The Effect Of PRMT5on Osteogenic Differentiation Of Bone Mesenchymal Stem Cells

ObjectiveTo observe the protein arginine methyl transferase5(PRMT5) changes duringthe osteoblast differentiation of bone marrow mesenchymal stem cells (BMSCs) invitro. To detect PRMT5expression from gene and protein level during thisdifferentiation, it will provide an experimental basis for the further study on theeffect of epigenetics to BMSCs osteoblast differentiation.Methods1. BMSCs were isolated and cultured from SD rats through whole bone marrowadherent culture. Cell growth was observed under an inverted microscope, MTTmethod was used to portray the growth curve and detect the ability of cellproliferation. Identify BMSCs according to morphology、flow cytometer (FCM)and induced-differentiation potentiality.2. Detect PRMT5changes during the osteoblast differentiation of BMSCs. TheBMSCs were cultured in6well plates, cells were divided into experiment groupand control group, experiment group were cultured by osteogenic differentiationmedium, control group were cultured by10%FBS-DMEM. The real timefluorescence quantitative polymerase chain reaction (qPCR) was used to detectthe expression of PRMT5mRNA when cultured3days,6days,12days and24days,draw trend lines and compare the differences between two groups.3. To detect PRMT5expression from protein level during osteogenic differentiation,the methods to deal with BMSCs were the same as mentioned above. Westernblot technique was used to detect the expression of PRMT5protein when cultured3days,6days,12days and24days, compare the differences between two groups.Result1. BMSCs grew well with strong proliferative ability, by looking under themicroscope BMSCs grew in whirl manner. growth curve were accord with thetypical characteristics of BMSCs. Surface antigens were normal expressedaccording to the FCM, BMSCs expressed CD29and CD44at99.93%and 99.46%respectively, while expressed CD31and CD45both at0.01%. After21days of osteogenic induction, alizarin red staining verified the formation ofmineralized nodules, so BMSCs were identified.2. Agarose gel electrophoresis was used to analyse the amplification products ofPCR, the results showed that the PCR products were target genes. qPCR resultsshowed that the expression of PRMT5mRNA decreased during osteogenicinduction in experiment group, while there were no obvious changes in thecontrol group.3. The gel imaging analysis system was used to detect the protein bands on PVDFmembranes after the antibody were incubated, the photos showed that theexpression of PRMT5protein decreased during osteogenic induction inexperiment group, while there were no obvious changes in the control group.Conclusion1. The SD rat BMSCs can be successfully isolated and cultured by whole bonemarrow adherent culture.2. The expression of PRMT5in BMSCs decreased during osteogenic differentiation,it may play a role in inhibiting the osteogenic differentiation.