Effect Of Bone Morphogenetic Proteins On Induces Mouse Liver Stem Cells Differentiating Into Mature Hepatocytes

Objective: The mouse fetal liver stem cells HP14.5were selected as seedcells and the stem cells were infected with the constructed recombinantadenovirus expressing bone morphogenetic proteins (BMP2-15)、Hepatocyte Growth Factor (HGF)、and green fluorescent protein (GFP).To explore the differential regulation of mouse fetal liver stem cells tomature hepatocytes after induced with recombinant adenovirus-mediatedmouse bone morphogenetic proteins2-15(rAdBMP2-15).Our purpose is tolay the theoretical and experimental foundation of liver stem celltransplantation in the clinical application of end-stage liver disease.Methods:1. The mouse fetal liver stem cells were infected with rAd BMP2-15、rAdHGF and rAdGFP. Then, to investigate the differential regulation ofliver stem cells, the potential BMP factors which capable of inducingmouse liver stem cells into hepatic parenchymal cells were preliminaryselected by fluorescent element enzyme gene report assay at day1,4and7 point.2. BMP9,the most significant potential inducible factor was selectedfor further research. HP14.5cells were grouped as3teams. Theexperimental group expressing BMP9; the positive control group HGF andthe negative control group GFP. HP14.5cells were infected with differentcytokines, The production albumin (ALB) were tested by fluorescentelement enzyme gene report assay at day1,4,7and10,and albumin(ALB),alpha fetoprotein (AFP) and cell keratin18(CK18) were tested byhalf quantitative PCR, Real-time PCR, cellular immune fluorescence at day7.3.The living functions of HP14.5cells were observed after inducedwith BMP9by PAS sraining、urea synthesis of the cells and ICG uptakeexperiment test at4,7and10days post infection.Results:1. BMP2、BMP9and BMP10can effectively increased Alb-Glucactivity, and BMP9is the most effective one among them.2. The expression of Alb-Gluc after1,4,7and10days induction withBMP9and HGF，Luciferase assay was gradually increased and peak at7day，and Alb-Gluc activity to stabilize at10day, compared to GFP negativecontrol group, tested by fluorescent element enzyme gene repor（tP<0.05）.3.7days later, the mRNA expression of AFP induced with BMP9、HGF was obviously decreased compared to the GFP negative control group，and the expression of Alb and CK18were increased compared to theGFP negative control group tested by RT-PCR and Real-Time PCR（P<0.05）.4. The specific marker of mature hepatocyte Alb and CK18havestrong expression after7days induction which were tested by cellularimmune fluorescence assay, and the negative result were observed withthe GFP negative control group.5. BMP9group、HGF positive group have acquired functionalcharacteristics of hepatocytes which could stored glycogen，the positiverate was the highest at10day. However, less inductive activity was foundin GFP negative control group.6. The detection of urea synthesis function demonstrated that themeasured values of urea nitrogen in cell cultural supernatants in BMP9group and HGF positive control group were increasing by the day, whilethere was not significant changes in control group.7. Analysis of ICG intake test, demonstrated that the intake of ICGwas increasing with the time of cell induction in BMP9group and HGFpositive control group, the positive rate of cells was the highest at day10.The positive rate of cells was around82%and80%in BMP9group andHGF positive control group, respectively, while the rate was around15%inGFP negative control group. Conclusion: BMP2、BMP9and BMP10, the potential differentiationinducible factors in BMPs family were selected from BMP2-15, whichcould induce mouse liver stem cells into mature hepatocytes. BMP9hasthe most effective function of inducing the differentiation of HP14.5cellsinto the mature hepatocyte-like cells, also these cells have syntheticmetabolism functional of mature hepatocytes.