功能化单壁碳纳米管/NR2B-shRNA干扰质粒复合物的制备及其用于慢性疼痛的基因治疗  

PREPARATION OF FUNCTIONALIZED SINGLE-WALLED CARBON NANOTUBES/NR2B-SHRNA INTERFERENCE PLASMID COMPLEX AND ITS APPLICATION IN GENE THERAPY FOR CHRONIC PAIN

作　　者：甘丽[1] 阎雪彬[1,2] 章饶香[1] 黄东[1] 

机构地区：[1]中南大学湘雅三医院麻醉科,长沙410013 [2]中南大学材料科学与工程博士后流动站,长沙410083

出　　处：《中国疼痛医学杂志》2013年第4期220-224,共5页Chinese Journal of Pain Medicine

基　　金：国家自然科学基金(项目编号:81172546);中南大学研究生科研创新项目(项目编号:2011ssxp198);湖南省科技厅科技计划项目(项目编号:2010WK3021)

摘　　要：目的：评价鞘内注射功能化单壁碳纳米管shRNA干扰质粒复合物对福尔马林炎性痛小鼠的基因沉默效果。方法：先将整合了NR2B目的基因的pYr-1.1-hU6-EGFP（带绿色荧光）质粒进行扩增,抽提,消毒浓缩,浓度纯度测定,以获得高纯度、高浓度的NR2B-shRNA干扰质粒;将难溶于水的单壁碳纳米管用表面活性剂十六烷基三甲基溴化铵（CTAB）和超声振荡法制备成功能化单壁碳纳米管（f-SWNT）混悬液,通过凝胶成像系统检测功能化单壁碳纳米管与NR2B-shRNA的结合情况;选取雄性清洁级成年C57BL/6小鼠40只,体重18~23 kg,随机分为5组（n=8）：空白组（C组）,假手术组（S组）,实验组（A组）,阳性对照组（P组）,阴性对照组（N组）;分别行鞘内注射,7天后建立福尔马林炎性痛小鼠模型,测定术后1 h自发痛行为学改变,取脊髓L4~6节段采用RT-PCR法检测NR2BmRNA表达。结果：获得能够稳定表达的NR2B-shRNA干扰质粒;表面活性剂十六烷基三甲基溴化铵（CTAB）修饰单壁碳纳米管能形成较稳定的功能化单壁碳纳米管混悬液;功能化单壁碳纳米管能与NR2B-shRNA干扰质粒很好地结合,其结合最佳质量比为1：3.5;对于福尔马林所致的Ⅰ相急性痛,所有组间的急性期伤害性反应无差别（P〉0.05）,阳性对照组（P组）和实验组（A组）小鼠对福尔马林所致的Ⅱ相持续痛的伤害性反应显著降低（P〈0.02）;阳性对照组（P组）和实验组（A组）小鼠脊髓NR2BmRNA表达均低于其他对照组（P〈0.05）。结论：功能化单壁碳纳米管能与NR2B-shRNA干扰质粒很好地结合形成功能化单壁碳纳米管/NR2B-shRNA干扰质粒复合物;鞘内注射功能化单壁碳纳米管用作基因载体能有效转染NR2B-shRNA干扰质粒复合物行NR2B基因沉默,抑制小鼠脊髓NR2B基因表达,减轻福尔马林Ⅱ相痛行为学。Objective： To investigate the gene silencing effect of intrathecally injecting functionlized single-walled nanotubes/NR2B-shRNA interference plasmid compound on inflammatory pain of murine. Methods： First amplify, extract, concentrate and determinate the purity of pYr-I.I-hU6-EGFP （with green fluorescence protein） plasmid integrated with NR2B gene in order to obtain high purity NR2B- shRNA interference plasmid. Disperse single-walled nanotubes （SWNTs） with surfactant cetyltrimethyl ammonium bromide （CTAB） and ultrasound dispersing method. Test the combination of single-walled carbon nanotubes with NR2B-shRNA interference plasmid by conducting agarose gel electrophoresis.Forty male C57BL/6 mice, weighing about 18 - 23 g, were randomly divided into 5 groups （n = 8 each）： blank control group （group C）, sham operation group （group S）, the experimental group （group A）, the positive control group （group P） and the negative control group （N）, then seven days later, the formalin inflammatory pain mice model were established and spontaneous pain behavioral changes were observed one hour after operation, then lumbar segments of spinal cords were taken in each group for detecting the expression of NR2BmRNA. Results： Get stably expressed NR2B-shRNA interference plasmid; Surfactant cetyltrimethyl ammonium bromide （CTAB） modified single-walled carbon nanotubes can form a stable suspension of functionalized single-walled carbon nanotubes in deionized water. The optimum mass ratio between functionalized single-walled carbon nanotubes （f-SWNT） and NR2B-shRNA interference plasmid is 1：3.5. The nociceptive response in the acute phase was indistinguishable among all groups for nociception with regard to the flinching response （P 〉 0.05）, the nociceptive response in the tonic phase significantly decreased in the experimental group （group A） and the positive control group （group P） compared to other groups （group C, group S and group N） （P 〈 0.02）; the expre

关 键 词：NR2B-shRNA干扰质粒 单壁碳纳米管 十六烷基三甲基溴化铵 NMDA 受体2B亚基 炎性痛 

分 类 号：R402[医药卫生—临床医学]