The Proteomics Study Of Cerebrospinal Fluid From Patients With Ependymoma

Ependymoma is a kind of CNS tumor. Recently the researches of ependymoma are mainly focused on genomic study, which reveals that this disease is related with the allelomorphic gene deletion. However, most of these researches were limited in some specific areas and can not disclose the formation and nature of ependymoma comprehensively.Proteomics technique is performed on the biosystem or cell level, other than single protein. The current technology can trace the disease-related proteins after analyzing the proteomic transmutation. There are no reports about the proteomics study of cerebrospinal fluid from patients with ependymoma in the domestic. In this paper, a method of Two-dimensional Gel Electrophoresis with high reproducibility and resolution is established. The conclusions can provide data for setting up more advanced experiment methods, and improve the relevant preciseness and reproducibility, and then identify the different protein'spots by means of mass spectrum.Objective: To evaluate the differences of cerebrospinal fluid proteins extracted between ependymoma patients and normal controls according to the result of the Two-dimensional Gel Electrophoresis.Method: The cerebrospinal fluid proteins from patients with ependymoma and normal controls are precipitated by three kinds of sediment methods, followed by dissolving the sediment with lysate and OD value. Three kinds of sediment methods were compared according to OD values. Standard curves are plotted by Bradford method, and then the protein concentration of the cerebrospinal fluid is calculated. Next the cerebrospinal fluid protein is separated by IEF and SDS-PAGE electrophoresis. And the 2-DE gel is stained by Silver Nitrate. In the end, the 2-DE gel maps are analyzed by special software to compare the difference between cerebrospinal fluid proteins of ependymoma patients and normal group.Results: 1. Comparison of the cerebrospinal fluid proteins from three different sediment methods: the protein precipitated by acetone of TCA. 2. Measuring the protein concentration by Bradford method: the standard curve is produced from BSA, on which the protein concentration is calculated to determine the sample quantity for electrophoresis. 3. The results of 2-DE: Cerebrospinal fluid proteins are separated by 2-DE, and then analyzed after silver staining. Each sample of different groups is electrophoresised twice to guarantee the reproducibility of these methods. The results are very close and the number of protein'spots distributed at the range of pH4-7and MW10-200kD is the most. After detecting the gels spots, the 2-DE gels of the normal controls are compared. Under the same condition, the detected spots are:The spots of the normal controls are 239±20, the spots of ependymoma patients are 264±32, the matching spots are 186±28 pairs, and the matching rate is 73.9%.The difference of %vol between the matching gels is analyzed through software. By defining %vol at 1.5, the changes of gels are recorded. There are 19 different spots in the first group, include 12 up-regulation'spots and 7 down-regulation'spots.Conclusion: Ependymoma is a kind of CNS tumor. Its pathogenesis is not well known, and there are no other effective treatments except operations. Proteomics technique can contribute to recognizing the occurrence and development of ependymoma, finding the tumor-related proteins, determining the target of drug treatment, and diagnosing and treating for the tumors.1. It is the first time to evaluate the differences of cerebrospinal proteins extracted from ependymoma patients and normal controls by the method of proteomics. The Two-dimensional Gel Electrophoresis system is established to effectively segregate the proteins.2. After performing the research on the techniques of Two-dimensional Electrophoresis, a stable experimental process of 2-DE is established.3. There are some different expressions of the cerebrospinal fluid proteins between the two contrast groups. The proteins'spots with obviously changed relative amount in 2-DE atlas are discovered through the special software. These spots indicate that some tumor-related proteins probably exist in the cerebrospinal fluid. These 2-DE atlases are high reproducible and will provide references for the future study of the CNS's tumor.