慢病毒介导的聚腺苷二磷酸核糖聚合酶-1siRNA对大鼠脑梗死后神经血管单元的影响

Effects of Lentivirus-mediated poly( ADP-ribose) polymerase-1 siRNA transfer on neurovascular unit following cerebral infarction in rats

目的观察慢病毒介导的聚腺苷二磷酸核糖聚合酶-1(PARP-1)siRNA对大鼠脑梗死后神经血管单元(NVU)的影响。方法 SD大鼠132只随机分为假手术组(n=32)、脑梗死组(n=30)、空病毒组(n=32)和PARP-1组(n=38)。空病毒组和PARP-1组大鼠分别于侧脑室注射空病毒和携带目的基因的病毒5μl进行RNA干扰,14 d后进行脑梗死模型制作。造模术24 h后依据Longa 5分法进行神经功能评分。采用TTC染色检测脑梗死体积,HE染色观察脑组织病理改变,伊文思蓝(EB)通透率检测计算脑组织EB含量,脑组织干湿重法测定脑组织含水量,电镜观察NVU超微结构的改变。结果假手术组与空病毒组间大鼠PARP-1 mRNA表达水平的差异无统计学意义(P=0.244)。与空病毒组比较,PARP-1组RNA干扰后3 d、5d、8 d PARP-1 mRNA表达水平均明显下降(均P<0.05)。假手术组大鼠无神经功能缺损,无脑梗死。与假手术组比较,脑梗死组和空病毒组大鼠缺血侧脑组织EB含量和脑组织含水量明显增加(均P<0.05)。与脑梗死组和空病毒组相比,PARP-1组大鼠神经功能评分显著降低,脑梗死体积明显减小,脑组织EB含量和脑组织含水量明显减少(均P<0.05)。HE染色显示,假手术组大鼠海马神经细胞染色均匀,细胞排列紧密整齐且结构清晰;脑梗死组和空病毒组大鼠海马神经细胞肿胀、破裂、轮廓模糊,染色较深;PARP-1组神经细胞排列整齐,染色均匀。电镜可见,假手术组海马NVU超微结构清晰完整;脑梗死组和空病毒组神经细胞变性,线粒体肿胀、嵴减少,髓鞘变薄、分层,血管内皮细胞凋亡、基底膜不连续,突触数量减少、结构破坏;而PARP-1组海马NVU超微结构损伤明显减轻。结论抑制PARP-1的表达,可明显改善脑梗死大鼠的神经功能,缩小脑梗死体积,减轻脑梗死后NVU的损伤。

Objective To investigate the effects of Lentivirus-mediated poly（ ADP-ribose） polymerase-1（ PARP-1） siRNA transfer on neurovascular unit（ NVU） in rats following cerebral infarction（ CI）. Methods One hundred and thirty-two rats were randomly divided into sham group（ n = 32）,CI group（ n = 30）,control siRNA group（ n = 32）,PARP-1 group（ n = 38）. Lateral ventricle of rats in control siRNA group and PARP-1 group were injected with 5 μl control siRNA or PARP-1 siRNA for RNA inference. CI model was performed 14 days later. Then,24 h later,neurological evaluations were performed according to Longa five-point scale. TTC straining was conducted to assess CI volume. HE straining was used to examine cerebral pathological changes. Evans blue（ EB） leakage was conducted to assess the level of EB in brain. Brain water content was measured by dry-wet method. Electron microscopy was used to observe ultrastructure of NVU. Results No significant differences of PARP-1 mRNA were observed between sham group and control siRNA group（ P = 0. 244）. Compared with control siRNA group,PARP-1mRNA was significantly decreased in PARP-1 group at 3 d,5 d,8 d after RNA interference（ all P 0. 05）. No neurological deficits and CI were observed in sham group. Compared with sham group,EB leakage and brain water content were significantly increased in CI group and control siRNA group（ all P 0. 05）. Compared with CI group and control siRNA group,neurobehavioral scores,CI volume,EB leakage and brain water content of PARP-1 group were significantly decreased（ all P 0. 0 5）. HE staining showed that neurons in hippocampus of sham group was stained evenly,cells were arranged orderly and apparented clearly; hippocampal neurons of the CI group and control siRNA group were swollen,fractured,indistinct and stained deeply; neurons in PARP-1 group were well-distributed and stained evenly. The electron microscope showed that ultrastructures of NVU in sham group were distinct and normal; neurons in the CI group and control siRNA group were denatured,mitochondria was swollen,mitochondrial cristae was fractured,myelin became thinner and delaminated,endothelialcell and basal lamina were deformed,synapse was reduced and destroyed; while ultrastructure deficits of NVU were ameliorated in PARP-1 group.Conclusion PARP-1 inhibition may improve neurological function,ameliorate CI volume,alleviate NVU deficits following CI.