The Variation Of Klf4 And Nanog Expression And Promoter Methylation During Inducing Differentiation Of Rat Bone Marrow CD90~+Lin~-Stem Cells To Hepatic Cells In Vitro

1.Background and ObjectiveBone marrow mesenchymal stem cells belong to a kind of adult stem cell which have capacities for self-renewal and pluripotency.It has been proved both in animals and clinical trials that under given conditions bone marrow mesenchymal stem cells can differentiate into mature hepatic cells.Pan Runhua,the researcher of our group,has found that the expression of Oct4 and frequency of promoter methylation of Oct4 vary according to the differentiation of rat bone marrow CD90+Lin-Cells to Hepatic Cells in vitro.However,Oct4,Klf4 and Nanog should be geared to a group of pluripotent genes which play a significant role in maintaining pluripotency of stem cells.Based on the achievement of Pan Runhua,the purpose of this study is to observing the variation of Klf4 and Nanog expression and promoter methylation during inducing differentiation of rat bone marrow CD90+Lin-cells to hepatic cells in vitro,in order to discovering its epigenetics mechanism more.2.Materials and methods2.1 Regents and instrumentsHigh sugar DMEM medium(America,Hyclone),EpiTect Bisulfite Kit(Germany,Qiagen),0.25%trypsin-EDTA(America,Gibco),Gel Extraction Kit(America,Omega),Recombinant human hepatocyte growth cytokines(America,Peprotech),Australian premium fetal bovine serum(Israel,Biolnd),TaKaRa EpiTaq HS(Japan,Takara),Penicillin-Streptomycin(America,Gibco),Biospin DNA extraction kit(China,Bori-tech),RNasin(America,Promega),RT-PCR kit(Germany,DBI),dexamethasone(America,Gibco),RNA extraction kit(Japan,Takara),Reverse Transcription kit(Germany,DBI),Ultraviolet Spectrophotometer UV-1206(Japan,SHIMODZU),Gel scanning system DF-23B(Britain,UVP),Stratagene Mx3000P Real time PCR instrument(America,Agilent),ABI9700 PCR instrument(America,ABI).2.2 Experimental cellsThe bone marrow mesenchymal stem cells,CD90+Lin-surface antigen cells,were extracted from vista male rats by immunomagnetic activated cell sorting.2.3 Experimental methods2.3.1 Resuscitation,culture and inducement of cellsThe CD90+Lin-bone marrow mesenchymal stem cells that are resuscitated are cultivated in the culture solution-thehigh sugar DMEM medium,fetal bovine serum and penicillin-streptomycin.The solution are changed every 3 days until the cells amount to 80%,they become sub-culture cells by using 0.25%trypsin-EDTA.According to the Pan Ruihua's study,the third generation of cells are induced with thehigh sugar DMEM medium,fetal bovine serum,penicillin-streptomycin,25?g/L recombinant human hepatocyte growth cytokines and 0.1nmol/L dexamethasone.The medium are replaced every 3 days until to 14 days.The cell profiles are recorded by microscope.2.3.2 Gene expression detection by quantitative real time PCR reactionAccording to specification of RNA extraction kit(Trizol-Chloroform-Isopropanol),the cultivated cells from 0-day,7-day and 14-day are extracted for their whole RNA.Using 1?g whole RNA as template,the first strand of cDNA is accomplished by PCR instrument according to specification of RT-PCR kit.Finally,the results are calculated by relative quantitative method.2.3.3 Klf4 and Nanog promoter methylation detectionAccording to specification of DNA extraction kit,the cultivated cells from 0-day,7-day and 14-day are extracted for their whole DNA.In accordance with specification of EpiTect bisulfite kit,the DNA are modified with bisulfite.Finally,the PCR products which are conducted to PCR reactions after modified with bisulfite are analyzed by agar gel electrophoresis.2.3.4 Data analysisStatistical analysis software SPSS version 20.0 is used to calculate results.The measurement data are showed as x±s.The expression of Alb,Nanog mRNA and Klf4 mRNA are calculated by one-way ANOVA for their differences,by LSD testing for their intercomparison.The frequencies of promoter methylation are calculated by ?2 testing.P<0.05 means different statistical significantly.3.ResultsDuring inducing differentiation of rat bone marrow CD90+Lin-cells to hepatic cells in vitro,the relative expression of Alb mRNA were increased gradually,with significant differences among the groups(day 0,7 and 14)(p=0.001).During day 0 to day 7 of Rat bone marrow CD90+Lin-cells differentiation to hepatic cells in vitro,it was obvious that the relative expression of Klf4 and Nanog mRNA and the methylation frequency of the first CpG sites in Nanog promoter decreased(p<0.001),while the methylation frequency of the first and sceond CpG sites in Klf4 promoter and second CpG sites in Nanog promoter were increased(P<0.001).However,the variation of methylation frequency of the third CpG sites in Klf4 promoter was no significance(p=0.247).During day 7 to day 14,it was evident that the relative expression of Klf4(p<0.001)and Nanog mRNA(p=0.045)and the methylation frequency of the first CpG sites in Klf4 promoter increased(p=0.007),while the methylation frequency of the second and third CpG sites in Klf4 promoter and second CpG sites in Nanog promoter decreased(p<0.001).However,the variation of methylation frequency of the first CpG sites in Nanog promoter was no significance(p=0.109).4.ConclusionsIt revealed that during inducing differentiation of rat bone marrow CD90(+)Lin(-)cells to hepatic cells in vitro,the variation of KIf4 and Nanog Promoter Methylation had influences not only on the expression of Klf4 and Nanog but also other differentiation-related genes.These might be the key mechanism to regulate differentiation ofRBMMSCs to Hepatic Cells in vitro.