Bahi Amine, Boyer Frederic, Kolira Manoj, Dreyer Jean-Luc
Institute of Biochemistry, University of Fribourg, Fribourg, Switzerland.
J Neurochem. 2005 Mar;92(5):1243-55. doi: 10.1111/j.1471-4159.2004.02961.x.
The tetraspanin CD81 is induced in the mesolimbic dopaminergic pathway after cocaine administration. To further investigate its role, a regulatable lentivirus (Lenti-CD81) bearing the CD81 gene under the control of a tetracycline-inducible promoter and lentiviruses expressing short hairpin RNA (shRNA) targeted against CD81 (Lenti-CD81-shRNAs) have been prepared. Infection of HEK293T cells in vitro with Lenti-CD81-shRNAs resulted in 96.5% gene silencing (from quantitative real-time PCR(qRT-PCR) and immunocytochemistry). In vivo delivery of Lenti-CD81-shRNA into the nucleus accumbens or ventral tegmental area resulted in 91.3 and 94% silencing of endogenous CD81, respectively. Stereotaxic injection of Lenti-CD81 into these regions, resulting in CD81 overexpression, induced a four- to fivefold increase in locomotor activity after chronic cocaine administration, which returned to basal levels when Lenti-CD81-shRNA had been coinjected or when CD81 expression was blocked by doxycycline. Furthermore, silencing endogenous CD81 in vivo resulted in a significant decrease in locomotor activity over controls, again suppressing cocaine-induced behaviour.
可卡因给药后，四跨膜蛋白CD81在中脑边缘多巴胺能通路中被诱导表达。为进一步研究其作用，制备了一种可调控的慢病毒（Lenti-CD81），其携带在四环素诱导型启动子控制下的CD81基因，以及表达靶向CD81的短发夹RNA（shRNA）的慢病毒（Lenti-CD81-shRNAs）。体外将Lenti-CD81-shRNAs感染HEK293T细胞，通过定量实时聚合酶链反应（qRT-PCR）和免疫细胞化学检测，导致96.5%的基因沉默。体内将Lenti-CD81-shRNA注射到伏隔核或腹侧被盖区，分别导致内源性CD81沉默91.3%和94%。立体定位注射Lenti-CD81到这些区域，导致CD81过表达，在慢性可卡因给药后诱导运动活性增加4至5倍，当同时注射Lenti-CD81-shRNA或用强力霉素阻断CD81表达时，运动活性恢复到基础水平。此外，体内沉默内源性CD81导致运动活性相对于对照组显著降低，再次抑制了可卡因诱导的行为。
