脊髓背角神经元miR-9在糖尿病神经痛大鼠钙稳态调节蛋白1过表达中的作用  

Role of microRNA9 in spinal dorsal horn neurons in over-expression of calcium homeostasis modula-tor 1 in rats with diabetic neuropathic pain

作　　者：刘文捷[1] 姜军[1] 李安超 何万友[2] 胡啸玲[1] 郭曲练[3] 

机构地区：[1]南华大学附属第一医院麻醉科,衡阳市421001 [2]佛山市第一人民医院麻醉科,528000 [3]中南大学湘雅医院麻醉科,长沙市410008

出　　处：《中华麻醉学杂志》2017年第8期954-957,共4页Chinese Journal of Anesthesiology

基　　金：湖南省卫生厅资助项目（B2013-039）

摘　　要：目的评价脊髓背角神经元微小RNA9（miR-9）在糖尿病神经痛大鼠钙稳态调节蛋白1（CALHM1）过表达中的作用。方法健康雄性SD大鼠93只，2月龄，体重180～200 g，采用腹腔注射1%链尿佐菌素（STZ）60 mg/kg的方法制备大鼠糖尿病模型。实验Ⅰ 采用随机数字表法分为2组：正常对照组（C组，n＝10）和糖尿病神经痛组（DNP组，n＝83）。分别于STZ注射前、注射后1、2、3、4、5、6周时测定机械缩足反应阈（MWT）；于STZ注射后6周时采用原位杂交法测定脊髓背角miR-9表达。实验Ⅱ STZ注射后6周时取糖尿病神经痛大鼠，分离、培养脊髓背角神经元，以5×106个/ml的密度接种于培养孔（2 ml/孔），采用随机数字表法分为2组（n＝18）：对照组（C组）和miR-9反义寡核苷酸组（ASO组）。ASO组加入miR-9反义寡核苷酸的单链核苷酸序列：5′-UUCUCCGAACGUGUCACGUTT-3′，终浓度为100 pmol/L。于孵育48 h时采用qRT-PCR法测定miR-9和CALHM1 mRNA的表达，于孵育72 h时采用Western blot法测定CALHM1的表达。结果实验Ⅰ 与C组比较，DNP组STZ注射后2～6周时MWT降低，脊髓背角miR-9表达上调（P〈0.05）。实验Ⅱ 与C组比较，ASO组脊髓背角神经元miR-9、CALHM1及其mRNA表达下调（P〈0.05）。结论脊髓背角神经元miR-9可能介导了糖尿病神经痛大鼠CALHM1的过表达。ObjectiveTo evaluate the role of microRNA9 （miR-9） in spinal dorsal horn neurons in over-expression of calcium homeostasis modulator 1 （CALHM1） in rats with diabetic neuropathic pain （DNP）.MethodsNinety-three healthy male Sprague-Dawley rats, aged 2 months, weighing 180-200 g, were used in the study.Diabetes mellitus was induced by intraperitoneal 1% streptozocin （STZ） 60 mg/kg.The experiment was performed in two parts.Experiment Ⅰ The rats were divided into control group （group C, n=10） and DNP group （n=83） using a random number table.The mechanical paw withdrawal threshold （MWT） was measured before STZ injection and at 1, 2, 3, 4, 5 and 6 weeks after STZ injection.The expression of miR-9 in the spinal dorsal horn was determined using the in situ hybridization at 6 weeks after STZ injection.Experiment Ⅱ The rats with DNP were selected at 6 weeks after STZ injection, and the spinal dorsal horn neurons were isolated and cultured.The neurons were seeded in culture plates at the density of 5×106 cells/ml （2 ml/well） and divided into 2 groups （n=18 each） using a random number table： control group （group C） and miR-9 antisense oligonucleotide group （group ASO）. The neurons were cultured in normal culture atmosphere in group C. In group ASO, the single nucleotide sequence of miR-9 antisense oligonucleotide sequence 5′-UUCUCCGAACGUGUCACGUTT-3′ was added with the final concentration of 100 pmol/L.The expression of miR-9 and CALHM1 mRNA was detected using quantitative real-time polymerase chain reaction at 48 h of incubation.The expression of CALHM1 was detected by Western blot at 72 h of incubation.ResultsExperiment Ⅰ Compared with group C, the MWT was significantly decreased at 2-6 weeks after STZ injection, and the expression of miR-9 in the spinal dorsal horn was up-regulated in group DNP （P〈0.05）. Experiment Ⅱ Compared with group C, the expression of miR-9 and CALHM1 protein and mRNA in spinal dorsal horn neurons was significantly down-regulated in gr

关 键 词：微RNAs 钙通道 脊髓 神经元 糖尿病 神经痛 

分 类 号：R587.2[医药卫生—内分泌]