| Glioma occurs in 40～50% of brain primary neoplasm, more than 50% are all malignant glioma. Although the survival time of low grade glioma patients last several years, they always recurrent and progress to high grade in post-operation.It was report that 20～40% patients with malignant neoplasm would occur brain metastasis. Brain metastasis are ten times more than malignant glioma. Many patients around 40～50% with brain metastasis always have just single focus. It is often difficult to make exact diagnosis of brain metastasis and glioma just depending on clinic material. For they have the same imaging characters such as enhancing, edema around the tumor, necrosis in the centre of the neoplasm.In the development of proteomics, especially the SELDI-TOF-MS technology which is based on the development of mass technology and protein chips is abroad used in clinic. Combined biomarker detected by SELDI-TOF-MS has higher sensitivity and specificity than single biomarker in diagnosis of tumor. So we analysis CSF of patients with brain malignant glioma or brain metastasis directly using SELDI-TOF-MS in order to set up CSF protein fingerprint model for distinguished diagnosis between brain malignant glioma and brain metastasis. At the same time we culture the glioma cell in free serum median, collect the median, analysis the median by SELDI-TOF-MS and compare the result with the CSF protein fingerprint in order that we can obtain some secreted protein fingerprints that are only or abroad distributed in CSF of malignant glioma.OBJECTIVETo set up CSF protein fingerprint model for distinguished diagnosis between high-grade glioma and brain metastasis. Based on CSF protein fingerprint of malignant glioma, brain metastasis and non-tumor diseases of brain, to select potential biomarker peaks combined with the protein fingerprint of the median.MATERIAL AND METHODS1.1 MATERIAL: sixty-one CSF: twenty-five CSF of glioma(fifteen CSF of glioma III, nine CSF of glioma IV, one CSF of glioma III in post-operation, one CSF of glioma IV in post-operation). Seventeen CSF of metastasis, one CSF of patient with suspect brain metastasis. Eighteen CSF of non-tumor diseases of brain. One median of primarily cultured glioma cell, six median of secondarily cultured glioma cell which had been passaged for 36 times (there median of the cell which had been cultured 24hours, 48hours and 72hours. The others of the cell which wass cultured with protease inhibitor were obtained at the same three time points.). Two median of secondarily cultured glioma cell which had been passaged for 40 times.(one median of the cell which had been cultured 24hours, The others of the cell which wass cultured with protease inhibitor wass obtained at the same time point.)1.2 Facility: Surface Enhanced Laser Desorption/Ionization-Time of Flight-Mass Spectrometry(SELDI-TOF-MS), (PBS II~+, Ciphergen Inc. America).1.3 protein chip: CM10 which has a layer of anti-water coat on the surface is attributed to WCX-2. So it is provided with both advantage of WCX-2 and H4.1.4 Internet application and the evaluation standard of the biomarker: Internet application: spss10.1 software, Ciphergen Software 3.1, Biomarker Wizards and ZUCI PDAS software. The evaluation standard of the biomarker: sensitivity and specificity.2 METHODS2.1 Sample preparation: diluted the sample using dilute solute in equal volume. Adjusted the sample concentration in binging buffer. Arrays processed with 50%saturated SPA solution being prepared in 50% acetonitrile and 0.5%TFA.2.2 Divided the sample into different group2.2.1 CSFGroup 1: malignant glioma (23) VS brain metastasis (17);Group 2: malignant glioma (23) VS non-tumor disease of the nervous system (18)Group 3: brain metastasis (17) VS non-tumor disease of the nervous system (18)Group 4: pre-operation of malignant glioma(2) VS post-operation of malignant glioma2.2.2 The median of glioma cellThe median of the primary glioma cell VS the median of the secondary glioma cell.Compared the median of different time points of the secondary glioma cell.The median of the glioma cell which was culture with protease inhibitor VS the median of the glioma cell which was culture without it.2.3 The methods of modelingSelected the different M/Z in Statistics.Analyzed the account in Bioinformatics software, selected M/Z for the model.Evaluated the sensitivity and the specificity of the model.2.4 Selected the potential secreted protein for identificationMade sure the secreted protein M/Z value in the median.Based on the secreted protein M/Z value, selected the around protein M/Z value in the CSF of patients with glioma but not in patients with brain metastasis or non-tumor disease of the nervous system.Detected the potential protein M/Z value in the CSF of the patient in post-operation.RESULT3.1 Comparing the CSF protein fingerprint of glioma with metastasis, 170 peaks had been detected. Analyzed the account by ZUCI PDAS software,3912.7851 and 8930.644m/z showed very good power for discriminating glioma from metastasis. The model which was constituted by 3912.7851 and 8930.644 m/z separated glioma patients from metastasis with the specificity of 82.35%, sensitivity of 88.00%.3.2 the distribution of protein peaks in the median at different time pointsDetected 51 peaks in median of glioma cell which had been cultured in free serum median for 24 hours. While detected 25 peaks with protease inhibitor during the process of culture.Detected 48 peaks in median of glioma cell which had been cultured in free serum median for 48 hours. While detected 80 peaks with protease inhibitor during the process of culture.Detected 26 peaks in median of glioma cell which had been cultured in free serum median for 72 hours. While detected 24 peaks with protease inhibitor during the process of culture.3.3 the secreted protein profile of glioma cellThe glioma cell which had been passaged 40times had been cultured in free serum for 24 hours and the median was collected. Through analyzed the median we obtained 7 protein peaks which lied at: 2153.78, 4100.91, 9112.07,13672.26,59122.69,79992.74, 88860.08The glioma cell which had been passaged 40 times had been cultured in free serum with protease inhibitor for 24 hours and the median was collected. Through analyzed the median we obtained 12 protein peaks which lied at: 3231.11, 4311.71, 4361.23, 5883.80, 5995.94, 6521.67, 8424.99,9116.45, 10050.51, 13679.32, 59066.09, 88974.01。At last we selected 11 secreted protein peaks which lied at 2153.78, 3231.11, 4311.71, 4361.23, 5883.80, 5995.94, 8424.99, 9116.45, 10050.51, 59066.09, 88974.01。3.4 selecting the highly abundant secreted protein peaks in CSF of patients with malignant glioma.9075～9120: highly expressed in thirteen CSF of patients with glioma, only two CSF of patients with metastasis and one CSF of patients with non-tumor nervous system disease. Comparing to post-operation highly expressed in pre-operation. Highly expressed in median of both primary glioma cell and secondary glioma cell.8.1KD～8.5KD: highly expressed in twenty-one CSF of patients with glioma, eight CSF of patients with metastasis and five CSF of patients with non-tumor nervous system disease. Comparing to post-operation highly expressed in pre-operation.10020KD～10070KD: highly expressed in ten CSF of patients with glioma, six CSF of patients with metastasis and five CSF of patients with non-tumor nervous system disease. Highly expressed in median of glioma cell which had been passaged for 40 times.Conclusion1. The model constituted of 3912.7851, 8930.644 is able to distinguish brainmetastasis and malignant glioma.2. The secreted protein peaks such as 9100Da, 8182Da, 10050Da in CSF of patient with glioma are the potential biomarker for glioma. As to the peak 8930Da which consist the model is also the potential biomarker for glioma.
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