热休克蛋白对多巴胺能神经细胞的保护作用研究

Heat shock protein protects dopaminergic nerve cells

目的探讨热休克蛋白(HSP)对蛋白酶体抑制剂处理的多巴胺能神经细胞的活力以及细胞内包涵体形成的影响。方法将PC12细胞进行热处理,免疫印迹法鉴定热休克蛋白表达,并确定最佳热处理条件;再应用高选择性的蛋白酶体抑制剂Lactacystin处理PC12细胞。MTT方法检测细胞活力,免疫荧光细胞化学染色观察细胞内包涵体形成的变化。结果免疫印迹法证实PC12细胞热处理2h后HSP70水平即开始迅速升高,一直持续至24h,其中4h的HSP70水平表达较高,故选其为最佳观察条件。未经热处理的对照组经5μM、10μM、15μM和20μMLactacystin处理24h后,PC12细胞的活力显著降低,呈剂量依赖性;热处理组的细胞活力比对照组显著升高,两者有显著性差异(P<0.01)。免疫荧光染色显示对照组细胞的胞浆内包涵体明显增多,而热处理组胞浆内包涵体明显减少。结论热休克蛋白能显著增强细胞活力,减少细胞内包涵体形成,对多巴胺能神经元可能有一定的保护作用。

Objective To investigate the effects of heat shock protein on vitality d the dopaminergic nerve cells treated with proteasoreal inhibitor and on the formation of intracellular inclusions. Methods PC12 cells were expesed to 41.5℃ water for 4 hrs （ heat-treated group） or to room-temperature （ control group）, and were then exposed to a highly specific proteasomal inhibitor Laetacystin at rations concentrations （5, 10, 15 and 20 μM）. Cell vitality was measured by MTT assay. The formation of inclusions was determined with immunofluorescence cytochemistry staining. The expression of heat shock protein was analyzed by Western Blot. Results Western Blot analysis demonstrated that HSP70 was observed in PC12 cells from 2 hrs to 24 hrs of heating, peaking at 4 hrs. After exposed to Lactacystin at various concentrations for 24 hrs, cell vitality decreased in a dose-dependent manner in the control group. Cell vitality in the heat-treated group was significantly higher than that in the control group （P 〈 0.01 ）. In addition, immunofluorescence cytochemistry staining showed that PC12 cells had significantly fewer intracellular inclusions in the heat-treated group than the control group. Conclusions HSP may increase the vitality of the dopaminergic neuron cells and reduce the formation of intracellular inclusions, thus providing a protective effect dopaminergic nerve cells.