大鼠神经病理性痛时脊髓lncRNA与kindlin-1/Wnt3a信号通路的关系  

Relationship between spinal lncRNA and kindlin-1/Wnt3a signaling pathway in a rat model of neuropathic pain

作　　者：金宇林 徐海平 宋兴荣 赵柏松 Jin Yulin;Xu Haiping;Song Xingrong;Zhao Baisong(Department of Anesthesiology,Guangzhou Women and Children′s Medical Center,Guangzhou 510623,China)

机构地区：[1]广州市妇女儿童医疗中心麻醉科,510623

出　　处：《中华麻醉学杂志》2021年第7期835-839,共5页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金(81701109)。

摘　　要：目的探讨大鼠神经病理性痛时长链非编码RNA(lncRNA)与kindlin-1/Wnt3a信号通路的关系。方法实验Ⅰ7日龄SD大鼠,体重15~20 g,雌雄不拘,断头处死后取脊髓背角,提取并培养原代星形胶质细胞,加入LPS 1μg/ml诱导星形胶质细胞活化24 h。采用PCR免疫共沉淀法筛选与kindlin-1结合的lncRNA,采用荧光原位杂交法观察星形胶质细胞中lncRNA FOXF1-AS1的定位,采用生物素标记磁珠法检测lncRNA FOXF1-AS1与kindlin-1的结合情况。实验Ⅱ清洁级健康雄性SD大鼠30只,体重250~280 g,10~12周龄,采用随机数字表法分为5组(n=6):假手术对照组(C组)、神经病理性痛组(NP组)、lncRNA FOXF1-AS1过表达组(F组)、lncRNA FOXF1-AS1过表达+kindlin-1 shRNA组(FK组)和lncRNA FOXF1-AS1过表达+Wnt抑制剂组(FW组)。采用坐骨神经慢性压迫法建立大鼠神经病理性痛模型。F组于术前28 d时鞘内注射lncRNA FOXF1-AS1过表达慢病毒10μl,其余各组鞘内注射空载病毒10μl;FK组于术前21 d时鞘内注射kindlin-1 shRNA干扰腺病毒10μl,其余各组鞘内注射空载病毒10μl;FW组于术后1~3 d时鞘内注射Wnt抑制剂IWP-210μl,其余各组于相同时点鞘内注射人工脑脊液10μl。分别于术前1 d、术后4和7 d时测定机械缩足反应阈(MWT)和热缩足潜伏期(TWL)。于术后7 d时痛阈测定结束后处死大鼠取脊髓组织,采用Western blot法检测kindlin-1、Wnt3a和胶质纤维酸性蛋白(GFAP)表达,采用ELISA法检测TNF-α和IL-1β含量。结果实验Ⅰ表达于星形胶质细胞胞浆内的lncRNA FOXF1-AS1与kindlin-1具有结合作用。实验Ⅱ与C组比较,NP组术后4和7 d时MWT降低,TWL缩短,脊髓kindlin-1、Wnt3a和GFAP表达上调,TNF-α和IL-1β含量升高(P<0.05);与NP组比较,F组术后4和7 d时MWT降低,TWL缩短,脊髓kindlin-1、Wnt3a和GFAP表达上调,TNF-α和IL-1β含量升高,FK组和FW组术后4和7 d时MWT升高,TWL延长,脊髓TNF-α和IL-1β含量降低,FK组脊髓kindlin-1、Wnt3a和GFAP表达下调,FW组脊髓kinObjective To investigate the relationship between spinal long chain noncoding RNA(lncRNA)and kindlin-1/Wnt3a signaling pathway in a rat model of neuropathic pain(NP).Methods The experiment was performed in two parts.ExperimentⅠSprague-Dawley rats of both sexes,aged 7 days,weighing 15-20 g,were selected.Rats were sacrificed,the dorsal horn of spinal cord was removed,and the primary astrocytes were extracted and cultured.Lipopolysaccharide 1μg/ml was added to induce the activation of astrocytes for 24 h.The lncRNA binding to kindlin-1 was identified using PCR immunoprecipitation method.The localization of lncRNA FOXF1-AS1 in astrocytes was observed by fluorescence in situ hybridization,and the binding between lncRNA FOXF1-AS1 and kindlin-1 was detected by biotin-labeled magnetic bead method.ExperimentⅡThirty clean-grade healthy male Sprague-Dawley rats,aged 10-12 weeks,weighing 250-280 g,were divided into 5 groups(n=6 each)using a random number table method:sham operation control group(group C),NP group,lncRNA FOXF1-AS1 overexpression group(group F),lncRNA FOXF1-AS1 overexpression plus kindlin-1 shRNA group(group FK)and lncRNA FOXF1-AS1 overexpression+Wnt inhibitor group(group FW).NP was induced by chronic constrictive injury in anesthetized animals.In group F,lncRNA FOXF1-AS1 overexpression lentivirus 10μl was intrathecally injected at 28 days before operation,and vector virus 10μl was intrathecally injected in the other groups.In FK group,kindlin-1 interfering shRNA interference adenovirus 10μl,and vector virus 10μl was intrathecally injected in the other groups.In group FW,Wnt inhibitor IWP-210μl was intrathecally injected at 1-3 days after operation,artificial cerebrospinal fluid 10μl was intrathecally injected at the same time point in the other groups.Mechanical withdrawal threshold(MWT)and thermal withdrawal latency(TWL)were measured at 1 day before operation,at 4 days and 7 days after operation.The animals were sacrificed at the end of measurement of pain threshold at 7 days after operation,and t

关 键 词：神经痛 长链非编码RNA 整合素结合蛋白 WNT蛋白质类 脊髓 

分 类 号：R614[医药卫生—麻醉学]