RNA干扰应用于抑制脊髓损伤急性期白细胞介素6受体表达的实验(英文)

Inhibitory effect of RNA interference on the expression of interleukin-6 receptor in the acute phase of spinal cord injury

背景:由于中枢神经损伤后局部微环境病理生理机制极其复杂,尤其急性期炎性反应及继发胶质瘢痕形成对有效轴突再生、局部神经细胞再生排列及局部干细胞增殖迁徙影响极大,成为早期阻碍神经修复的根本原因,故如何有效拮抗损伤区急性期炎性损伤因素,优化神经再生微环境是目前脊髓损伤修复的重要策略之一。目的:应用大鼠脊髓损伤模型,设计构建并筛选最佳白细胞介素6受体α表达抑制shRNA腺病毒表达载体。设计:重复测量实验。单位:深圳市第二人民医院脊柱外科。材料:选用健康Wistar大鼠40只,雌雄不拘,鼠龄8～10周。兔抗鼠GFAP一抗购自Santa Cruz公司,siRNA构建真核表达质粒pGenesil(携带绿色荧光表达系统)购自武汉晶赛生物工程技术公司。方法:实验于2006-11在武汉同济医学院基础医学部免疫教研室开放实验室及深圳市第二人民医院医学实验中心完成。针对白细胞介素6受体α基因编码序列设计并合成三对9bp茎环结构19bp干扰序列特异性shRNA模板,体外构建带绿色荧光蛋白的重组腺病毒表达载体;并建立大鼠急性脊髓损伤模型,损伤后12h局部注射编码shRNA重组腺病毒,通过实时荧光定量RT-PCR及Western blot技术,观察RNA干扰后损伤脊髓局部白细胞介素6R表达抑制效果,筛选最佳抑制白细胞介素6R表达的腺病毒表达载体。主要观察指标:RNA干扰后损伤脊髓局部IL-6RRNA表达、蛋白表达水平抑制效果。结果:序列测定证实白细胞介素6R-shRNA重组腺病毒表达载体构建成功,通过实时荧光定量RT-PCR,Western blot技术筛选出最佳白细胞介素6R-shRNA效应腺病毒表达载体,在mRNA及蛋白表达水平抑制白细胞介素6R基因表达效率分别为49%和56%。结论:构建并筛选成功的白细胞介素6R-shRNA效应腺病毒表达载体,在大鼠急性脊髓损伤模型中能高效抑制白细胞介素6R基因表达。

BACKGROUND： Pathophysiological mechanism of local microenvironment is complex after central nerve injury; especially,both inflammatory reaction at an acute phase and formation of secondary glial scar have tremendous effects on effective regeneration of axon, regeneration and arrangement of local nerve cells, proliferation and migration of local stem cells;therefore, it becomes a basic reason for blocking nerve repair in an early period. Thus, how to effectively resist inflammatory factors in injured region at an acute phase and how to optimize microenvironment of neural regeneration are the most important strategies for repairing spinal cord injury in recent years. OBJECTIVE： To design, establish and screen the best expression of interleukin-6 receptor （IL-6R） α to inhibit shRNA adenovirus expression vector by using spinal cord injury models. DESIGN： Duplicative measurement study. SETTING： Department of Spine Surgery, the Second People＇s Hospital of Shenzhen. MATERIALS： A total of 40 healthy Wistar rats, either gender, 8-10 weeks old, were selected in this study. Rabbit-anti-rat glial fibrillary acidic protein （GFAP） antibody Ⅰ was provided by Santa Cruz Compan; siRNA eukaryon expression plasmid pGenesil （cohtaining green fluorescent expression system） was provided by Wuhan Jingsai Bioengineering Company. METHODS： The experiment was carried out in Immune Opening Laboratory, Basic Medical Faulty, Tongji Medical College, Huazhong University of Science and Technology, and Medica Laboratory Center, the Second People＇s Hospital of Shenzhen in November 2006. Three pairs of shRNA template which composed of 19 bp reverse repeated motif of IL-6 receptor （IL-6R） α target sequence with 9 bp spacer were designed and synthesized, then the recombinant adenovirus expression vectors with green fluorescence protein were constructed in vitro respectively. The acute spinal cord injury models were completed, and the adenovirus recombinants were regionally injected post 12 hours after spinal cord injury;in addition, the inhibitory effect of RNA interference （RNAi） on expression of IL-6R in local region after spinal cord injury were detected by using real-time fluorescence quantitative reverse transcription polymerase chain reaction （RT-PCR） and Western blot so as to screen adenovirus expression vector which had the best inhibitory effect on expression of IL-6R. MAIN OUTCOME MEASURES： Inhibitory effect of RNAi on expressions of IL-6R RNA and protein in local region after spinal cord injury. RESULTS： Sequence analysis showed that IL-6R-shRNA recombinant adenovirus expression vector was successfully constructed, and optimal IL-6R-shRNA recombinant adenovirus expression vector was screened by using real-time fluorescence quantitative RT-PCR and Western blot. The IL-6R expressions were 49% and 56% at the levels of mRNA and protein, respectively. CONCLUSION： The IL-6R——shRNA recombinant adenovirus expression vector is successfully constructed and screened.The gene expression of IL-6R can be highly inhibited after acute spinal cord injury.