目的 探讨特异性siRNA 沉默Nogo-66 受体（NgR）对宫内感染所致早产大鼠脑损伤修复的影响及作用机制。方法 孕15 d Sprague-Dawley 大鼠分别应用RU486 和LPS 诱导早产，随机选取RU486 诱导的早产大鼠为对照组，将LPS 诱导的宫内感染致脑损伤早产大鼠随机分为模型组、空载体组和NgR-siRNA 组，每组36 只。对照组和模型组仅给予常规饲养，空载体组和NgR-siRNA 组均于出生后第1 天（P1）经侧脑室1 次性注入慢病毒空载体和NgR-siRNA 慢病毒载体后常规饲养。各组分别于P3、P7、P14 时随机选取8 只早产大鼠断头取脑。RT-PCR 检测NgR mRNA 表达，Western blot 测定活性RhoA 蛋白表达，免疫荧光组化检测小胶质细胞活化程度和少突胶质前体细胞（OPCs）形态，苏木精-伊红染色观察脑组织病理学改变，P30 时行动物行为学评分。结果 P3 时，NgR-siRNA 组脑组织NgR mRNA 表达量、活性RhoA 蛋白水平显著低于模型组和空载体组（P < 0.05）；各组NgR mRNA 表达量与活性RhoA 蛋白水平均呈正相关性（分别r=0.792、0.747、0.827、0.825，P < 0.05）。免疫荧光组化结果显示，NgR-siRNA 组P3 时小胶质细胞CD11b 荧光强度值较模型组和空载体组明显减弱（P < 0.05）；各组O4 抗体标记的OPCs 细胞形态主要呈现三极突起形态。病理学结果显示，对照组脑室周围白质结构正常，染色清晰；模型组和空载体组白质结构疏松，纤维紊乱，可见软化灶；NgR-siRNA 组白质结构疏松，纤维紊乱相对较轻，胶质细胞增生不明显，无明显软化灶。行为学评分显示，NgR-siRNA 组的悬吊实验评分、活动总路程、平均速度和跨格次数大于模型组和空载体组，而斜坡实验时间及中心区活动时间和路程明显少于模型组和空载体组（P < 0.05），但同对照组比较差异均无统计学意义（P > 0.05）。结论 NgR 特异性siRNA 可有效沉默宫内感染所致脑损伤早产大鼠NgR 基因表达，在脑损伤后的修复中具有显著神经保护作用。
Abstract
Objective To investigate the effect of Nogo-66 receptor (NgR) silencing with specific small interfering RNA (siRNA) on brain injury repair in preterm rats with brain injury caused by intrauterine infection and related mechanism of action. Methods The pregnant Sprague-Dawley rats (with a gestational age of 15 days) were selected, and premature delivery was induced by RU486 or lipopolysaccharide (LPS). The preterm rats delivered by those treated with RU486 were selected as the control group. The preterm rats with brain injury caused by intrauterine infection induced by LPS were divided into model, empty vector, and NgR-siRNA groups, with 36 rats in each group. The rats in the control and model groups were given routine feeding only, and those in the empty vector and NgR-siRNA groups were given an injection of lentiviral empty vector or NgR-siRNA lentivirus via the lateral ventricle on postnatal day 1 (P1) and then fed routinely. On P3, P7, and P14, 8 rats in each group were randomly selected and sacrificed to harvest the brain tissue. RT-PCR was used to measure the mRNA expression of NgR. Western blot was used to to measure the protein expression of active RhoA. The immunofluorescence histochemistry was used to determine the degree of activation of microglial cells and the morphology of oligodendrocyte precursor cells (OPCs). Hematoxylin and eosin staining was used to observe the pathological changes in brain tissue. The behavioral score was evaluated on P30. Results On P3, the NgR-siRNA group had significantly lower mRNA expression of NgR and protein expression of active RhoA in brain tissue than the model and empty vector groups (P < 0.05). In each group, the mRNA expression of NgR was positively correlated with the protein expression of active RhoA (P < 0.05). The results of immunofluorescence histochemistry showed that on P3, the NgR-siRNA group had a significantly reduced fluorescence intensity of the microglial cells labeled with CD11b compared with the model and empty vector groups (P < 0.05). The OPCs labeled with O4 antibody in the four groups were mainly presented with tripolar cell morphology. The results of pathological examination showed a normal structure of white matter with clear staining in the periventriclar area in the control group, a loose structure of white matter with disorganized fibers and softening lesions in the model and empty vector groups, and a loose structure of white matter with slightly disorganized fibers, slight gliocyte proliferation, and no significant necrotic lesions in the NgR-siRNA group. As for the behavioral score, compared with the model and empty vector groups, the NgR-siRNA group had a higher score in the suspension test, a longer total activity distance, and greater mean velocity and number of squares crossed, as well as a shorter time of slope test and a shorter time and distance of activity in the central area (P < 0.05), while there were no significant differences in these parameters between the NgR-siRNA and control groups (P > 0.05). Conclusions NgR silencing with specific siRNA can effectively silence the expression of NgR in pertem rats with brain injury caused by interauterine infection and has a significant neuroprotective effect in brain injury repair.
关键词
Nogo-66 受体 /
siRNA /
宫内感染 /
早产 /
大鼠
Key words
Nogo-66 receptor /
Small interfering RNA /
Intrauterine infection /
Premature delivery /
Rats
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基金
安徽省高等学校省级自然科学研究重点项目（KJ2013A252）。
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