| Objective：Proteomics, with the two-dimensional gel electrophoresis (2-DE), massspectrometry(MS) and bioinformatics as key technologies, has provided a goodplatform for bio-scientific research. It demonstrated the changes of protein expressionlevels and interactions between proteins dynamically and wholely. Research ofdifferential proteomics in ulcerative colitis(UC) was carried out to investigate UC-associated proteins and potential biomarkers for diagnosis. The ulcerative colitis modelswith immune sensitization plus the method of local acetic acid stimulation will beestablished and then evaluated in order to reveal the pathogenesis and search biomarkersand therapeutic targets for UC.Methods:(1) UC-diseased and normal colon tissues were taken from9UC patients.Two-dimensional gel electrophoresis(2-DE) was performed to seprate proteins from twogroups. PD quest software was applied to analyze2-DE images, and part of thedifferentially expressed proteins between two groups were identified by matrix-assistedlaser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS).(2)Sixty SPF-grade healthy SD rats were divided into4groups randomly. Based onsensitization of mucous membrane of colon, the control group was irrigated withphysiological saline for two times; the other3groups were injected with the acetic acidat the dose of3%,5%,8%separately for two times; in addition, the interval of twoenemas lasted one week. At the end of1st,2nd,3rdweek after irrigation, the1/3rats alivewere selected and killed from each group. The gross morphology and the pathologicslices of colon from each group were observed. Meanwhile, the concerning expressionlevel of serum TNF-α, IL-4were also evaluated. Results:(1)Two-dimensional maps of two groups were successfully established.Gel-analysis software detected27different spots, an average of1720±35inUC-diseased group, and1648±32in healthy controls. Ten spots were selected, seven ofthem were identified by MALDI-TOF-MS. Functional analysis of these proteinsrevealed that they were related to cell cycle regulation, signal transduction, apoptosis,stress response, cytoskeleton and enzymehydrolysis.(2)①The common situations asfollows: within the1stweek after modeling, the rats of each group totally had thesymptoms with different level, such as: loose stools, spirit drooping, decrease of eating,weight loss etc. However, the group with acetic acid at low dose was recovered withinthe2ndweek, and the group with acetic acid at medium, high dose had the abovementioned symptoms for3weeks. The rats of control group had the good spirit, eating,and stools, while kept weight growth slowly within1st~3rdweek.②The score ofgross morphology: within the1st~2ndweek after model establishment, the group withacetic acid at high dose got higher score than the one with acetic acid at mediumdose(P<0.01), but both groups got higher scores than the one with acetic acid at lowdose and control group. Within the3rdweek after model establishment, both groups withacetic acid at high dose and the one with acetic acid at medium dose got higher scorethan low dose group and control group(P<0.01); there was no significant differenceabout the scores between the group with acetic acid at high dose and the one with aceticacid at medium dose(P>0.05); also there was no significant statistic difference about thescores between the group with acetic acid at low dose and the control group(P>0.05).③Histological changes: within the1st,2ndweek, the high dose group had the classicsymptoms such as ulcer, crypt abscess, acute and chronic inflammatory cell infiltrationetc. Within the1stweek, the low dose group had the symptoms of mucosa mildinflammation, and began to improve within the2ndweek. At the end of the3rdweek, thegroup with acetic acid at medium dose still had ulcerative lesions; but the group withacetic acid at low dose had the symptoms such as tissue recovery, disorderedarrangement of gland structure. Within the1st~3rdweek after model establishment, the rats of control group had the symptoms of hyperemia and edema of mucosa, but noappearance of ulcer.④Detection of serum cytokine levels: Within the1st~3rdweek,the two groups with acetic acid at high and medium dose had higher level of serumTNF-αthan the low dose group and control group(P<0.01). Meanwhile, the two groupswith acetic acid at high and medium dose had lower level of IL-4than the low dosegroup and control group(P<0.01).Conclusion:(1) Seven different proteins were identified between UC-diseased colonand normal colon, including PHB, HSP, Alpha-1antitrypsin, VIM, Caspase-1,Cytokeratin20, FLNa. These proteins may be the candidate biomarkers for UC;Proteomic technology was evaluated an effective way for screening biomarkers.(2) Theanimal model with UC was successfully induced by immune sensitization combinedwith local acetic acid stimulation. Therefore, this method can be regarded as effective tostudy human UC. |
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