脂多糖建立帕金森病细胞模型的探索

Exploration of establishment of lipopolysaccharide-induced PC12 cell model of Parkinson's disease

目的探讨脂多糖(lipopolysaccharide,LPS)诱导PC12细胞建立帕金森病(Parkinson disease,PD)炎症细胞模型的方法。方法 PC12细胞培养后加入LPS,分别为1000 ng/mL、600 ng/mL、200 ng/mL、50 ng/mL共5组,各组加入LPS 24 h,观察PC12细胞形态变化,采用CCK8法检测细胞活力;Hoechst荧光染色法检测核凋亡情况;细胞免疫组化方法检测酪氨酸羟化酶(tyrosine hydroxylase,TH)的表达情况。结果单纯PC12细胞组,细胞生长较密,神经突起细长;在LPS组24 h时,50 ng/mL组细胞形态基本无明显变;200 ng/mL组细胞突起变短,600ng/mL组细胞突起短缩明显伴有肿胀;1000 ng/mL组细胞圆缩明显增多。CCK8试验显示LPS浓度分别为50 ng/mL、600 ng/mL、200 ng/mL、1000 ng/mL时,细胞活性下降到92.3%、72.3%、49.5%和15.2%。Hochest核染色可以看到,50ng/mL偶见到核固缩,200 ng/mL有部分核产生固缩情况,1000 ng/mL几乎均表现核萎缩,凋亡。以600 ng/mL浓度时,核固缩情况占据40%左右。选择最佳建模浓度为600 ng/mL,免疫组化显示在LPS浓度600 ng/mL时,TH可有表达。说明PC12细胞在脂多糖诱导下可以有一定向多巴胺能细胞分化的能力。结论 600ng/mL浓度的LPS 24 h诱导PC12细胞所建立的PD模型能够很好地模拟PD病理改变,可作为研究PD免疫炎症发病机制的细胞模型。

Objective To explore a method of a cell model of Parkinson＇s disease（PD） by lipopolysaccharide（LPS） on PC12 cells.Methods PC12 cells were exposed to LPS（1000 ng/mL、600 ng/mL、200 ng/mL、50 ng/mL） for 24 hour. The change of cell morphology were watched and cell viability was measures by CCK8. Hoechst fluorescence staining method was used to confirm the nuclear apoptosis.The expression of tyrosine hydroxylase was detected by mmunohistochemical.Results The PC12 cell growth was dense and obvious protrusion.The morphology of PC12 cell made great change after giving LPS for 24 h. The morphology in 50 ng/mL was not significantly changed. The cell protuberances were shortened in 200 ng/m. Some of cells in 600 ng/mL were swelling, There was a significant increase of nuclear apoptosis in the 1000 ng/m I. CCK8 assay showed that the PC12 cell activity was significant reduced to 92.3%, 72. 3%, 49.5% and15.2% separately after 50 ng/mL, 600 ng/mL, 200 ng/mL, 1000 ng/mL LPS treating for 24 h. Hochest nuclear staining show that I have seen nuclear fixation were seen at random in 50 ng/mL; partial nucleation production and shrinkage can be seen in 200 ng/mL; there has almost all the nuclear atrophy and apoptosis in 1000 ng/mL. So the appropriate concentration was 600 ng/mL because of its nuclear shrinkage was almost about 50%. At this situation, TH also could be detected by immunohistochemical. The PC12 cells LPS-induced has ability of differentiation to dopaminergic cells. Conclusion The model establishes in 600 ng/mL LPS which induces for 24 h can simulate the pathololgical characteristic of PD very well. Thus, LPS as a neurotoxin can successfully establish PD cell model, which also enable the elucidation of the immune inflammation etiology.