CAJ | 학술논문

目的观察移植转染胶质源性神经营养因子(GDNF)基因人羊膜细胞(HACs)对创伤性脑损伤(TBI)大鼠海马神经元的影响.方法采用改进Feeney法制作TBI致海马神经元损伤模型.TBI 24 h后于挫伤灶边缘移植,移植处距硬脑膜4 mm和2 mm深浅两点移植,共5μl含5×10~5个细胞.TBI+GDNF组移植转染GDNF基因HACs;TBI+eGFP组移植转染eGFP基因HACs;TBI+PBS组注射5μl PBS;假TBI组未行移植操作.移植后12 d Morris水迷宫检测结束后制片并行焦油紫染色,检测海马及移植针道靶点周围组织,取移植针道靶点周围组织行RT-PCR检测GDNFmRNA水平.结果免疫荧光法检测转染GDNF基因HACs荧光显微镜下呈红色荧光.TBI+GDNF组大鼠学习记忆功能明显好于TBI+eGFP组和TBI+PBS组,TBI+eGFP组和TBI+PBS组大鼠损伤侧海马神经元显著缺失,胞体缩小,深染.TBI+GDNF组部分海马神经元缺失.RT-PCR检测TBI+GDNF组GDNFmRNA高水平表达.结论移植转染GDNF基因HACs对TBI大鼠海马神经元有保护作用.
목적 관찰이식전염효질원성신경영양인자(GDNF)기인인양막세포(HACs)대창상성뇌손상(TBI)대서해마신경원적영향.방법 채용개진Feeney법제작TBI치해마신경원손상모형.TBI 24 h후우좌상조변연이식,이식처거경뇌막4 mm화2 mm심천량점이식,공5μl함5×10~5개세포.TBI+GDNF조이식전염GDNF기인HACs;TBI+eGFP조이식전염eGFP기인HACs;TBI+PBS조주사5μl PBS;가TBI조미행이식조작.이식후12 d Morris수미궁검측결속후제편병행초유자염색,검측해마급이식침도파점주위조직,취이식침도파점주위조직행RT-PCR검측GDNFmRNA수평.결과 면역형광법검측전염GDNF기인HACs형광현미경하정홍색형광.TBI+GDNF조대서학습기억공능명현호우TBI+eGFP조화TBI+PBS조,TBI+eGFP조화TBI+PBS조대서손상측해마신경원현저결실,포체축소,심염.TBI+GDNF조부분해마신경원결실.RT-PCR검측TBI+GDNF조GDNFmRNA고수평표체.결론 이식전염GDNF기인HACs대TBI대서해마신경원유보호작용.
Objective To investigate the effect of grafting transfecting GDNF gene human amniotic cells (HACs) on hippocampal neuron following traumatic brain injury (TBI) in rats. Methods The model of hippocampal neuronal death following TBI was built up by improved Feeney's weight drop techniques. 5 μl transfecting HACs were injected into the margin of contusion 24 h after injured by microsyringe and stereotactic frame. Groups consisted of transfecting GDNF gene HACs group(TBI + GDNF), transfecting eGFP gene HACs group(TBI +eGFP), PBS group (TBI + PBS)and sham TBI group. The spatial memory performance was evaluated on 5 consecutive days by Morris water maze on 7 days after transplantation. After the probe trial, the rats were sacrificed for hippocampal neuron and brain tissue around target morphological analysis by cresyl violet staining under light microscopy. Expression of GDNF was detected by PCR in mRNA level. Results Transfecting GDNF gene HACs showed red fluorescence by immunofluorescence. The rats of TBI + GDNF group exhibited less escape latencies than those of TBI + eGFP group and TBI + PBS group, but had longer escape latencies than those of sham TBI group. Cresyl violet staining showed hippocampal neuronal loss, shrinkage and dark staining of neurons in TBI + eGFP group and TBI + PBS group. Compared with other groups, GDNF expression of TBI + GDNF group obviously increased in mRNA leveL Conclusion Grafting HACs transfected by GDNF gene plays a role in protecting against hippocampal neuronal death following TBI.