原发性帕金森病患者皮肤活检术检测自噬功能研究

Skin biopsy for assessment of autophagy dysregulation in the pathogenesis of Parkinson's disease

目的运用皮肤活检术分别从自噬以及分子伴侣介导的自噬两个方面评价原发性帕金森病(parkinson’s disease,PD)患者皮肤成纤维细胞的自噬水平的变化,初步探讨自噬在PD发病机制中的作用。方法收集PD患者23例及正常对照20例皮肤标本,并对PD病人进行UPDRSⅡ、UPDRSⅢ、Hoehn&Yahr分级法量表评分,运用皮肤活检术收集PD组的皮肤标本。通过免疫组化法检测微管相关蛋白轻链3(microtubule associated protein 1light chain3,LC3)、Beclin-1的表达,用蛋白质印迹法(Western blot)检测LC3Ⅱ/LC3Ⅰ的比值变化以及Beclin-1。结果与正常对照组相比,PD患者皮肤成纤维细胞Beclin-1、LC3表达上调,差异有统计学意义(P<0.05),LC3Ⅱ/LC3Ⅰ比值升高,差异有统计学意义(P<0.05)。PD患者皮肤成纤维细胞巨自噬水平明显上调,自噬水平异常调控可能参与PD的发生、发展过程。结论通过皮肤活检术检测PD患者皮肤自噬水平用于帕金森病的早期诊断具有一定的临床价值。

Objective To assessment the function of chaperone‐mediated autophagy （CMA） ,and macroautophagy in Par‐kinson’s disease patients using skin biopsy ,and explore the role of autophagy in pathogenesis of Parkinson’s disease. Methods The study samples included 23 PD patients and 20 normal controls. Clinical data of all PD patients were collected ,and graded by UPDRSⅡ ,Hoehn ＆ Yahr scales. Punch skin biopsies were taken from PD patients. Skin biopsy specimens were immuno‐histochemically stained with anti‐LC3 ,anti‐Beclin‐1 and anti‐LAMP2a antibodies. The expression of microtubule‐associated protein1 light chain 3（LC‐3） ,beclin‐1 ,heat shock cognate protein70 （HSC‐70） ,lysosome‐associated membrane protein 2a （LAMP2a） were detected by Western blot.Results Beclin‐1 ,LC3 immune positive cells optical density value in skin fibro‐blasts of PD was significantly more than that of the healthy controls （P〈0.05）. Western blot result showed LC3Ⅱ /LC3Ⅰ , Beclin‐1 expression was significantly higher than that of control subjects （P〈0.05）.Conclusion Macroautophagy activity was higher in PD patients’ fibroblasts ,and dysregulation of macroautophagy may participate in the process of PD.