TO901317 Promotes GF To Induce Differentiation Of Human Bone Marrow Mesenchymal Cells Into Dopamine Neurons

Objective:1.To explore the concentration and time of addition of liver X receptors(LXRs)agonist TO901317 promotes GF to induce the differentiation of human bone marrow derived mesenchymal stem cells(hBMSCs)into dopaminergic(DA)neurons and the best period to induce differentiation.2.To observe the function of DA neurons induced differentiation of hBMSCs by TO901317 and GF in vitro.3.Preliminary study on the mechanism of TO901317 promoting the differentiation of hBMSCs into DA neurons with GF.4.To observe the therapeutic effect of differentiated DA neurons on Parkinson’s disease(PD)rats.Method:1.Determine the concentration of TO901317,the addition time and period to induce hBMSCs differentiation into DA neurons with GF.(1)Determine the concentration of TO901317.According to the concentrations of added TO901317(0.125,0.25,0.5,1,2μM),h MBSCs were divided into five groups.Growth factors(GF)were added to each group to induce hBMSCs differentiation.Twelve days after induced differentiation of cultured hBMSCs,the expressions of Tuj1 and Tyrosine hydroxylase(TH)was detected by immunofluorescence.Calculate the TH positive cell rate.(2)Determine the time to join TO901317.After determining the concentration of TO901317,on the basis of GF treatment to hBMSCs,the concentration of TO901317 was added on the first day,third day and ninth day with GF to culture cells 12 days,respectively.Tuj1 and TH were detected by immunofluorescence.Calculate the TH positive cell rate and confirm the time to add TO901317 to GF.(3)Determine the period of TO901317 to induce hBMSCs differentiation into DA neurons.After determining the best concentration and adding time of TO901317,the period of treating hBMSCs with TO901317 combined with GF was three days,six days,nine days and twelve days,respectively,and then immunofluorescence was used to detect Tuj1 and TH.Calculate the rate of TH positive cells and determine the time course of TO901317 to induce hBMSCs differentiation.2.The function of induced-cells was tested in vitro.After determining the best induction and differentiation scheme of TO901317 combined with GF treatment of hBMSCs,the cells were divided into Control group,GF group and LXR + GF group.Dopamine release was detected by ELISA.The expressions of neun and nestin were detected by immunofluorescence.The expression of TH was detected by immunofluorescence and western blot.3.Preliminary study on the mechanism of TO901317 to influence the differentiation of hBMSCs.Immunofluorescence was used to detect the expressions of LXRα and LXRβ in each group.Real-time polymerase chain reaction(q PCR)was used to detect the expression of ABCA1 m RNA.4.The effect of TO901317 combined with GF-treated cells on Parkinson’s disease induced by 6-hydroxydopamine(6-OHDA)in SD rats.The Parkinson’s disease(PD)models were established by Sprague-Dawley(SD)rats by injecting 6-OHDA into the lateral ventricle.Then SD rats were divided into control group,model group,and cell transplantation treatment group.Immunofluorescence and western blot were used to detect TH expression,and HE was used to detect cell morphological and pathological changes,and apomorphine-induced behavioral detection.Results:Our results showed that the best solution of TO901317 combined with GF induces hBMSCs to differentiate into DA neurons was that 0.5μM TO901317 added to GF to induce hBMSCs to differentiate for 6 days.Its differentiation rate can reach 90%.The experiments found that the differentiated cells in LXR+GF group had the ability to express TH and release dopamine in vitro.While the induced-cells were transplanted into the substantia nigra of PD model rats.Compared with the model group,the expression of TH in the striatum of the cell treatment group increased significantly,and the behavior of PD rats induced by apomorphine was significantly improved.After long-term use of TO901317,LXRα and LXRβ decreased significantly,and ABCA1 m RNA increased significantly.Conclusions:1.TO901317 could not affect the proliferation of hBMSCs,but it can affect the differentiation of hBMSCs.2.TO901317 promotes GF to induce the differentiation of hBMSCs into DA neurons.The method was that 0.5μM TO901317 is added with GF to induce hBMSCs to differentiate for 6 days.3.The DA neurons induced by TO901317 combined with GF have the corresponding neuronal function,which is the ability to release dopamine.4.The mechanism by which TO901317 induce hBMSCs to DA neurons may be related to the activation of the LXR-ABCA1 signaling pathway.5.The induced-cells had the potential to treat PD.