GDNF基因转染的细胞侧脑室内移植对缺血性脑损伤大鼠的神经保护作用

目的 研究胶质源性神经营养因子GDNF基因转导的细胞侧脑室内移植治疗局灶性缺血性脑损伤大鼠的可行性,并探讨GDNF的神经保护作用机制.方法 体外培养SH-SY5Y细胞株,并用分子克隆技术转导入GFP-GDNF基因,使其可持续分泌绿色荧光蛋白GFP和GDNF.取75只体重150～180 g的健康雄性大鼠,分为移植缺血组、注射缺血组、缺血对照组和正常组等.在立体定向仪介导下向移植缺血组大鼠侧脑室注入转导GDNF基因的SY5Y细胞,向注射缺血组大鼠侧脑室内注射入5U/10μL的GDNF,24 h后采用线栓法对移植缺血组、注射缺血组和缺血对照组动物进行手术,建立局灶性脑缺血损伤模型（即大脑中动脉阻断,MCAO）,在缺血后2 d时用Longa五分法评测行为学改变,TTC染色判断梗塞体积大小,用Western-blot法检测凋亡相关蛋白Bcl-2/Bax的表达水平.结果 在缺血损伤两天后,移植缺血组动物的肢体功能恢复效果优于缺血对照组,TTC染色亦提示移植缺血组脑梗死体积小于缺血对照组和注射缺血组（P＜0.01）.缺血灶周围脑组织凋亡相关蛋白Bcl-2和Bax含量均明显增高,移植缺血组Bcl-2/Bax的比值高于缺血对照组和注射缺血组.结论 GD-NF对缺血性脑损伤大鼠有神经保护作用,用GDNF基因转导的细胞移植治疗是一种可行的途径,效果优于经侧脑室直接给药.GDNF可调节凋亡相关蛋白Bcl-2/Bax的表达,这可能为其神经保护作用的机制之一.

Neuroprotective Effect of GDNF- transfected Cells Transplanted into Lateral Cerebral Ventricle on Focal Cerebral Ischemia in Rats

Objective To investigate the neuroprotective effect of GDNF-transfected cells transplanted into lateral cerebral ventricle on focal cerebral ischemia in rats. Methods By using gene clone technique,the GFP-GDNF gene was transfected into SH -SY5Y cell lines,made it secret GFP and GDNF. Then GDNF-transfected cells were injected into lateral cerebral ventricle of adult male Sprague -Dawley rats,then compared with adult male Sprague - Dawley rats only injected with GDNF. Another two groups were cerebral ischemia rats and the control. 24 hours later,rats underwent operation (middle cerebral artery occlusion) to build up focal cerebral ischemia model. Another 24 hours later,animals were evaluated neurologically by Longa's method. Then rats were decapitated for 2,3,5 - triphenyltetrazolium chloride staining and Western-blot detection,then compared the results between the four groups. Results Animals injected with GDNF-transfected cells had a better functional recovery and smaller infarction volume than those injected with GDNF (P < 0. 05). After ischemic injury, both Bcl - 2 and Bax protein expression increased significantly. Bcl -2/Bax ratio in rats injected with GDNF-transfected cells was higher than that of rats injected with GDNF. Conclusions GDNF can protect rats from cerebral ischemic injury. GDNF transfected cells transplanting technique is feasible, which has a better therapeutic effect than injecting GDNF into lateral cerebral ventricle directly. GDNF can regulate the expression of Bcl-2 and Bax protein.