修复作用。方法将前期实验中构建好的针对NgR的特异性小干扰RNA系列siNgR199慢病毒重组体，按感染复数=3体外转染已培养好的大鼠皮层神经元细胞。荧光电镜观察慢病毒重组体的转染效率，采用实时荧光定量PCR检测NgR基因沉默效率，验证慢病毒重组体的有效性。建立大鼠重度脊髓损伤模型，大鼠分组后分别于大脑皮层运动区注射生理盐水、慢病毒重组体，采用BBB评分法观测大鼠后肢运动恢复情况，采用神经示踪法观察脊髓损伤区神经纤维生长情况。结果慢病毒重组体体外转染神经元细胞的效率〉99％，NgR基因沉默效率为61％。大鼠模型给药后8周，慢病毒重组体组大鼠脊髓损伤区可观察到神经纤维生长通过，而生理盐水组大鼠脊髓损伤区未见神经纤维生长通过，BBB评分结果提示慢病毒重组体组大鼠后肢运动功能恢复优于生理盐水组（P〈0．01）。结论慢病毒siNgR199重组体能够促进脊髓损伤区神经纤维的生长，在一定程度上促进脊髓损伤后大鼠后肢运动功能恢复。Objective To evaluate the effects of lentiviral vector-mediated RNA interfere gene Nogo receptor（ NgR）of rat cortical neurons in repairing spinal cord injury. Methods The recombinant-lentiviral vector with small inferring RNA siNgR199 which had been constructed was transfeeted into rat cortical neuron cells in vitro in 3 multiplicity of infection（MOI）. The infection rate was determined with fluorescent microscope, and the target gene was detected by PCR analysis. Then, the recombinant was injected into the cortical motor area of the rats with severe spinal cord injury, and the saline was also injected into other rats with severe spinal cord injury as a match control. The functional recovery of the rats＇ hindlimb was assessed using BBB score and the nerve fiber of the injured region was observed by nerve tracing. Results The rate of recombinant infecting rat cortical neuron in vitro exceeded 99%. PCR analysis confirmed that the effect of lentiviral vector-mediated RNA interfereing gene NgR of rat cortical neurons in vitro was 61%. Although all rats with spinal cord injury were observed to have the hindlimb functional recovery, these rats injected with recombinant had better hindlimb functional recovery than others showing by more BBB score （ P 〈 0. 01 ）. Moreover, it was found that some nerve fiber passed the injured spinal cord region of the rats which were injected with recombinant. Conclusion The recombinant lentiviral vector with siNgR199 which had been constructed is able to promote the growth of nerve fiber and the functional recovery of the rats＇ hindlimb.

关 键 词：慢病毒属 RNA干扰 脊髓损伤 NOGO受体 

分 类 号：R686[医药卫生—骨科学]