A Study On Expression Of Human Gene Fxyd6 And Membranous Ion Channel Proteins FXYD2 And FXYD4 In Bile Duct And Cholangiocarcinoma

Objective:1. To validate the expression difference of human gene fxyd6 in normal, benign and malignant bile duct tissues. To observe the subcellular localization of fxyd6 gene in human cholangiocarcinoma.2. To investigate expressions of homologous membranous ion channel proteins FXYD2 and FXYD 4 in murine normal extrahepatic bile duct.3. To observe expression of FXYD2 protein in paraffin sections of human cholangiocarcinoma.4. To explore the relationship between FXYD protein family and bile duct diseases as well as the function of new gene fxyd6.Methods:1. Two pairs of fxyd6 gene primers(Pl/P2 and P3/P4) were designed. The RT-PCR were performed using total RNA isolated from normal, benign and malignant bile duct tissues as templates. The expression difference between them was identified by RT-PCR , and IS-RT-PCR were applied to detect the subcellular localization in paraffin sections of human cholangiocarcinoma. 2 . Western Blotting and immunofluorescence were used to observe expressions of FXYD2 and FXYD4 proteins of murine extrahepatic normal bile duct.3. Western Blotting and immunofluorescence were used to determine different expressions of FXYD2 protein in human bile duct and cholangiocarcinoma.4. The image analysis of results with Image-Pro Plus Analysis Software was used to semiquantitatively determine the different expressions between normal and carcinoma tissues. Statistical analyses were done by paired,two-tailed Student's t test. A P value smaller than 0.05 was considered assignificant.Results: The main results of the study are as follows:1. fxyd6 gene was expressed in human normal bile duct, congenital biliary cyst and carcinoma tissues which was highly expressed in cholangiocarcinoma samples and lowly expressed in normal and benign ones.2. fxyd6 gene has a plasma subcellular localization in paraffin sections of human cholangiocarcinoma cell.3. FXYD2, 4 proteins have a unique expression in murine extrahepatic normal bile duct.4. FXYD2 protein was expressed in human cholangiocarcinoma at significantly higher levels than in normal bile duct tissues.Conclusions:1. fxyd6 gene may act as an essential component of the malignant transformation process in human cholangiocarcinoma.2. Na-K-ATPase y subunit and CHIF, each of these auxiliary subunits produce a distinct functional effect on Na,K-ATPase that is adapted to the physiological needs of the bile duct which is expressed in murine.3. FXYD2 protein may play an important part in progress of human cholangiocarcinoma inducing abnormal growth.