移植胶质细胞源性神经营养因子工程细胞至侧脑室治疗血管性痴呆大鼠的实验研究

Experimental research on the effect of lateral ventricle transplantation of neurotrophic factor-transfected cells derived from Glia cell line on vascular dementia in rats

目的研究胶质细胞源性神经营养因子（GDNF）基因转导的细胞侧脑室移植对血管性痴呆大鼠学习、记忆功能的影响及对大鼠海马区发育调节脑蛋白（Drebrin）表达的影响。方法取104只健康雄性SD大鼠，随机分为移植组、注射组、对照组和假手术组。移植组、注射组及对照组采用左侧颈总动脉结扎加右侧颈总动脉反复夹闭的方法建立血管性痴呆大鼠模型，假手术组仅分离而不夹闭双侧颈总动脉。每组取6只大鼠在移植治疗后3d、7d及10d处死，用于观测荧光强度。每组20只大鼠在移植治疗3d后采用Morris水迷宫法检测大鼠的学习记忆功能；治疗4d后处死大鼠，用酶联免疫吸附测定法（ELISA）检测颞叶脑内GDNF水平，用RealtimePCR法和Westernblot法分别检测海马区DrebrinmRNA和蛋白的水平。结果移植组大鼠移植治疗后3d在侧脑室周围见较强荧光分布，治疗后7d荧光强度减弱，治疗后10d大鼠各脑区均未检测到绿色荧光。治疗后移植组大鼠学习记忆能力明显改善，逃避潜伏期（34．89±4．15）s短于注射组（43．86±6．95）S和对照组（50．89±3．66）S，而第三象限穿梭次数（11．00±1．49）次多于注射组（9．26±1．38）次和对照组（8．04±1．12）次，（均P〈0．05）。同时，移植组大鼠脑内GDNF水平（315．71±27．43）、DrebrinmRNA（5．54±0．35）与Drebrin蛋白水平（0．55±0．05）均高于注射组（GDNF：256．26±19．90；DrebrinmRNA：3．10±0．33；Drebrin蛋白：0．43±0．06）和对照组（GDNF：141．95±21．33；DrebrinmRNA：1．32±0．23；Drebrin蛋白：0．26±0．06）（均P〈0．05）。结论GDNF工程细胞可在大鼠脑内存活7d左右。通过向侧脑室内移植GDNF工程细胞来治疗血管性痴呆具有一定可行性，效果优于直接向侧脑室内注射GDNF。GDNF治疗血管性痴呆的作用可能与调节神经可塑性有关。

Objective To investigate the effect of lateral ventricle transplantation of neurotrophic factotransfected cells derived from Olia cell line on vascular dementia in rats and gene expression of Drebrin in hippocampal region. Methods By using gene clone technique, the GDNF gene was transfected into SH-SYSY cell lines. 104 adult male Sprague-Dawley rats weighing （200 ±20） gram were divided into groups： transplanted group, injected group, control group, all of which accepted operation by permanent ligation of left common carotid artery and clipping right common carotid artery repeatedly to build up model of vascular dementia, and sham operation group which accepted no ligation or clipping. 6 rats from each group were decapitated on the third day, seventh day and tenth day after transplanting treatment were for fluorescence detection. The rest 20 rats in each group were used to detect learning and memory functions by Morris water maze on the third day and decapitated on the fourth day after transplanting treatment. Then GDNF level in temporal lobe were detected by enzyme-linked immunosorbent assay （ELISA）, while Drebrin mRNA and protein levels in hippocampal region were detected by real time-PCR and Westernblot respectively. Results There was strong fluorescent light detected around lateral ventricle of rats in transplanted group on the third day after transplantation, which faded on the seventh day and disappeared on the tenth day. The learning and memory functions of rats in transplanted group were improved significantly. The escape latency was shorter in transplanted group than in injected group and control group [（34.89 ±4. 15） s vs （43.86±6.95） s, （50.89±3.66） s, both P〈0.051, while shuttle times through the third quadrant were more often in transplanted group than in injected group and control group [（11.00± 1.49） vs. （9.26 ± 1.38）, （8.04±1.12）, both P〈0.051. GDNF level and Drebrin mRNA and protein levels were higher in transplanted group than in iniected group and control group 〈GDNF （315.71±27.43） vs （256.26±19.90）, （141.95±21.33）, Drebrin mRNA.. （5.54±0.35） vs. （3.10±0.33）, （1.32±0.23）, Drebrinprotein： （0.55±0.05） vs （0.43±0.06）, （0.26±0.06）, all P〈0. 051. Conclusions GDNF-transfected cells could survive in the lateral cerebral ventricle of rats for about seven days. The method for treating vascular dementia through the technique of transplanting GDNF-transfected cells is certain feasible, which has a better therapeutic effect than GDNF-injection directly into lateral cerebral ventricle. The therapeutic effect of GDNF on vascular dementia may be related to its action of regulating neural plasticity.