Effect Of Parathyroid Hormone On The Osteogenic Potential Of Rat Bone Marrow Mesenchymal Stem Cells

Objective:1.Through isolation culture and identification of the rat bone marrow mesenchymal stem cells, to provide qualified source of cells for experiment.2.To investigate effect of the different concentrations parathyroid hormone on the osteogenic potential of rat bone marrow mesenchymal stem cells and possible mechanism of action.In order to provide the experimental basics and basis of parathyroid hormone in treatment of osteoporosis.Methods:1.By adherent culture method,the BMSCs were isolated, cultured and identified.2.Different concentrations of PTH1-34(0,10-10,10-9,10-8mol/L) applied to the BMSCs discontinuously.In 48 hours as a cycle, the first 6 hours dosing, after 42 hours without medicine.Using the MTT method detect the proliferation of the bone marrow mesenchymal stem cells.3.Different concentrations of PTH1-34(0,10-10,10-9,10-8mol/L) applied to the BMSCs discontinuously.In 48 hours as a cycle, the first 6 hours dosing, after 42 h without medicine.Using the PNPP method detect the activity of alkaline phosphatase(ALP),using the ria method detect the content of osteocalcin(BGP) and the real-time quantitative rt-pcr detect the expression of Bunx2 m RNA.4.Data are presented as x±s.analysis of experimental data, all statistical analysis is performed with windows SPSS 22.0. The experiment is determined by One-way ANOVE.The different between random two groups are determined by LSD-t.Differences are considered significant at a value of P<0.05.Results:1.Under the inverted phase contrast microscope, primary cell culture showed a spindle after 3 days to stick wall,5 days later,the distribution of colony was distributed.10-14 days cells covered the bottle wall.Third generation BMSCs growth curve results show that the first 1-2 days incubation period,3-5 days for the logarithmic growth phase,after entering the plateau, most in the G1/G0 phase of the cell cycle determination.The flow cytometry instrument is used to detect the P3 generation of BMSCs surface antigen CD29,CD44 expression positive and CD45 negative expression.BMSCs differentiate into induced osteoblast,we can see the calcium nodules after the alizarin red staining,BMSCs differentiate into induced the fat cells,we can see the lipid droplets after the oil red O staining.2.The different concentrations of PTH1-34(0,10-10,10-9,10-8mol/L)applied to the BMSCs discontinuously,the proliferation was no obvious change,there is no significant difference among all groups(P>0.05).3.The different concentrations of PTH1-34(0,10-10,10-9,10-8mol/L)applied to the BMSCs discontinuously,the ALP activity,BGP content and Runx2 m RNA expression were increased compared with blank control group,of which 10-8mol/L PTH1-34 group was the highest,and in a concentration dependent manner,the blank control group and the experimental groups,between the experimental groups, the difference was statistically significant(P<0.05).Conclusions:1.Bone marrow mesenchymal stem cells can be successfully isolated and purified in vitro,and have the ability of self-renewal and multi differentiation.2.PTH1-34 can promote the osteogenic potential of rat bone marrow mesenchymal stem cells.3.PTH1-34 may be through raising the expression of Bunx2 m RNA,then promote the osteogenic potential of rat bone marrow mesenchymal stem cells.