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

Objective:To study the directional differentiation of bone marrowmesenchymal stem cells (BMSCs) into parathyroid cells in vitro. Studieshave shown that bone marrow mesenchymal stem cells is an kind of cellswith easy separation, culture, expansion, and possessing multipledifferentiation potential, which can differentiate into bone cells, fat cells,cardiac cells and stem cells. It can act as the cell source of multiple organreconstruction and damage repair. Currently, there are only few literaturesabout differentiation of mesenchymal stem cells into parathyroid cells invitro. Is an important human parathyroid glands, parathyroid cells is themain function of parathyroid hormone secretion, the main function is toregulate the body and affects the metabolism of calcium and phosphoruspeople. Which can lead to inadequate secretion of hypoparathyroidism.Hypoparathyroidism, which is the most common complications afterthyroid surgery, can cause throat and diaphragm spasm, and evensuffocation death, seriously threatening the health and life human being.There is no effective treatment today, and it has been a difficulty medically. The study on the directional differentiation of bone marrowmesenchymal stem cells into parathyroid cells is of great significance inthe clinical treatment of parathyroid damage and hyperparathyroidism.Therefore, we cultured mesenchymal stem cells in vitro, induced it todifferentiate into parathyroid cells directionally, and studied thedifferentiation method, which laid a certain foundation for further clinicaltreatment of parathyroid damage.Method:1-week-old SD rats were sacrificed by cervical vertebra luxation,and placed in75%ethanol for15min. The rat femur and tibia were takenout under sterile conditions. en BMSCs was separated and purified, andcultured for passage in vitro. BMSCs were identified using flowcytometry to detect cell surface markers (CD44, CD45, CD90). BMSCswere induced to differentiate to neural cells, indicating that BMSCsobtained with this method can differentiate into cells of trans-germinallayer, with multi-directional differentiation potentials. Cut rat parathyroid,placed it in RPMI1640culture medium (containing15%fetal bovineserum) at4℃, cut into pieces, rinsed, and cultured the parathyroid withcentrifugation method as a positive control group. Then the parathyroidcell lysate was prepared. Then it was divided into two control groups. Thefirst group was added with parathyroid cell-free lysates containing100ng mL-1Activin A,100ng mL-1SHH,100μL mL-1and DMEM-F12 culture medium containing0.2%FBS and cultured. The second groupwas not added with parathyroid cell free lysates, increased the serumconcentration to2%after1day culture, further increased the serumconcentration to5%after another1day and continued to culture. Bonemarrow mesenchymal stem cells were induced to differentiate toparathyroid cells. Cells were collected at5d,12d,19d and26d,respectively. The differentiated parathyroid cells were identified bydetecting parathyroid cell surface markers (GCM2, CASR, PTH) usingmethods of immunocytochemistry, immunofluorescence, Western blot,and PCR.Results:①BMSCs with better uniformity after passage can be observedunder the microscope; BMSCs cultured in vitro were induced todifferentiate into neuron-like cells, in which GFAP, MAP2showedpositive expression with immunocytochemistry and immunofluorescencestaining;②The control group with the parathyroid cell-free lysate couldgain parathyroid cells with better differentiation; the expression ofmarkers CASR, GCM2, PTH in differentiated parathyroid cells and thatof the positive control group were made a statistical analysis (p <0.05).The differentiation effect of control group without parathyroid cell-freelysate was not obvious. Conclusion:The rat bone marrow mesenchymal stem cells were extractedsuccessfully, after culturing in vitro, Activin A, SHH, parathyroidcell-free lysates, and0.2%FBS DMEM-F12were added to inducedifferentiation of parathyroid cells to gain a better effect, laying a certainexperimental foundation to repair parathyroid damage clinically and treathypoparathyroidism.