| Background:Both multiple sclerosis (MS) and neuromyelitis optica (NMO) are demyelinating diseases of the central nervous system and their underlying mechanisms are unknown. The primary pathological process is demyelinating change, and the clinical manifestations vary with the involved part of the central nervous system.Although multiple sclerosis and neuromyelitis optica both belong to demyelinating diseases of the central nervous system, there are many differences in the pathogenesis, clinical manifestation, clinical characteristics, treatment protocols, etc between them, particularly after the discovery of autoantibody NMO-IgG in the serum of the NMO patients, the current tendency is that NMO is an isolated disease rather than a subtype of MS. However, the difficulty in the collection of the tissue sample of central nervous system impedes the further investigation of pathological mechanisms of MS and NMO. Moreover, as an important component part of internal environment, the body fluid contains polypeptides, proteins and enzymes which play very important roles in the physiological processes, and the changes of the proteins and polypeptides in quality and quantity can reflect the pathophysiological processes under disease accurately, meanwhile, the complex immune responses in MS and NMO result dynamic changes of the types and quantity of the proteins in cerebrospinal fluid, serum, brain tissue and various subcellularorganelle. In consideration of these factors, the information about the occurrence, progression and the compensatory mechanisms of body to disease (e.g. nerve protection, tissue remodeling, inflammatory reaction etc) may be obtained through the study on the changes of special proteins in serum and cerebrospinal fluids of the patients with MS or NMO.The conception of proteome is an integral and dynamic conception, which mainly includes the qualitative analysis and quantification of total proteins and the functional analysis of massive proteins. Proteomics is a science studying the proteome expressed in cell, tissue and individual on the basis of the changes of the types and quantity of proteins as well as the time the space of local presence and analyzing life activities comprehensively from the structural and functional aspects. The most significant feature of the proteomics study is its capability of high throughput comprehensive analysis to the protein components in body fluid, tissue or cells under physiological and pathological conditions, which contributes to the overall understanding about the complex and treacherous diseases of nervous system.This experiment includes the proteomics study on the serum and cerebrospinal fluids from the patients with MS or NMO, protein isolation and identification, the analysis and discussion about the proteomics characteristics of the serum and cerebrospinal fluids from the patients with MS or NMO as well as the comparative study on these diseases in order to explore the pathological mechanisms of them further and provide some auxiliary evidence for the diagnosis and differential diagnosis of MS and NMO. Part One Proteomics Study On The Serum And Cerebrospinal Fluids From MS PatientsPurpose:To establish the 2-D electrophoresis pattern of the serum and cerebrospinal fluids from MS patients, observe the differential expression through the comparison of the 2-D electrophoresis results of the serum and cerebrospinal fluids between the MS group and normal control group, identify the proteins with differential expression and discuss the significance.Method:1. Collect the samples of serum and cerebrospinal fluids from MS group and the normal control group, conduct total protein isolation and quantify the protein by Bradford method.2. The 2-D electrophoresis of the sample with equal amount of serum and cerebrospinal fluid is run in triplets, in which, the first dimension is isoelectric focusing electrophoresis and the second is SDS-PAGE gel electrophoresis.3. Following silver staining, the software ImageMaster 2D Platinum5.0 is used to analyze the images.4. Select the interested proteins spots of both groups (the difference of the volume percent for the spots in the 2-D electrophoresis patterns of two groups is over twofold or the spots with the difference of presenting or not presenting), remove the spots from the gel for sampling, digest the samples by enzymolysis and identify the proteins by high-sensitivity HD-ms/ms mass spectrometry.5. The online software MASCOT is utilized to search the proteins.Result:Ⅰ. Results of serum: 1. There are 414 protein spots in the 2-D electrophoresis pattern of the serum from MS group and 476 proteins in the 2-D electrophoresis pattern of the serum spots from the normal control group in total.2. Comparison of the 2-D electrophoresis pattern of serum between the MS group and the normal control group showed that there are 345 matched protein spots,159 proteins spots in MS group are up-regulated compared with