帕金森病患者血浆增强α-突触核蛋白聚集体稳定性的研究

Increased stability ofα-synuclein aggregates in blood plasma of patients with Parkinson's disease

目的探讨帕金森病(PD)患者血浆孵育形成的α-突触核蛋白(α-Syn)聚集体的稳定性。方法收集自2020年6月至2020年12月于桂林医学院附属医院神经内科确诊的10例PD患者外周血液样本及同期10例健康体检人员外周血液样本,用140μL 0.01 mol/L PBS将1 mg重组人α-Syn溶解后分别与70μL健康人员、PD患者血浆及PBS于37℃环境下恒温振荡孵育7 d(依次命名为HC、PD及PBS组);预成型原纤维(PFF)组采用10μgα-Syn PFF用于后续实验。利用不同浓度胰蛋白酶(胰蛋白酶:α-Syn浓度比分别为1∶80、1∶40、1∶20)、不同浓度蛋白酶K(PK)(1.0、1.5、2.0μg/mL)消化各组蛋白后采用Western blotting法检测消化前后α-Syn水平的差异。结果(1)胰蛋白酶对PFF的消化作用:随着胰蛋白酶浓度的增加,PFF逐渐减少,当胰蛋白酶与α-Syn PFF蛋白浓度比提高至1∶20时,相对分子质量>35000处的α-Syn聚集体基本被消化,与其他浓度组比较差异有统计学意义(P<0.05)。(2)胰蛋白酶对α-Syn聚集体的消化作用:在相对分子质量<25000处,当胰蛋白酶与蛋白浓度比为1∶20时,与HC和PD组比较,PBS组α-Syn表达水平降低更为显著,差异有统计学意义(P<0.05)。在相对分子质量35000~40000处,3种胰蛋白酶浓度下3组α-Syn表达水平均明显降低;与HC和PD组相比,PBS组降低均更为显著,差异均有统计学意义(P<0.05)。在相对分子质量>40000处,当浓度升高到1∶20时,PD组α-Syn才出现明显减少,被消化程度表现为PBS组>HC组>PD组,组间差异有统计学意义(P<0.05)。(3)PK对PFF的消化作用:当PK浓度为1.0、1.5及2.0μg/mL时,相对分子质量>35000处蛋白均已被基本消化,相对分子质量<25000处α-Syn单体明显减少并出现小分子片段;与阴性对照(PK浓度为0μg/mL)比较,差异均有统计学意义(P<0.05)。(4)PK对α-Syn聚集体的消化作用:在相对分子质量<25000处,PBS组α-Syn单体被消化成更小的片段,而HC和PD组α-Syn单体未见明显消化,同一浓度下组间差异有统计学意义(P<0.05)。在相对分子质量35000~40000处,随着PK浓度增加,PBS组α-Syn二聚体被消化程度明显增加;HC组α-Syn二聚体的量随着PK浓度增加而增加;而PD组α-Syn二聚体的量随PK浓度的增加变化不明显;同一浓度下组间差异有统计学意义(P<0.05)。在相对分子质量>40000处,随着PK浓度增加,PBS、HC及PD组α-Syn均出现一定程度减少,同一浓度下组间差异有统计学意义(P<0.05)。结论PD患者血浆孵育形成的α-Syn聚集体的稳定性高于HC血浆及PBS孵育形成的α-Syn聚集体。

Objective To investigate the stability ofα-synuclein(α-Syn)aggregates formed by incubation with blood plasma of patients with Parkinson's disease(PD).Methods Peripheral blood samples were collected from 10 patients diagnosed as having PD in our hospital from June 2020 to December 2020 and 10 healthy control subjects(HC)at the same time period.The 1 mg recombinant humanα-Syn was dissolved in 140μL 0.01 mol/L phosphate buffer solution(PBS),and then,incubated with plasma from HC and PD patients and PBS at 37℃for 7 d(HC group,PD group and PBS group).Preformed fiber(PFF)group was used for subsequent experiment with 10μg PFF.After digestion with different concentrations of trypsin(concentration ratios of trypsin/α-Syn=1:80,1:40,and 1:20)and protease K(PK,1.0,1.5,and 2.0μg/mL),the differences ofα-Syn levels before and after digestion were detected by Western blotting.Results(1)Effect of trypsin on PFF digestion:PFF gradually decreased with the increase of trypsin doses;when trypsin/α-Syn ratio=1:20,α-Syn aggregates with molecular weight greater than 35000 were almost completely digested,and its digestion was significantly different as compared with that in other concentration ratios(P<0.05).(2)Effect of trypsin on digestion ofα-Syn aggregates:at the relative molecular weight<25000,when the concentration ratio of trypsin/α-Syn=1:20,as compared with HC and PD groups,the PBS group had significantly more obvious decrease(P<0.05).At the relative molecular weight 35000-40000,theα-Syn levels in PBS,HC and PD groups were significantly decreased as compared with those before digestion at all concentration ratios;as compared with HC and PD groups,the PBS group had significantly more obvious decrease(P<0.05).At molecular weight>40000,α-Syn decreased significantly in PD group only when the concentration ratio of trypsin/α-Syn=1:20,and the degrees of digestion was PBS group>HC group>PD group,with significant differences among groups(P<0.05).(3)Effect of PK on PFF digestion:when PK concentration was 1.0,1.5 or 2.0μg/mL,α-Syn was basically digested at relative molecular weight>35000,andα-Syn monomer was reduced and small fragment appeared at relative molecular weight<25000;as compared with negative controls(0μg/mL PK),these changes in groups of PK concentration of 1.0,1.5 or 2.0μg/mL were significantly different(P<0.05).(4)Effect of PK on digestion ofα-Syn aggregates:at relative molecular weight<25000,α-Syn in PBS group was digested into smaller fragments whileα-Syn in HC and PD groups was not digested;significant differences was noted among groups at the same concentration(P<0.05).At relative molecular weight of 35000-40000,with the increase of PK concentration,the amount ofα-Syn dimer in PBS group decreased(increased digestibility),that in HC group increased,however,that in PD group did not change obviously;significant difference was noted at the same concentration among the three groups(P<0.05).At relative molecular weight>40000,with the increase of PK concentration,α-Syn in PBS,HC and PD groups decreased to a certain extent,and significant difference was noted among groups at the same concentration(P<0.05).Conclusion The stability ofα-Syn aggregates formed by incubation with plasma from PD patients is higher than that formed by incubation with HC plasma and PBS.