Olfactory Ensheathing Cells On Neural Stem Cells And Cholinergic Neurons In Vitro Experimental Study

Objective: To investigate effects of olfactory ensheathing cells (OECs)on the proliferation and differentiation from neural stem cells (NSCs) to cholinergic neurons, and the most appropriate concent of OECs were co-cultured with NSCs in vitro. That provide a theoretical basis for cells transplantation for the treatment of AD.Methods:1. Mechanical isolation the primary NSCs from hippocampus of neonatal rats (in 24h after birth), the cells are suspended and cultured in serum free medium (containing bFGF, EGF and B27) for long-term survival in vitro. Then identified them. 2. OECs were isolated from postnatal 1-2 days rat olfactory bulbs. The primarly cultured OECs were with the treatment of mothod based on the rates of attachment, arabinoside cytosine(A ra-c) and the combination of both. Then identified them. 3. After 7 days of OECs were furified culture, centrifugation to remore FBS, re-again added into the maintenance nedium, after mechanical army to adjust respectively cells density were 1×104 /ml, 1×106/ml, 1×108 /ml .Take 1ml trickle-down a culture plate that was coated by 0.1% PLL,to collect the third-generation NSCs after centrifugation,remove the original culture medium, and re-again added into the maintenance nedium, after mechanical army to adjust respectively cells density was 5×104 /ml,then take 1ml trickle-down OECs'culture plate. NSCs and 1×104 /ml, 1×106/ml, 1×108 /ml OECs were co-culture respectively. NSCs in the control group were left intact.To research the proliferation of NSCs. We used immunocytochemical techniques to detect ChAT-positive neurons induced by different concentration of OECs.Results:1. The primery and passage cells that mechanized isolation from hippocampus of neonatal rats could divide and proliferate continuously, then become new nerve bulbs. NSCs can survive for long time in vitro. They show multipotential of differentiate into neurons and glial cells and the ability of self-renewing and proliferating. The NSCs are positive for Nestin expression.2. The OECs display a very characteristic morphological appearance. Most of OECs were bipolar or tripolar with long and silm processes. The OECs purification by the method based on the combination of rates of attachment and arabinoside cytosine(Ara-c) are more efficient than the others. Positive staining of NGFRp75 and GFAP were observed in OECs from neonatal rat olfactory bulbs by immunocytochemistry.3.①1×10 4 /ml,1×10 6 /ml, 1×108 /ml OECs were co-culture respectively with NSCs. In the 1×106/ml OECs and NSCs co-culture group, take 1ml NSCs trickle-down the OECs culture plate 24 hours later, began to some cells adherent, clone cell balls gradually expand 48 hours later, cells began to increase, after 3 days, cells increased significantly. Compared with the control group is significant(P<0.05).The cells reached a peak 7 days later, compared with the control group , 1×104 /ml OECs , 1×108 /ml OECs are significant(P<0.05). In the 1×104 /ml OECs and NSCs co-culture group, in the 1×108 /ml OECs and NSCs co-culture group, after 24 hours cells adhearde, There are cells migrate out of the clone balls 3 days later, that cells are radial form. After 5 days, the cells increased significant, compared with the control group is significant(P<0.05). At 3 days after co-culture, three different concentration OECs are to promote the proliferation of NSCs. NSCs and 1×106/ml OECs group, the population of NSCs is increased significantly, it is more than 1×104 /ml OECs group and 1×108 /ml OECs group, and proliferation is the most significant.②NSCs are induced respectively with 1×104 /ml,1×106/ml,1×108 /ml OECs . In 1×106/ml OECs group, OECs can be seen that re-adherent growth 24 hours later, NSCs re-formed nerve cell balls,OECs adhered growth under suspended nerve cell balls; the NSCs began to attached to the OECs 5 days later, clone balls gradually expand, and small protrusions grow; after 7 days, there are slender protrusions grow around NSCs, it can be seen that a large number of neuron-like cells and OECs co-growth. 1×104 /ml OECs group cells and 1×108 /ml OECs group NSCs adherent late ,and differentiation slower than 1×106/ml OECs group .OECs are induced by different concentrations.NSCs 7 days later, compared with control group, ChAT positive cells are more by immunocytochemistry ,and cells larger, protrusions longer, that show neurons have a higher maturity. ChAT positive cell rate respectively is 4.60%,5.96%,4.62%.ChAT-positive cells rate are increased in each group.In which 1×106/ml OECs group, the effect to promote the differentiation of cholinergic neurons is the most obvious, it was found OECs could promote the differentiation of NSCs into cholinergic neurons, compared with the control group was significant(P<0.05).Conclusions:1.The OECs are isolated and cultured from olfactory bulbs of postnatal rats. The method of purification for OECs through combining the rates of attachment and arabinoside cytosine(Ara-c) is an simple, economical and effective for the enrichment of OECs.2. OECs can induce NSCs proliferation and differentiation from NSCs to cholinergic neurons.