Oecs And Nscs Joint Experimental Study Of Transplantation In The Treatment Of Alzheimer's Disease

Objective:To investigate the effects of proliferation and differentiation after Neural Stem Cells (NSCs) and Olfactory Ensheathing Cells (OECs) Co-transplantated into the brain of Alzheimer's disease (AD) rats,and provide a theoretical basis for cells co-transplantion for the treatment of AD.Methods:1. Isolation olfactory ensheathing cells from olfactory bulb of neonatal rat(in 5-7d after birth).Then they were purified and identified.2. Isolation neural stem cells from hippocampi of neonatal rat(in 24h after birth).Then subcultured after formation primary balls of cloned neural cells,and identified them by immunocytochemistry and immunofluorescence.The NSCs were labeled with 5-bromodeoxyuridine before transplantation.3. Aβ1-42 injected into the area CA1of hippocampi with stereotaxic apparatus of rat's brain to established AD model.We apply the Morris water maze and the HE staining to identify the AD model.4. Normal saline,NSCs and NSCs+OECs were separately transplanted into the hippocampi of AD rats.After transplanted seven days,fourteen days,twenty-one days and twenty-eight days,using the Morris water maze detected the rats'learning and memory, and using the immunohistochemical staining detects the expression of BrdU,neuron specific enolase(NSE) and choline acetyltransferase(ChAT) in the slice of rat brain organization,and then analyses the transplanted cells' proliferation and differentiation.Results:1. When the olfactory ensheathing cells were cultured 4-7d,their processes become long and slim,and most of them were bipolar and tripolar.Their purity were more than 90%.The immunocytochemical staining of NGFRp75 and GFAP were positive.2. A lot of big nerve bulbs were formed when neural stem cells were primary cultured about 7d.After subcultured the immunofluorescence staining of Nestin and BrdU were positive,and the immunocytochemical staining of Nestin,NSE, GFAP and ChAT were positive.3. After making the AD model succeeds, the rats'activity reduced. Compared with the previous,the water maze escaped latency and the times of crossing the platform reduced obviously (P﹤0.01),and their learning and memory decreased significantly.HE staining showed that the cellular levels reduced,and the arrangement of the cells were disordered,and Some of nucleuses were condensation and deep staining.4. After transplantation we used the Morris water maze detected the rats'learning and memory.Starting from the fourteenth day,the average escaped latency of the neural stem cells transplantation group were lower significantly than the average escaped latency of the saline group(P﹤0.01).The average escaped latency of the OECs and NSCs co-transplantation group were lower significantly than the saline group at each time point(P﹤0.01). The average escaped latency of the OECs and NSCs co-transplantation group were lower significantly than the neural stem cells transplantation group at the twenty-first day and the twenty-eight day(P﹤0.01).So the learning and memory of the OECs and NSCs co-transplantation group improved obviously.5. After transplantation the rat brain tissue slice immunohistochemistry staining showed that the survival cells and the NSE-positive cells of the OECs and NSCs co-transplantation group were more than the neural stem cells transplantation group(P﹤0.01).The number of survival,NSE-positive cells and ChAT-positive cells of the neural stem cells transplantation group's each time point were no obvious difference. The number of survival,NSE-positive cells and ChAT-positive cells of the OECs and NSCs co-transplantation group had a obvious growth trend from 7d to 21d and it had significant statistic difference(P﹤0.01).But the growth trend become slow from 21d to 28d(P﹤0.05).Conclusions:1. Isolation neural stem cells and olfactory ensheathing cells respectively from hippocampi and olfactory bulb of neonatal rat,and then transplantation to the AD rats, olfactory ensheathing cells can promote neural stem cell survival, proliferation and differentiation of cholinergic neurons.2. Transplantation of olfactory ensheathing cells and neural stem cells can significantly improve the AD model rats'learning and memory.