those in the normal control group, among which, the differences between 42 proteins spots are over twofold, and 186 proteins spots in MS group are down-regulated. For the unmatched proteins spots in these two groups, there are 69 spots in MS group and 131 spots in the normal control group.3. Compared with the 2-D electrophoresis pattern of serum from the normal control group, the number of unexpressed proteins spots in MS group is 81.4. Two differential proteins are identified through HD-ms/ms mass spectrometry and MASCOT network searching:immunoglobinλ, and keratin 83, the former only presents in the 2-D electrophoresis pattern of serum from the patients with multiple sclerosis, and the expression of the latter in MS group is far over twofold of that in the normal control group.Ⅱ. Results of CSF:1. There are 118 protein spots in the 2-D electrophoresis pattern of CSF from MS group and 350 proteins in the 2-D electrophoresis pattern of CSF from the normal control group in total.2. Comparison of the 2-D electrophoresis pattern of CSF between the MS group and the normal control group showed that there are 51 matched protein spots,38 proteins spots in MS group are up-regulated compared with those in the normal control group, among which, the differences between 24 proteins spots are over twofold, and 13 proteins spots in MS group are down-regulated. For the unmatched proteins spots in these two groups, there are 67 spots in MS group and 299 spots in the normal control group.3. Three differential proteins are identified through HD-ms/ms mass spectrometry and MASCOT network searching:prealbumin, keratinl and keratin 9, prealbumin only presents in the 2-D electrophoresis pattern of CSF from the patients with multiple sclerosis, keratinl only presents in the 2-D electrophoresis pattern of CSF from the normal control group and the expression of keratin 9 in MS group is far over twofold of that in the normal control group.Conclusion:1. The expression of a variety of proteins in the 2-D electrophoresis pattern of serum was significantly different in the Multiple sclerosis patients comparison to normal control group.2. Ig lamda chain and keratin 83 have been identified as significantly different serum proteins. Enhanced expreesion of Ig lamda chain in serum may be related to Immune mechanism of MS.3. The expression of a variety of proteins in the 2-D electrophoresis pattern of CSF was significantly different in the Multiple sclerosis patients comparison to normal control group.4. Prealbumin, keratinl and keratin 9 have been identified as significantly different CSF proteins.Part Two Proteomics Study On The Serum And Cerebrospinal Fluids From NMO PatientsPurpose:To establish the 2-D electrophoresis pattern of the serum and cerebrospinal fluids from NMO patients, observe the differential expression through the comparison of the 2-D electrophoresis results of the serum and cerebrospinal fluids between the NMO group and normal control group, identify the proteins with differential expression and discuss the significance.Method:1. Collect the samples of serum and cerebrospinal fluids from NMO group and the normal control group, conduct total protein isolation and quantify the protein by Bradford method.2. The 2-D electrophoresis of the sample with equal amount of serum and cerebrospinal fluid is run in triplets, in which, the first dimension is isoelectric focusing electrophoresis and the second is SDS-PAGE gel electrophoresis.3. Following silver staining, the software ImageMaster 2D Platinum5.0 is used to analyze the images.4. Select the interested proteins spots of both groups (the difference of the volume percent for the spots in the 2-D electrophoresis patterns of two groups is over twofold or the spots with the difference of presenting or not presenting), remove the spots from the gel for sampling, digest the samples by enzymolysis and identify the proteins by high-sensitivity HD-ms/ms mass spectrometry.5. The online software MASCOT is utilized to search the proteins.Result:Ⅰ. Results of serum:1. There are 472 protein spots in the 2-D electrophoresis pattern of the serum from NMO group and 476 proteins in the 2-D electrophoresis pattern of the serum spots from the normal control group in total.2. Comparison of the 2-D electrophoresis pattern of serum between the NMO group and the normal control group showed that there are 374 matched protein spots, 175 proteins spots in NMO group are up-regulated compared with those in the normal control group, among which, the differences between 57 proteins spots are over twofold, and 199 proteins spots in MS group are down-regulated. For the unmatched proteins spots in these two groups, there are 98 spots in NMO group and 102 spots in the normal control group.3. Compared with the 2-D electrophoresis pattern of serum from the normal control group, the number of unexpressed proteins spots in NMO group is 52.4. One differential proteins are identified through HD-ms/ms mass spectrometry and MASCOT network searching:haptoglobin Hp2, and the expression of which in NMO group is far over twofold of that in the normal control group.