The Experimental Study Of Bone Marrow Side Population Cells Proliferation And Differentiation Into Hepatocytes In Vitro

Objective1. To explore the possibility of BMSP amplification in vitro.2.To explore the role and mechanism of HGF in BMSP transdifferentiation into hepatocyte.Methods1. Isolated bone marrow cells from rat femur,tibia and fibula were obtained to isolateBMSP by FACS sorting.Culture BMSP with low sugar DMEM+10%FBS medium. CellsABCG-2gene expression levels was detected by RT-PCR.2. Culture BMSP with HGF and detecte expression levels of ALB, AFP at3,7,14,21daysby RT-PCR, Western Blot and other methods.Results1. About2-10×108bone marrow cells in suspension were obtained from femur, tibia,and fibula of SD rat, BMSP were obtained from this cell suspension by cell sorting.2. Side population cells in culture grew adherently after about6hour.Cells morphologywas spindle shape.With prolonged incubation time, the cell growth curve showed increasewith the most number of cells in the seventh days,and then entering a plateau. The cellsfrom seventh days were subcultured1:2.Cells grew to the tenth generation and remainstrong proliferation ability.3. Induced differentiation experiments showed a certain concentration of HGF hassignificantly promoted BMSP transdifferentiation into hepatocytes. RT-PCR analysisshowed that3day cultured of BMSP was not expression AFP and ALB mRNA in thecontrol group and the HGF factor group.AFP mRNA was expressed at the seveth dayculture whereas ALB mRNA was not expressed.AFP and ALB mRNA were not expressedin the control group. AFP mRNA expression decreased on the fourteenth day.ALB mRNAwas expressed on the fourteenth day.The control group AFP and ALB mRNA were not expressed. ALB mRNA was expressed at the seveth day after cultured,AFP mRNA wasnot expressed,the control group AFP and ALB mRNA were not expressed. There is astatistically significant difference between factor group and control group, P＜0.05.Conclusion1. BMSP were obtained to isolate by FACS sorting,they can be amplified and maintainedstem cell characteristics in vitro within a period of time,and the cultured BMSP still havestrong self-renew and proliferation ability.2. BMSP can gradually differentiate into hepatocytes under the influence of HGF.