The Clinical Prevent And Differential Proteomics Study Of Benign Bile Duct Cicatrix Stricture

Objective1. To evaluate value of magnetic resonance cholangiopancreatography onprevention of complications in laparoscopic cholecystectomy.2. To disscuss the application of three approach of laparoscopic cholecystectomy.3. To explore the method of cultivation and identification for human benign biliaryduct cicatrix fibroblasts.4. To investigate the people benign bile duct scar stricture fibroblasts and embryoniclung fibroblasts protein expression differences and to identify the differentialprotein.5. To analyze the function of TAGLN and FKBP1A in the fibroblasts of benign bileduct scar.Method1. The clinical data of1079patients who would be performed by LC from January2006to June2010in our hospital were retrospective analysed. According to performing MRCP or not in the different period, the patients were divided into non-MRCP group and MRCP group. The data of bile duct injuries and bile duct stone residue were contrasted between two groups.2. From Jan to Dec2009,150cases laparoscopic cholecystectomy were performed in our hospital. The clinical data and surgical outcomes were retrospectively analysed.3. The benign biliary duct cicatrix tissue which sampled from clinic was cultured for fibroblasts by primary explant culture. The performance of fibroblasts was observed with light microscope, TEM and SEM. The cells’growth was observed with making cell growth curve. The cultrued cells were identified by vimentin immunofluorescence. The cells’vimentin mRNA was dectedted by RT-PCR. 4. Human benign bile duct stricture fibroblast cell line and human embryonic lung fibroblasts were cultured in vitro and total protein of two fibroblasts was extracted and quantified. Then the protein was anlysised by two-dimensional gel electrophoresis, image analysis, obtained differentially expressed proteins. Peptide mass spectrometer is used to draw the quality fingerprint spectrum, through the computer software and network information to identify differentially expressed proteins. The expression of FKBP1A, NME1TAGLN and Moesin were analysis by western blot and immune cell fluorescence in two fibroblasts.5. To build pcDNA3.1TAGLN eukaryotic expression plasmid, and screen FKBP1A siRNA interference fragment, then they were transfected to two fibroblasts. The cell proliferation and apoptosis were determined by MTT method and flow cytometry instrument. The expressions of FKBP1A, TAGLN, smadl, smad5were detected by real-time quantitative PCR and immune protein imprinting in two fibroblasts.Results1. The intraoperative and postoperative bile duct injury were found in5patients and the residual common bile duct stones were found in27patients in non-MRCP group. The intraoperative and postoperative bile duct injury and the residual common bile duct stones were not found in MRCP group. The data of bile duct injuries and bile duct stone residue were statistic difference between two groups (P<0.05). There were found double gallbladder in1patient by MRCP, Mirizzi syndrome in8patients, variant cystic duct in34patients, accessory hepatic duct in28patients, and complicating common bile duct stones in27patients in MRCP group. The diagnostic accuracy was87.5%,94.1%,89.3%and88.9%, respectively.2. There were46patients which were performed laparoscopic cholecystectomy by tri-apporach,54patients by bi-apporach, and50patients by transumbilicar approach. All operations were successful. There were significant indifferences about operating time, blood loss, and recovery time of bowel function in the three groups. There were significant differences about postoperation hospital and pain in the three groups (P<0.05). They have not complications such as abdominal pain, jaundice, biliary leaks, bleeding and incisional hernia of abdominal wall when they are followed-up post-discharge.3. The cells could climb and adherence from the tissue after5days, and connect total after14days in primary culture. The isolated fibroblast could grow, proliferate and passage in vitro. Microfilaments were observed in the cell plasm under TEM which was identified as fibroblast. The cell was positive in vimentin by immunofluorescence. The mRNA of vimentin was positive by RT-PCR.4.28differences proteins were found by differential proteomics analysis.9species up-regulated and19species in human benign bile duct scar stricture fibroblasts. Its function is associated with cell metabolism, apoptosis, etc. The differential proteomics, FKBP1A, NME1TAGLN and Moesin were analysised by Western blot and immune cell fluorescence.5. The proliferation of benign bile duct scar fibroblasts is restrained and apoptosis occurred after transfecting pcDNA3.1-TAGLN and FKBP1A-siRNA. Smad1, smad5of the fibroblast at the genetic level (P<0.05) and the expression of protein levels were increased. TAGLN at the genetic level (P<0.05) and the expression of protein levels were increased in the cells after transfecting FKBP1A-siRNA.Conclusion1. It could prevent the bile duct injury and residual common bile duct stones that the patients with LC were perfomed preoperative MRCP.2. Laparoscopic cholecystectomy can safe perform by the three approachs, however, different patients should have been performed by different approach according to practical situation. The citerion by which to choose different approach should be safe operation.3. The fibroblast of human benign biliary duct cicatrix was conveniently succeeded.4. The protein expression human benign bile duct scar stricture fibroblasts and embryonic lung fibroblasts KMB17is different. FKBP1A, NME1and TAGLN, Moesin maybe become clinical prediction molecular markers forbile duct scar stricture formation and molecular targets for the treatment of bile duct scar stricture.5. TAGLN and FKBP1A in benign bile duct scar fibroblasts play an important role. FKBP1A regulated the expression of TAGLN by the TGF-β/smads signal pathway.