基因转染人羊膜细胞对颅脑创伤大鼠海马的作用

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.

The effects of human amniotic cells transfected with GDNF gene on hippocampal neuron following traumatic brain injury in rats

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.