The Study On Folate Induction Of The Bone Marrow Stromal Cells In Vitro And In Vivo

The study on Folate induction of the bone marrow stromal cells in vitro and in vivoObjective:The aim of our experiment is to study on the influence of differentiation induced by Folate on the bone marrow stomal cells into nerve cells and the cell proliferation and differentiation after Bone Marrow Stromal cells transplantation to treat cerebral ischemia rats, meanwhile, investigate the mechanism of transplantation. It mainly consisted of two aspects:â‘ To study on the influence of differentiation induced by Folate on the Human bone marrow stomal cells (HBMSCs) into nerve cells and explore the secretion mechanism of HBMSCs;â‘¡To observe the effect of HBMSCs after induced by Folate on the proliferation and differentiation of endogenous neural precursor cells and the nerve function in rats after cerebral ischemia and reperfusion by tail vein injection, evaluate the therapeutic value of supernatant from HBMSCs, and explore the effect mechanism of transplantation of HBMSCs after cerebral infarction in vivo.Methods:HBMSCs harvested from adult normal human marrow, then were passaged and purified in culture medium. HBMSCs were passaged five times before experiment, then purified HBMSCs were pre-induced by 1mM BME, then induced by BHA,2%DMSO,and three different dosage of Folate(4mg/L,40 mg/L,400mg/L). Using the immunocytochemical method detected the expression of the NSE and the GFAP and the MTT method observed the influence of proliferation of HBMSCs at different time points. Sixteen Wistar rats made into models of middle cerebral artery occlusion (MCAO) and reperfusion were divided into four groups including sham-operated group, ischemia control group, low-concentration supernatant group and high-concentration supernatant group. Different of HBMSCs were injected by vein BrdU that labeled the proliferating neural precursor cells were injected intraperitoneally in rats models after cerebral ischemia and reperfusion for 24 hours. In the end, the positive cells labeled by BrdU, BrdU+NSE (Neuron specific enolase) and BrdU+GFAP (Glial fibrillary acidic protein) were counted by immunohistochemistrical method at the 7th day after ischemia and reperfusion, the nerve function were assessed by NSS among ischemia control group, low-concentration supernatant group and high-concentration supernatant group at three time points(1d,4d,7d).Results:After 3h induced by Folate, the HBMSCs' cell body became round, strong light refraction, thin protuberance and the protuberance gradually increased with the increase time and connected and crisscrossed with each other. After 8h the cell immunochemistry showed, in the control group the positive cells, mainly GFAP positive, were about 70 percent, meanwhile, in the middle and high dose of Folate the positive cells, mainly NSE, were about 90 percent. The differences between the control and the low dose group and the middle and high dose group were significant (P<0.01). After HBMSCs induced by Folate of 40 mg/L treatment, the number of positive cells labeled by BrdU, BrdU+NSE and BrdU+GFAP in the ependyma/subventricular zone and hippocampus become increased at the 7th day after ischemia and reperfusion. At same time, compared with the ischemia control group and low-concentration group, the number of three kinds of positive cells in high-concentration supernatant group increased significantly in above zones (P<0.05).Conclusion:â‘ The different doses of Folate can promote the HBMSCs proliferated and differentiated into nerve cells, especially the middle dose of 40 mg/L.â‘¡The HBMSCs after induced by Folate can promote the proliferation and differentiation of endogenous neural precursor cells after cerebral ischemia.