Ⅱ. Results of CSF:1. There are 155 protein spots in the 2-D electrophoresis pattern of CSF from NMO group and 350 proteins in the 2-D electrophoresis pattern of CSF from the normal control group in total.2. Comparison of the 2-D electrophoresis pattern of CSF between the NMO group and the normal control group showed that there are 82 matched protein spots,64 proteins spots in MS group are up-regulated compared with those in the normal control group, among which, the differences between 47 proteins spots are over twofold, and 18 proteins spots in NMO group are down-regulated. For the unmatched proteins spots in these two groups, there are 73 spots in NMO group and 268 spots in the normal control group.3. Three differential proteins are identified through HD-ms/ms mass spectrometry and MASCOT network searching:transferrin, keratin 1 and keratin 9. The expression of transferrin, keratin 1 and keratin 9 in NMO group is far over twofold of that in the normal control group. Conclusion:1. The expression of a variety of proteins in the 2-D electrophoresis pattern of serum was significantly different in the NMO patients comparison to normal control group.2. Haptoglobin Hp2 has been identified as significantly different serum proteins. Enhanced expreesion of haptoglobin Hp2 in serum may be related to Immune regulation mechanism of NMO.3. The expression of a variety of proteins in the 2-D electrophoresis pattern of CSF was significantly different in the NMO patients comparison to normal control group.4. Transferrin, keratinl and keratin 9 have been identified as significantly different CSF proteins.5. Enhanced expreesion of Itransferrin in CSF may be related to blood-brain barrier injury of NMO.Part Three Proteomics Study On The Serum And Cerebrospinal Fluids From MS and NMO PatientsPurpose:To compare the 2-D electrophoresis pattern of the serum and cerebrospinal fluids between MS and NMO patients, identify the proteins with differential expression and discuss the significance.Method:1. Collect the samples of serum and cerebrospinal fluids from MS group and NMO group and the normal control group, conduct total protein isolation and quantify the protein by Bradford method.2. The 2-D electrophoresis of the sample with equal amount of serum and cerebrospinal fluid is run in triplets, in which, the first dimension is isoelectric focusing electrophoresis and the second is SDS-PAGE gel electrophoresis.3. Following silver staining, the software Image Master 2D Platinum5.0 is used to analyze the images.4. Select the interested proteins spots of both groups (the difference of the volume percent for the spots in the 2-D electrophoresis patterns of two groups is over twofold or the spots with the difference of presenting or not presenting), remove the spots from the gel for sampling, digest the samples by enzymolysis and identify the proteins by high-sensitivity HD-ms/ms mass spectrometry.5. The online software MASCOT is utilized to search the proteins.Result:I. Results of serum:1. There are 414 protein spots in the 2-D electrophoresis pattern of the serum from MS group and 472 proteins in the 2-D electrophoresis pattern of the serum spots from NMO group in total.2. While at the same time expressed in the 2-D electrophoresis pattern from normal control group, for the unmatched proteins spots in the following two groups, there are 52 spots in MS group and 81 spots in NMO group.3. Two differential proteins are identified through HD-ms/ms mass spectrometry and MASCOT network searching:haptoglobin and keratin 83. The expression of the former in MS group is far over two fold of that in NMO group and the expression of the latter in NMO group is far over twofold of that in MS group.Ⅱ. Results of CSF:1. There are 118 protein spots in the 2-D electrophoresis pattern of CSF from MS group and 155 proteins in the 2-D electrophoresis pattern of CSF from tNMO group in total. 2. While at the same time expressed in the 2-D electrophoresis pattern from normal control group, for the unmatched proteins spots in the following two groups, there are 14 spots in MS group and 45 spots in NMO group.3. TWO differential proteins are identified through HD-ms/ms mass spectrometry and MASCOT network searching:prealbumin and keratin 1, prealbumin only presents in the 2-D electrophoresis pattern of CSF from the patients with multiple sclerosis, keratin 1 only presents in the 2-D electrophoresis pattern of CSF from the NMO group.Conclusion:1. The expression of a variety of proteins in the 2-D electrophoresis pattern of serum and CSF was significantly different in the Multiple sclerosis patients comparison to NMO group.2. Haptoglobin, keratin 83, Prealbumin and keratinl have been identified as significantly different proteins. |
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