【Abstract】ObjectiveTo explore a rational and effective operative procedure of fresh tissue samples preservation for biliary tract carcinoma, and to establish a tissue bank with high quality, the authors had made a plan to store up the tissue samples of cholangiocarcinoma resected during operaion in deeplow temperature (cryopreservation), which is based on the clinical preponderance in the treatment of biliary tract diseases of the department of hepatobiliary surgery of General Hospital of P.L.A., and will be a good foundation for the systemic basic research of bile duct carcinoma. MethodsCases of biliary tract tumors confirmed by pathology were selected from the inpatients of Department of Hepatobiliary Surgery in General Hospital of P.L.A. from Jan. 2000 to June 2001. Fresh tissues were taken from the excised sample, which were stored in three different disinfectant tubes labelled cancer ( or tumor ) group, peritumor group and normal group. They were stored in liguid nitrogon container temporarily, and transferred into refrigerator for longtime storage as quickly as possible. The slices divided from the sample were grouped according to the dimensional space apart from the margin of tumor. Pathological diagnosis must be made on paraffin embedded samples. A part of the tissues was used to isolate total RNA by Trizol reagent for integrality judgment of it.ResultsTwentyone excised samples were stored including 4 intrahepatic bile duct carcinoma, 6 hepatic hilar cholangiocarcinoma, 7 extrahepatic bile duct carcinoma, and 4 duodenal ampulla carcinoma. The number and size of samples stored and duration of severance were not the same for reason of the difference of tumor volume, difficulty of operation and artificial jamming. The average duration was (47.60±43.87) min. The precipitated total RNA could be seen at the bottom of Eppendorf tube, of which the rate of A260/A280 was calculated about 1.6 to 1.8. It also could be seen in the 1%agarose gel electrophoresis for the obvious two bands of 28s and 18s, in which the28s band might be twofolds lighter than the 18s. Conclusion It is an important basic work for research of genes related to human disease to built a sample bank of human genetic resource. The present program for bile duct carcinoma tissue severance and storage is feasible and could supply the goodquality sample for further study. It must be reminded that the informed consent is needed and the left sample should be sufficient for postoperative pathological examination before the performance, which should be done by a fixed and experienced researcher group. Limitted to the operation of bile duct carcinoma, the time for beginning tissue severing should be in one hour after the sample excision, and samples should be divided into slices in short time to avoid decomposition of component during the following schedule.
Objective To obtain the full-length gene and functional domains of FXYD6 gene which is a cholangiocarcinoma related gene. Methods A new strategy with the integration of bioinformatics and molecular biology was used. Bioinformatical methods were used to analyze the full-length sequence, and to predict the functional domains of its protein. And the full-length sequence of FXYD6 was isolated by polymerase chain reaction from fetal hepatic, brain and spleen cDNA libraries, and then cloned in pGEM-T vector for sequence analyzing. Goldkey Sequence Analyzing Software was used to analyze the sequence of candidate domain without signal peptide.Results The full-length sequence of FXYD6 was isolated by Touch-down PCR from fetal hepatic and brain cDNA library, but was not from spleen cDNA library. The open reading frame Finder software was used in the National Center for Biotechnology Information website to find the most probable encoding regions of FXYD6 gene. And the +1 phase was selected as the template sequence, from 67 bp to 354 bp, to predict the functional domains by Goldkey Sequence Analyzing Software. The signal peptide was located from 1 amino acid (aa) to 17 aa， and the main domain was composed from 18 aa to 34 aa. The region between 35 aa and 57 aa was the transmembrane region. The FHYD peptide chain was highly conserved amino acids. Conclusion The study of full-length cDNA cloning of FXYD6 gene and its functional domains provides the basis for understanding the relationship between the structure and function of FXYD6. More work shall be performed on FXYD6 protein and its influence on the mechanism of cholangiocarcinoma.
【Abstract】Objective To investigate the anatomic structure and experimental methods of peribiliary vascular plexus(PVP) in rat’s hepatic portal bile duct.Methods To observe the morphological structure of PVP in rat’s hepatic portal bile duct after the hepatic artery was perfused with Chinese ink and transparency management, performed and to make three dimensional reconstruction of PVP’s spacial structure in MoticBuaa3Dvol software.Results The microvascular distribution and plane structure of PVP in rat’s hepatic portal bile duct could be shown clearly through Chinese ink perfusion and transparency management. The three dimensional structure of PVP could be reconstructed effectively by MoticBuaa3Dvol software, its effect was verisimilitude. Conclusion Chinese ink perfusion and vitrification is a simple and easy method in PVP’s morphologic study of rat’s hepatic portal bile duct. MoticBuaa3Dvol software is useful in microvascular study of hepatic portal bile duct.