Effects Of 17 ?-estradiol On Proliferation And Apoptosis Of Canine Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells(BMSCs)are a group of non-hematopoietic stem cells that exist in bone marrow cavity,which has the strong ability of self-renewal and multi-directional differentiation potential,otherwise,its advantages of convenient to achieve leading to it has been widely used in tissue injury and repair and genetic engineering field.Estradiol(E2)is one of the main steroid estrogen in vivo,and recent studies have found that the biological effects of E2 is widespread,not only participate in mediation related to reproductive function,but also involved in other processing of cells'regulation effect which has nothing to do with the reproductive function.To analyze the effects of characterization of E2 on canine BMSCs,the material of this experiment is isolated from bone marrow of 1 year old dogs by marrow puncture.And then we studied the effect of E2 on these cells by CCK-8,Annexin V-FITC/PI and RT-qPCR.The results are showed as follows:1 Isolation,cultivation and identification of canine BMSCs in vitroExperiment was designed to separate and purify the canine bone marrow mesenchymal stem cells using the whole bone marrow differential velocity adherent method and density gradient centrifugation method.The results showed that canine BMSCs were successfully cultivated by whole bone marrow differential velocity adherent and density gradient centrifugation.Compared with the former,primary cells from the latter method were more uniform after cultivation,the shorter time needed for primary cells proliferation into full confluency and with higher survival rate.The growth curves of undifferentiated cells in P3 and P8 were similar from the both methods.Immunohistochemistry(IHC)showed that the cells expressed CD 105,CD90 and CD29 respectively,while not expressed CD34 and CD31;and flow cytometry(FC)showed that the BMSCs 94.77%,89.53%and 93.87%expressed CD29,CD90 and CD105 of the 3th passage cells separated from density gradient centrifugation,which higher compared with the whole bone marrow differential velocity adherent method 89.27%,80.49%and 83.82%expressed CD29,CD90 and CD105.Otherwise,the BMSCs achieved from both methods can be induced into osteoblasts and adipocytes.These results indicated that BMSCs of dog could be isolated and cultivated by the both methods,and the density gradient centrifugation method is a better way to cultivate canine BMSCs compared to the whole bone marrow differential velocity adherent.2 Effects of 17p-estradiol on proliferation and apoptosis of canine BMSCs17?-estradiol was added to the medium at a concentration of 10-7mol/L,10-9mol/L,10-11mol/L,10-13mol/L,10-15mol/L.BMSCs viability and apoptosis rate was assessed the OD and flow cytometric scatterplot by the CCK-8 and Annexin V-FITC/PI assay on the third and fifth day after inoculating,respectively.After 3d,there is no significant effect was observed on cellular viability and apoptosis when the concertration of 17?-estradiol is 10-11mol/L,10-13mol/L,10-15mol/L,while the concertration is 10-7mol/L and 10-9mol/L,the viability of BMSC significantly inhibited(P<0.05),leading to significantly increase in cells' apoptosis(P<0.05),which showed high concertration of 17?-estradiol carried out cytotoxic effect on canine BMSCs.Training after 5 d,except the concentration 10-7 mol/L 17?-estradiol still can significantly promote the apoptosis(P<0.05),the others has no significant impact on the activity of BMSCs.RT-qPCR detection was applied to analysis the influence of 17?-estradiol on cell proliferation related genes(PCNA,CCND1 and CDKN1B),apoptosis related gene(Bcl-2,Bax,Caspase-3 and Caspase-8)and estrogen receptor-a(ER-a)mRNA expression.The results showed that,after 3d,10-11 mol/L 17?-estradiol group had a significantly higher amount of proliferation related genes PCNA and CCND1 expression compare to other groups(P<0.05),10-7 mol/L 17(3-estradiol group had a significantly higher amount of apoptosis related gene expression Bcl-2,Bax and Caspase-3 of the others(P<0.05).After 5d,17?-estradiol concentration of cells have the similar effect,without significant statistical difference.Analyze the mRNA expression of ER-a,result showed that:after 3d,10-7 mol/L 17?-estradiol can significantly inhibit its expression(P<0.05),but 10-11 mol/L promote its expression,compared with control group,the difference is verysignificant(P<0.01).In short,results show that high levels of 17?-estradiol through inhibiting the proliferation of BMSCs by related gene expression and promoting cell apoptosis related gene expression to suppress the proliferation of BMSCs.And the most obvious inhibitory effect concertration is10-7 mol/L.Suitable concentration(10-11 mol/L)of 17 ?-estradiol can promote the proliferation of BMSCs in different extent.