稳定表达GFP-Parkin融合蛋白的SH-SY5Y细胞系的建立和功能初探

Establishment of SH-SY5Y Cell Line Stably Expressing GFP-Parkin and its Preliminary Function

目的:建立稳定表达GFP-Parkin的SH-SY5Y细胞系,并检测Parkin对MPP^+引起的SH-SY5Y细胞损伤的保护作用。方法:将Parkin编码序列克隆到载体pEGFP-C1中,构建重组质粒pEGFP-Parkin,转染SH-SY5Y细胞,通过G418筛选,建立稳定表达GFP-Parkin的SH-SY5Y细胞系;荧光显微镜和Western印迹鉴定Parkin表达,MTT法检测Parkin对MPP^+致细胞损伤的保护作用。结果:酶切鉴定及测序结果表明重组质粒pEGFP-Parkin构建正确;荧光显微镜下可见细胞内有GFP-Parkin的融合表达,Western印迹检测发现相对分子质量79×103的蛋白条带;MTT结果显示Parkin能够减弱MPP^+对SH-SY5Y细胞的损伤,与对照组相比,存活率高约15%,差异显著(P<0.05)。结论:构建了稳定表达GFP-Parkin的SH-SY5Y细胞系,为进一步研究Parkin的细胞保护作用和其他功能机制奠定了基础。

Objective： To establish SH-SY5Y cell line stably expressing GFP-Parkin, and evaluate its protective effect against MPP＋ by MTT assay. Methods： The coding sequence of human Parkin was cloned into the vector pEGFP-C1 to construct recombinant plasmid pEGFP-Parkin. The pEGFP-Parkin plasmid was transfected into SHSY5Y cells using LipofectAMINE 3000, and positive-expression cells were screened by using G418. The expression of GFP-Parkin was identified with fluorescent microscopy and Western blot. The protective effect of Parkin against MPP＋ in SH-SY5Y cells was detected by MTT assay. Results： The constructed pEGFP-Parkin plasmid was confirmed by restriction enzyme digestion and DNA sequencing analysis. Fluorescence photographs and Western blot data revealed that the SH-SY5Y cells expressed GFP-Parkin protein stably. MTT assay showed that survival of Parkin group was 15% higher than that of control group after exposure to 1 mmol/L MPP＋ for 48 h（P〈 0.05）, indicating that Parkin significantly enhanced cell survival. Conclusion： SH-SY5Y cell line stably expressing GFP-Parkin has been established, laying a foundation for further investigating cytoprotective effects and other functional mechanisms of Parkin.