Human Peripheral Blood Mononuclear Cell Proteome Analysis Of Parkinson Disease With Ganyang Huafeng And Xuexu Shengfeng Syndromes

SUBJECT With the technology of two-dimensional polyacrylamide gel electrophoresis(2-DE), we extracted and separated total protein in Peripheral Blood Mononuclear Cell of the Parkinson Disease patients with Ganyang Huafeng Syndromes and Xuexu Shengfeng Syndromes, and the healthy persons. And we established the expression maps of proteomics in Ganyang Huafeng patients', Xuexu Shengfeng patients' and normal PBMC. According to the analysis and comparing of the differences amone the 3 groups' proteomics express, we studied the differential proteins in the PBMC of the Parkinson Disease patients with Ganyang Huafeng Syndromes and Xuexu Shengfeng Syndromes, which can be helpful to provide scientific evidence for approaching the nature of TCM Syndromes .METHODS The PBMC of the Parkinson Disease patients with Ganyang Huafeng Syndromes and Xuexu Shengfeng Syndromes, and the healthy persons was extracted with polyglucosan-diatrizoate meglumine density gradient centrifugation and split. The quantity of the total protein in each PBMC sample was determined with the BCA method.Two-dimensional polyacrylamide gel electrophoresis technology was applied to separate proteins. The protein spots were visualized with technique of Coomassie brilliant blue staining. After scanning the stained 2-DE gels, we use the PDQuest software to analysis the differential protein spots in 3 groups.Matrix-Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF-MS) and searching in SWISS-PORT and MSDB database were used to identify proteins.Finally, we verified FBX4 with RT-PCR method.RESULTS1. The expression maps of proteomics including good resolution and reproducibility in PBMC of the Parkinson Disease patients with Ganyang Huafeng Syndromes and Xuexu Shengfeng Syndromes, and the normal persons were obtained after Coomassie Brilliant Blue staining. The average spots' number of PBMC is (700±30) in healthy person, (602±15) in Parkinson Disease patients with Ganyang Huafeng Syndromes and (577±28) in Parkinson Disease patients with Xuexu Shengfeng Syndromes.we match the spots of three gels refer to the gel of heathy person group.The average matching spots of PD-GYHFs is(552±16) ,the average matching rate is 78.8%. The average matching spots of PD-XXSFs is (494±25) , the average matching rate is 70.6%.The deviation of spots among different gels is (2.85±0.48)mm in IEF direction, (2.69±0.37)mm in SDS-PAGE direction direction2. We selected 15 spots with differential expression to make mass-spectrum identification.The expressing quantity of the 15 protein reduced by turns in the PBMC's 2-DE maps of the healthy persons , PD-GYHFs and PD-XXSFs. (P<0.05) The function of the initially identified proteins related to cytoskeleton(Rho GDI-2,Talin-1,CAP-1 ),anti-oxidative stress(Prx-III),protein degradation(FBX4),signal transduction(hnRNP-K,Transgelin-2) and cell cycle(Rap1b,Rsu-1),etc.3.We verified FBX4 by RT-PCR. The expressing quantity of FBX4 reduced by turns in healthy persons , PD-GYHFs and PD-XXSFs. (P<0.05) The result is according to the proteomic experiment.CONCLUSION1. The expression maps of proteomics in PBMC of the Parkinson Disease patients with Ganyang Huafeng Syndromes and Xuexu Shengfeng Syndromes, and the healthy persons were established.2. This research revealed the protein expression difference of PBMC between the two syndromes of PD patients.These differencial proteins suggests that Parkinson's disease is due to polyprotein abnormal expression. The polyprotein differencial expression of PD-GYHFs and PD-XXSFs suggests that have different material foundation (different proteins) in a same disease with different TCM syndromes. This result settle scientific ground for essential research of TCM syndromes.3.The outcome of RT-PCR is according to the proteomic study.It shows the stability and accuracy of proteomics.