稳定表达α-Synuclein 4种选择性剪接异构体的PC12细胞对神经毒性剂MPP^+的敏感性研究

Study on the sensitivity of the PC12 cells expressing four α-synuclein isoforms to MPP^+

目的研究稳定表达α-synuclein 4种选择性剪接体的PC12细胞对神经毒性剂MPP+的敏感性。方法 PCR获得α-synuclein的4个选择性剪切体,克隆入pcDNA3.1载体,转染PC12细胞,经G418筛选获得稳定表达这4个剪切体的PC12细胞株,采用细胞毒的方法检测这4个稳定细胞株对神经毒性剂MPP+的敏感性。结果同全长相比,去除外显子5的选择性剪切体α-synuclein114和α-synuclein98对神经毒性剂MPP+的敏感性升高,表现为细胞活力的降低。结论去除外显子5的选择性剪切体α-synuclein114和α-synu-clein98对神经毒性剂MPP+的敏感性明显升高。抑制外显子5的选择性剪切,从而抑制选择性剪切异构体α-synucle-in114和α-synuclein98的产生可能是阻止神经元退行性丢失的一种策略。

Aim To study the sensitivity of the PC12 cells expressing four α-syn isoforms to MPP＋. Meth- ods Four α-syn isoforms was cloned by PCR method, followed by subcloning into the pcDNA3. 1 eukaryotic expression vector. Four obtained recombination plas- mids were transfected into PC12 cells using Lipo- fectamine 2000 respectively. The protein expressions of these four isoforms were identified by WB. Results When exposed to MPP ＋ , these cell lines which overexpressed exon 5-lacking form of α-syn isoforms showed the toxicity to PC12 cells. Conclusion The inhibition of alternative splicing to α-syn exon 5 can reduce the production of α-syn 112 and α-syn 98, which may be a good strategy for rescuing dopaminergic neurons from exposure to neurotoxic agents.