A Study On The Effects Of Olfactory Ensheathing Cells And It Expressing NGF And BDNF On Proliferation And Differentiation Of Neural Stem Cells

The first part: Isolation, culture and identification of GFP transgenemice neural stem cells[Objective] To establish the method of culture neural stem cells (NSCs)from green fluorescent protein (GFP) transgene mice cerebral cortex.[Methods] Cells were isolated from the cerebral cortex of E12-14 andE16-18 GFP transgenic mice or ICR mice, and dissociated by mechanicaltrituration and plated at 2.5×10~8/L cells per 35 mm dishes in DMEM/F12supplemented with 1％N2, 20μg/L of bFGF. The growth of NSCs was observedevery day with inverted microscope. [Results] At 1-2 days after primaryculture, cell aggregation and the neurosphere was observed; at 5-6 daysafter primary culture, neurosphere is consisted of about ten to hundredcells and the neurosphere growth floating and arrange tight among cells,some appear cellular necrosis because of alimentary deficiency. Thenumber and diameter of neurosphere isolated from E12-14 mice were morethan those isolated from E16-18 mice (P＜0.05). There were no noticeabledifference in the number and diameter of neurosphere and growth patternof NSCs between GFP transgenic mice and ICR mice. Immunocytochemistryshowed that the Nestin positive reactant located at the cytoplast of roundshaped NSCs. [Conclusions] 1. NSCs isolated from E12-14 mice cerebralcortex posses more passage and multiplication capacity. 2. GFP has no any effect on growth of NSCs. 3. The result of immunocytochemistrydemonstrated that the cultured cells were neural stem cells.The second part: Isolation, culture and identification of mice olfactoryensheathing cells[Objective] To establish the method of culture olfactory ensheathingcells (OECs) in vitro, and to investigate the morphological features andthe immunocytochemical properties of the cultured OECs. [Methods] OECswere isolated from postnatal 1-2 days ICR mice olfactory bulbs. Theprimarily cultured OECs were with the treatment of method based on therates of attachment, arabinoside cytosine (Ara-C) and bFGF. The purityof the OECs was evaluated according to the percentage of P75NTR-immunostaining cells. The morphological changes of the OECs wereobserved under a phase contrast microscope at different culture time.[Results] The OECs display a very characteristic morphological appearance.Most of OECs are bipolar or tripolar with long and thin processes. In theunpurified group the rapidly proliferating fibroblasts were in themajority after 7 days in culture. Whereas in purified groups, the purityof OECs was estimated to be over 88.7％. The non-OECs cells consists ofastrocytes (＜3％), neurons (＜1％), oligodendrocytes (＜1％) and fibroblas.Positive staining of P75NTR and CNPase was observed in OECs byimmunocytochemistry, but GFAP and Nestin was not. [Conclusions] Thepurification is necessary for primary culture of OECs from postnatal miceolfactory bulbs. The third part: A study on the effect of olfactory ensheathing cells onthe proliferation and differentiation of neural stem cells[Objective] To research the effects of OECs on the proliferation anddifferentiation of NSCs. [Methods] OECs and NSCs were separated andcultured in vitro. The proliferation and differentiation of NSCs wasobserved by using means of co-culture and two-chamber shared medium assay.[Results] At 4 days after culture, in co-culture group and two-chambershared medium assay group, the population of NSCs was increasedsignificantly compared with control (P＜0.05), and the differentiation ofNSCs have no changes between all groups. At 7 days after culture, thenumber of NSCs in co-culture group was more than other groups (P＜0.05);In control group, the number and percentage of GFAP-immunostaining cellswere more than those at 4 days (P＜0.05); In co-curule group, GFAP andCNPase immunostaining cells were increased significantly compared withthose at 4 days and other groups at 7 days (co-culture group＞two-chambershared medium assay group＞control group, 9＜0.05); And in two-chambershared medium assay group, the number and percentage of CNPase-immunostaining cells were more than control (P＜0.05). [Conclusions] 1.OECs could stimulate NSCs to further proliferate by using means ofco-culture and two-chamber shared medium assay. 2. With prolonged culturetime, NSCs trend to differentiate into astrocytes in control group. 3.NSCs trend to differentiate into oligodendrocytes in two-chamber sharedmedium assay group. The fourth part: A study on the effects of olfactory ensheathing cellsand it expressing NGF and BDNF on proliferation and differentiation ofneural stem cells[Objective] To research the primary mechanisms of proliferation anddifferentiation of neural stem cells affected by olfactory ensheathingcells. [Methods] For investigating the mechanism of proliferation anddifferentiation ofNSCs, cytokinesand its receptors of OECs and NSCs wasanalyzed by means of RT-PCR and immunocytochemistry. Two days aftertwo-chamber shared medium assay, semi-quantitation PCR was used toresearch the relative quantitation of gene expression of those cytokinesand receptors. To studythe effects of NGF and BDNF on the proliferationand differentiation of NSCs, NGF and BDNF signaling were blocked by anNGF or BDNF antibody. [Results] The result of RT-PCR showed that OECs couldstablely express mRNA ofβ-NGF, BDNF, PDGF-B, bFGF, EGF and trkB, butnot NT-4; NSCs could stablely express mRNA ofβ-NGF, BDNF, PDGF-B, trkAand trkB, but not NT-4, bFGF and EGF. Moreover, positive staining of NGF,BDNF and P75NTR was observed in cytoplasm of OECs by immunocytochemistry.Two days after two-chamber shared medium assay, the expression of PDGF-B(P＜0.05) trkA and trkB of NSCs was up-regulated; and the expression ofNGF and BDNF Of OECs was increased. Blocking NGFor BDNF signaling withan NGF or BDNF antibody didn't effect on the proliferation of NSCs, butboth could increase population of astrocytes (Blocking BDNF signaling wasmost notable, P＜0.05), and reduced population of oligodendrocytes(Blocking NGF signaling was most notable, P＜0.05). Those results indicatedthat NGF and BDNF possibly have no effects on growth of NSCs, but possiblyhave effects on differentiation into astrocytes and oligodendrocytes.[Conclusions] 1. OECs expressed a number of cytokines in particular couldact both in a paracrine and autocrine manner. 2. NSCs could expressneurotrophic factors and its receptors. 3. Two days after two-chamber shared medium assay, OECs and NSCs could affect each other possibly bythe correlation between NGF and BDNF and its receptors trkA and trkB. 4.NGF and BDNF reduced differentiation of NSCs into astrocytes, andincreased into oligodendrocytes in co-culture and two-chamber sharedmedium assay.