Effects of beta-arrestin 2 antigene RNAs on mu-opioid receptor desensitization in C17.2 neural stem cells from mice
10.3969/j.issn.1673-8225.2010.01.017
- VernacularTitle:β-arrestin 2抗基因RNA对小鼠C17.2神经干细胞μ阿片受体脱敏感化的影响
- Author:
Feng GAO
;
Shasha CHEN
;
Xijiang LIU
;
Mao XU
;
Hui YANG
;
Yuke TIAN
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2010;14(1):82-85
- CountryChina
- Language:Chinese
-
Abstract:
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% CO_2 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. [~(35)S]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 [~(35)S]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.
