Identification Of FHL1 In Slow Transit Constipation And Its Significance

ObjectiveConstipation is a pathological process of many diseases caused by a complex symptoms. Currently clinical constipation usually divided into three categories:①slow transit constipation (STC);②outlet obstruction constipation;③mixed constipation. The STC accounts for 45.5% of the chronic constipation, and it is a relatively common clinical type. In theory any part of the digestive tract slow down the transmission of its contents may lead to constipation. But in fact slow down the transmission of 95% occurred in the colon. Therefore, STC also known as colonic slow-transit constipation, which was first proposed in 1986. It is a kind of power weakened by colon, colonic transit time extension of the main features of intractable constipation,and the vulnerable population is children and the elderly. It can cause nausea, vomiting, loss of appetite, abdominal pain, abdominal distention, and even intestinal perforation, as well as cardio-cerebral blood vessel diseases. With the accelerated pace of modern life, higher living standards, diet changes and socio-demographic aging, the incidence of STC has increased year after year.It has become a common disease which impact on quality of life of modern people. STC is a kind of the most common intractable constipation, it is very early to commence the study of its cause, but because the incidence of STC affected by many factors, its etiology is complex, and therefore it has not been entirely clear so far. We have to find the level of protein molecules in the pathogenesis of slow transit constipation to achieve to provide the necessary foundation for its pre-early treatment.Proteomics is the science and methodology with which to simultaneously study the expression of all proteins (the protome, rather than individual protein) in a cell, tissue, or organ. Proteomic analysis of identified global proteins can provide expression profiles of the proteins and their PTMs in cell. So, direct investigation on protein profiles is not substitutional. Proteomics has attracted much attention in recent years, and the global alterations in protein expression are characterized by 2-DE and mass spectrometry,in combination with bioinformatics.We use two-dimensional electrophoresis technology for STC and normal colonic tissues of protein separation, through the software analysis and mass spectrometry-Bioinformatics methods of finding disease-related proteins in the protein level of the colon the mechanism of STC, and with western-blot to detect the differences in protein FHL1 of STC and provide the basis information for early diagnosis and treatment.Methods1.clinical data and specimen collectionThe specimens used in experiments were from Shengjing Hospital affiliated China Medical University, surgical resection line of the whole colon with STC in 8 cases, preoperative diagnosis in line with the RomeⅢcriteria, age 42-62 years, with a median age of 53 years; specimens of the control group,8 cases (due to colostomy and colon polyps,no history of constipation) age 37-65 years old, with a median age of 52 years. Specimens from the body were putted into liquid nitrogen immediately,24 hours later moved to the depth of -80℃refrigerator spare.2.proteomic analysis of colon between STC and the control group(1) Total protein of colon was extracted from STC and the control group,protein concentrations were determined by Bradford.(2)For isoelectric focusing, Ettan IPGphorⅡwas used. Samples were applied to pH 3-10,24cm linear IPG strips and two-dimensional electrophoresis was performed, using 12.5% second dimension gels. Analytical gels were G-250 stained and scanned. Protein patterns were analyzed using ImageMaster 2D Platinum 6.0 software to find differential spots.(3) Significant protein spots were selected for mass spectrometry analysis and bioinformatics analysis.3. The expression of FHL1 in STC and control group(1) Total protein of colon was extracted from slow transit constipation and the control group,protein concentrations were determined by Bradford. (2) By western-blot method to verify that the protein expression in the colon, further analysis of correlation of FHL1 with slow transit constipation.Results1.Analyze the differential protein expression between STC and control group The 2D gel protein spot patterns were gained through pH3～10,24cm strips, with good reproducibility and resolution.On average,1280±30 protein spots were detected in control group,and 1080±51 in slow transit constipation.On average,the per gel spots matching rate was 70%±5%, statistical analysis using t-test method, selected protein spots differentially expressed greater than 2-fold (ratio> 2), t-test p<0.05 as the study of protein spots. Between colonic slow transit constipation group and the control group, there are 20 different spots,11 of which increase the expression of proteins,9 of which decreased expression of proteins2.The results of Proteomic identification of between STC and control group 20 easily to picked sports that have good reproducibility and obvious differences are charactered by MS.14 strains proteins were identified.3.The expression of the FHL1 in STC and control group Western blot analysis showed that FHL1 protein in the colon of slow transit constipation was significantly reduced, and was consistent with two-dimensional electrophoresis analysis results.Conclusion1.Proteomic difference between STC and normal contrals can be well presented with 2-DE. The data compared between STC and nomal control,we get 14 kinds of protein expression changes.2.Western-blot results find the expression of FHL1 is down-regulated in STC.