摘要
背景:前期实验中采用新型基因沉默方法——抗基因RNA下调了细胞内β-arrestin 2基因的表达,实现细胞水平的基因敲除效应。目的:在前期实验基础上,观察抗基因RNA下调小鼠C17.2神经干细胞β-arrestin 2基因表达后其对DAMGO诱导μ阿片受体脱敏感化的影响。方法:小鼠C17.2神经干细胞应用含体积分数为10%胎牛血清、10mg/L青霉素和10mg/L氨苄青霉素的DMEM培养基,置于37℃,体积分数为5%CO2培养箱进行培养。待细胞长至80%汇合,用0.25%胰蛋白酶消化传代,每五六天传代1次。通过RT-PCR和免疫细胞化学法检测C17.2细胞μ阿片受体的表达。在脂质体介导下,将已被证实具有基因沉默效应的抗基因RNA片段转染C17.2细胞,荧光显微镜下观察转染24h后绿色荧光蛋白的表达。应用μ阿片受体选择性激动剂DAMGO和[35S]GTPγS结合实验检测C17.2细胞μ阿片受体与G蛋白的耦联能力。结果与结论:RT-PCR结果证实C17.2细胞内有μ阿片受体存在,而免疫细胞化学结果显示μ阿片受体主要在细胞膜和细胞浆中表达。荧光显微镜下观察可见转染质粒pGPU6/GFP/Arrb9的C17.2细胞中有绿色荧光蛋白的表达。[35S]GTPγS结合实验结果显示:未应用DAMGO进行处理的细胞中,[35S]GTPγS结合的刺激比率为(253±17)%;应用DAMGO预先对细胞进行处理后,刺激比率降为(113±14)%(P<0.05);而转染β-arrestin 2抗基因RNA的细胞在DAMGO刺激下,[35S]GTPγS结合的刺激比率则仍维持在(239±15)%,表明抗基因RNA下调β-arrestin 2的表达,可抑制μ阿片受体脱敏感的产生。
BACKGROUND： Antigene RNAs （agRNAs） could be a useful tool to downregulate beta arrestin 2 （Arrb2） gene expressions, and realize gene knock-out effect in cell levels. OBJECTIVE： To observe the effects of agRNAs on DAMGO-induced mu-opioid receptor （MOR） desensitization in C17.2 cells by using agRNAs complementary to transcription start sites of beta arrestin 2 （Arrb2） to downregulate the gene expression in mouse C17.2 cells. METHODS： Mouse neural stem cells C17.2 was cultured in Dulbecco＇s minimal essential medium containing 10% fetal bovine serum, 10 mg/L penicillin and 10 mg/L ampicillin with 5% CO2 at 37 ℃. The cells were passaged every 5 to 6 days after digestion with 0.25% trypsin when cells were 80% confluent. The expression of MOR on mouse C17.2 cells was detected by RT-PCR and immunocytochemical method. AgRNAs which could silence the expression of Arrb2 was transfected into C17.2 cells with Lipofectamine. The expression of green fluorescent protein gene was observed by fluorescence microscopy 24 hours after transfection. [35S]GTPγS binding was assessed by autoradiography to examine the ability of the MOR to couple to G proteins on stimulation with selective agonist DAMGO. RESULTS AND CONCLUSION： The expression of MOR on C17.2 cells was confirmed by RT-PCR. The receptors were expressed on cell membrane and in plasma determined by immnocytochemistry. The expression of green fluorescent protein gene could be observed in C17.2 cells transfected with plasmid pGPU6/GFP/Arrb9 using fluorescent microsocpe. The results of [35S]GTPγS binding showed that the stimulation ratio in cells with and without DAMGO stimulation or transfected with agRNAs were （113±14）%, （253±17）% and （239±15）% respectively. This indicated that agRNAs could downregulate the expression of beta-arrestin 2 and inhibit the desensitization of MOR.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2010年第1期82-85,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金资助项目(30700783)
高等学校博士学科点专项科研基金新教师项目(20070487117)
贝朗麻醉科学研究基金(2007)~~
