中华外科杂志
中華外科雜誌
중화외과잡지
CHINESE JOURNAL OF SURGERY
2010年
20期
1573-1576
,共4页
慢病毒属%RNA干扰%脊髓损伤%Nogo受体
慢病毒屬%RNA榦擾%脊髓損傷%Nogo受體
만병독속%RNA간우%척수손상%Nogo수체
Lentivirus%RNA interference%Spinal cord injury%Nogo receptor
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.
目的 觀察慢病毒介導RNA榦擾大鼠神經元Nogo受體(NgR)基因錶達對脊髓損傷的脩複作用.方法 將前期實驗中構建好的針對NgR的特異性小榦擾RNA繫列siNgR199慢病毒重組體,按感染複數=3體外轉染已培養好的大鼠皮層神經元細胞.熒光電鏡觀察慢病毒重組體的轉染效率,採用實時熒光定量PCR檢測NgR基因沉默效率,驗證慢病毒重組體的有效性.建立大鼠重度脊髓損傷模型,大鼠分組後分彆于大腦皮層運動區註射生理鹽水、慢病毒重組體,採用BBB評分法觀測大鼠後肢運動恢複情況,採用神經示蹤法觀察脊髓損傷區神經纖維生長情況.結果 慢病毒重組體體外轉染神經元細胞的效率＞99%,NgR基因沉默效率為61%.大鼠模型給藥後8週,慢病毒重組體組大鼠脊髓損傷區可觀察到神經纖維生長通過,而生理鹽水組大鼠脊髓損傷區未見神經纖維生長通過,BBB評分結果提示慢病毒重組體組大鼠後肢運動功能恢複優于生理鹽水組(P＜0.01).結論 慢病毒siNgR199重組體能夠促進脊髓損傷區神經纖維的生長,在一定程度上促進脊髓損傷後大鼠後肢運動功能恢複.
목적 관찰만병독개도RNA간우대서신경원Nogo수체(NgR)기인표체대척수손상적수복작용.방법 장전기실험중구건호적침대NgR적특이성소간우RNA계렬siNgR199만병독중조체,안감염복수=3체외전염이배양호적대서피층신경원세포.형광전경관찰만병독중조체적전염효솔,채용실시형광정량PCR검측NgR기인침묵효솔,험증만병독중조체적유효성.건립대서중도척수손상모형,대서분조후분별우대뇌피층운동구주사생리염수、만병독중조체,채용BBB평분법관측대서후지운동회복정황,채용신경시종법관찰척수손상구신경섬유생장정황.결과 만병독중조체체외전염신경원세포적효솔＞99%,NgR기인침묵효솔위61%.대서모형급약후8주,만병독중조체조대서척수손상구가관찰도신경섬유생장통과,이생리염수조대서척수손상구미견신경섬유생장통과,BBB평분결과제시만병독중조체조대서후지운동공능회복우우생리염수조(P＜0.01).결론 만병독siNgR199중조체능구촉진척수손상구신경섬유적생장,재일정정도상촉진척수손상후대서후지운동공능회복.
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 transfected 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.
