The Research Progress Of Stem Cells Differentiated Into Bile Duct Cells

Background An outstanding issue concerning biliary surgery is the difficulty todeal with bile duct injury and bile duct stricture. As tissue engineering rapidly advanced,a substitute made by the means of tissue engineering might be an outlet to solve theproblem.However, the artificial substitute stems from sufficient origin cells in which bileduct epithelial cells (BDECs) may be indispensable.BDECs were relatively easy to gain,but were difficult to culture and fail to subculture in vitro, which made its limited use increating a substituted bile duct. Thus, alternatives for BDECs such as steam cells wereincreasely concerned. Stem cells could be induced to either self-renew or differentiate inenvironments. So far, the research of introducing stem cells into hepatocytes is more andmore, in which process signs of BDECs could also be found. Many study also found thatonce stem cells was induced to primary hepatic cells to the liver cells, which could bedifferentiated to BDECs with the activation of the TGF pathway. Meanwhile, the fastchanging status of researches in biomaterials made it possible to create a tissueengineering bile duct as well.Of course, there were many research found that rhesus bonemarrow mesenchymal stem cells or SD rat bone marrow mesenchymal stem cells couldinduced to differentiate into BDECs, but that was minority, and the technology was notmature,transformation efficiency were low, and a long time shuld be taken. So we hopedto design a new induced scheme with more convenient, simple and relatively highconversion efficiency to provide enough seed cells for the construction of tissueengineering bile duct. Objective To investigate the feasibility of inducing rBMMSCs into BDECs viasimulating the embryonic processes.At the same time, through the study to compare thetransformation efficiency of rBMMSCs after induction by the scheme added VPA.Methods The rBMMSCs were isolated and sublimated in vitro by using the wholebone marrow adherence screening method. At the same time, the morphological changesof rBMMSCs were observed and photographed under inverted microscope. The growthactivity of the third generation of high purity rBMMSCs were tested by MTT assay.Surface immune antigens of rBMMSCs were detected by flow cytometry.3weeks afterthe induced liquid of the osteogenic and adipogenic respectively added, thedifferentiation ability identified by special staining. Then the rBMMSCs wererandomly divided into3groups due to the different induction methods: control group,cytokines group (XGF) and cytokines＆sodium valproate group (XGF+VPA)Morphological changes were observed under the inverted microscope, gene expressionchanges were detected by Real Times PCR, and related proteins were detected byimmunofluorescence and Western blot.Results The growth curve of3rd subcultured rBMMSCs was presented as a“reverse Z”. Mineralization nodules and lipid droplets were found in rBMMSCs afterbeing osteogenic and adipogenic inducted for3weeks.The result of cell surface antigentested by flow cytometry suggest that CD29,CD90positive,CD45negative.Afterdifferentiation, the morphology of rBMMSCs were gradually changed from a shape oflong shuttle into polygon.The expression of related bile duct genes of rBMMSCsdisplayed a rising trend, and the bile duct gene expression of the rBMMSCs activated byXGF+VPA increased the most.21days later, the protein of CK19,CK7,AFP and GGTwere detected by immunofluorescence both in XGF group and XGF+VPA groupcells.While cells in control group only expressed GGT fluorescence, and the amount offluorescence cells was significantly less than the other two groups.rBMMSCs stimulatedby induced liquid in XGF group also expressed CK19, CK7,AFP and GGT,and thenumber of cells expressing various types of protein were significantly less than group XGF+VPA;(Cells in XGF group expressed CK19, CK7,AFP and GGT, but the numberof cells expressing those proteins were significantly less than XGF+VPA group.Inducedcells in XGF group and XGF+VPA group were detected the expression of CK7proteinby the Western blot, while control group no CK7protein expression was found.And theamount of CK7protein expression in XGF group was significantly less than that inXGF+VPA group by comparing densitometry units of the protein bands.The results ofexpression of CK19protein were similar with CK7.All of the three groups had anincreased GGT protein expression, while the expression of cells in control group wassignificantly less than the other two groups. The results of Western blot were in line withimmunofluorescence results.)Conclusion By simulating the embryonic processes, rBMMSCs might bedifferentiated into bile duct cells, and the conversion efficiency could be improved byVPA.