博尔纳病病毒磷蛋白在PC-12细胞内的稳定表达及对其增殖的影响

Expressing Borna disease virus phosphoprotein in PC-12 cell and its influence on the cell proliferation

摘要:

【目的】建立博尔纳病病毒磷蛋白在神经源性PC-12细胞内的稳定表达体系，初步探讨博尔纳病病毒磷蛋白对PC-12细胞的生长是否有影响。【方法】培养PC-12细胞，用阳离子脂质体的方法将带有博尔纳病病毒磷蛋白基因的表达质粒转染到细胞内进行稳定表达，用荧光显微镜和RT-PCR的方法检测细胞内磷蛋白的表达，用MTT方法检测磷蛋白对细胞生长的影响。【结果】 转染细胞经培养10代后仍然表达目的蛋白，成功建立稳定表达体系。MTT检测显示博尔纳病病毒磷蛋白对PC-12细胞的生长具有明显的抑制作用，其生长明显滞后，但粘附能力增加。【结论】 通过本文建立的体系能在PC-12细胞内稳定表达博尔纳病病毒磷蛋白，该体系可用于进一步深入研究博尔纳病病毒磷蛋白的作用机制，进而为研究博尔纳病病毒持续感染中枢神经系统的机制提供基础。此外本文通过检测细胞的增殖活性发现博尔纳病病毒磷蛋白对PC-12细胞的生长具有明显的抑制作用，可能是博尔纳病病毒持续感染中枢神经系统的重要机制之一。

Abstract:

?Objective? To establish stable expressing system of Borna disease virus (BDV) phosphoprotein in PC-12 cells, and then study its influence on cell proliferation of PC-12 cells. ?Method? An expression plasmid with green fluorescence protein was cloned and identified to express BDV phosphoprotein. Cultured PC-12 cell was transfected with the recombinant plasmid by positive ion lipidsome method. Fluorescence microscopy was used to detect the expression of phosphoprotein in PC-12 cells, then G418 was added into cell culture medium to kill these cells without recombinant plasmid. We performed reverse transcriptase polymerase chain reaction (RT-PCR) in the 10th generation of treated cells to examine the expression of BDV phosphoprotein. The proliferation of treated cells and control cells was examined by methyl thiazolyl tetrazolium assay (MTT). ?Result? The recombinant plasmid was confirmed to be able to express BDV phosphoprotein and green fluorescence protein by both fluorescence and RT-PCR. BDV phosphoprotein expressed in PC-12 cell inhibited cell proliferation. ?Conclusion? We established a stable expressing system of BDV phosphoprotein in PC-12 cell. This cell model can be used to study the effect of BDV phosphoprotein on the centre nervous system without exposure to live virus.