The Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells Into Parathyroid Cells In Vitro

OBJECTIVE:By co-culture,in vitro culture of bone marrow mesenchymal stem cells(BMSCs),so that BMSCs differentiate into parathyroid cells orientation.BMSCs applied for the clinical treatment of hypoparathyroidism lay the theoretical and experimental basis.METHODS:(1)SD Rat parathyroid cell isolation,culture Select weighing about 150 g ± 20 g healthy male SD rats,whichever parathyroid,rinsed,cut into pieces,after digestion,seeded in culture flasks with RPMI 1640 culture medium(containing 15% fetal bovine serum)culture.(2)Preparation of SD rat parathyroid cell conditioned medium Select the third generation,the fusion rate of 80% of SD rat parathyroid cells,after washed,change 12 medium(containing 15% FBS),cell culture medium was collected every day.After the culture supernatant was centrifuged through 0.22μm microporous membrane filtration,frozen spare.Before use by 1 /: 2(v / v)ratio with12 medium(containing 15% FBS)and mixed.(3)SD rat BMSCs isolated,cultured and identified Select 1week-old healthy male SD rats,femur and tibia sterile.We used Whole bone marrow adherent culture screening method to obtain SD rat BMSCs.Take 4th generation SD rats BMSCs,join CD44,CD90,CD29,CD11 b complement antibody was detected by flow cytometry,SD rats BMSCs were identified.(4)The SD rat MSCs differentiation,identification SD rat BMSCs cultured rat parathyroid cells were co-cultured conditions,provided the experimental group and the control group.Cells were collected at 7d,14 d,21d and 28 d after by WB(Western blot),RT-PCR method to detect parathyroid cell surface markers(PTH,CASR,GCM2)expression.RESULTS:(1)were observed to form niform SD rat parathyroid cells.(2)SD rat BMSCs can be stably passaged and good uniformity.Flow cytometry third generation of SD rat BMSCs purity over 95%.(3)SD rat parathyroid cell-conditioned medium co-cultured SD rat BMSCs,differentiate into parathyroid cells,WB and RT-PCR to detect the expression of parathyroid cell surface markers,and its expression with the extension of co-culture time and increased.CONCLUSION:This experiment proved successful SD rat parathyroid cell conditioned medium and co-cultured SD rat BMSCs can be induced BMSCs to parathyroid cells.