GSK3β调节神经细胞自噬在帕金森病发病中的作用及机制研究

GSK3β to adjust the role of autophagy in Parkinson＇s disease nerve cells and mechanism research

目的探讨表达不同活性的糖原合成激酶3β（GSK3β）对自噬通路的调节是否会引起α-突触核蛋白自噬水平的变化及其对细胞生长的影响。方法筛选稳定表达A30P α-突触核蛋白的PC12细胞株为研究对象，通过对GSK3β的过表达及沉默，检测GSK3β转染后的表达情况和各组细胞中LC．3Ⅱ、Beclinl、α-突触核蛋白的表达，以及细胞增殖能力、细胞凋亡变化。结果（1）与GSK3β沉默组、空白质粒组、空白对照组相比，GSK3β过表达组LC-3Ⅱ及Beclin1的表达增加（P〈0．05），α-突触核蛋白的表达减少（P〈0．05），MTT及TUNEL法结果表明细胞的凋亡下降，细胞增殖增加（P〈0．05）。（2）与GSK3β过表达组、空白质粒组、空白对照组相比，GSK3β沉默组LC-3Ⅱ及Beclin1的表达减少（P〈0．05）；α-突触核蛋白的表达增加（P〈0．05），MTT及TUNEL法结果表明细胞的凋亡增加，细胞增殖下降（P〈0．05）。结论（1）激活GSK3β活性可提高细胞自噬水平，增强细胞对α-突触核蛋白的降解能力，从而促进细胞存活。（2）抑制GSK3β活性会下调细胞自噬水平，减弱细胞对α-突触核蛋白的降解能力，从而减少细胞存活。（3）自噬与帕金森病（PD）的发病密切相关，提高自噬水平降解细胞内病理性聚集的α-突触核蛋白是未来治疗PD的一个新途径。

Objective To screen stable express A30P a-synuclein PC12 cell as the research object, and to preliminarily investigate the activity of different glycogen systhesis kinase 3[3 （GSK313） for the regulation of autophagy pathway causes a-synuclein autophagy level change and its effects on cell growth. Methods The vector of A30P a-synuclein was used to transfect to PC12 cells. G418 was used to screen stable expressed ceils. Liposome method was used to transfect GSK3β-expressed plasmid, GSK3β silence plasmid, and blank plasmid into these cell lines, and stable expressed activity of GSK3β cell lines were screened. Western blot was used to detect the expression of GSK3β after transfection and verify the transfection efficiency. Western blot was used to test groups of ceils in LC-3 Ⅱ , Beclinl, and a-synuclein expressions. Methyl thiazolyl tetrazolium （MTT） method was used to detect the change of cell proliferation. Terminal dexynucleotidyl transferase （TdT） mediated dUTP nick end labeling （TUNEL） method was used to detect apoptosis. Results （1） When GSK3β expressed group was compared to GSK3β silence group, blank plasmidgroup, and blank control group, the expressions of LC-3 11 and Beclinl were significantly increased （ P 〈 0.05 ）, the expression of a-synuclein was significantly reduced （ P 〈 0. 05 ）, the results of MTT and TUNEL showed that the cells were the decrease of apoptosis and increase of cell proliferation, with a statistically significant difference （P 〈 0. 05 ）. （2） When GSK313 silence group was compared to GSK3β express group, blank plasmidgroup, and blank control group, the expressions of LC-3 11 and Beclinl were significantly reduced （ P 〈 0. 05）, the expression of a-synuclein was significantly increased （ P 〈 0. 05）, the resuits of MTT and TUNEL showed that the ceils were the increase of apoptosis and decrease of cell proliferation, with a statistically significant difference （P 〈 0. 05）. Conclusions （1） Activation of GSK3β activity can improve the level of cell autophagy, enhance the ability of alpha-synuclein degradation, and promote cell survival. （2） Inhibition of GSK3β activity can reduce the level of cell autophagy, weaken the ability of alpha-synucleindegradation, and reduce cell survival. （3） Autophagy is closely related to the pathogenesis of Parkinson＇s disease （PD）. The improvement of the level of autophagy and enhancing of the degradation of asynuclein is a new way of the future treatment of PD.