Effect Of Basic Fibroblast Growth Factor, Bovine Pituitary Extract And Forskolin On Proliferation And Dose-effect Relationship Of Olfactory Ensheathing Cells In Vitro

Part I: Isolation,Culture,Purification and Biological Characteristics of Olfactory Ensheathing Cells from the neonatal rat Olfactory Bulbs.Objcctive:To culture and purify the olfactory ensheathing cells(OECs) from the postnatal day 2 rat olfactory bulb in vitro,and to investigate the morphological changes and the immunocytochemical properties of the cultured OECs.Methods:The OECs were dissociated from the postnatal rat olfactory bulbs.The primarily cultured OECs were purified based on the differing rates of attachment of the various harvested cell types and with the treatment of cytosine arabinoside and briefly exposing to trypsin.The morphological changes of the cultured OECs were observed under a phase contrastmicroscope at different culture time.The OECs were identified by NGFRp75 immunocytochemistry.The purity of the OECs was evaluated according to the percentage of NGFRp75 immunostaining cells.Results: The OECs displayed a very characteristic morphological appearance. Most of OECs were bipolar or tripolar with long and slim processes. In the unpurified group the rapidly proliferating fibroblasts were in the majority after 7 days in culture. The OECs purification by the method based on the differing rates of attachment of the various harvested cell types with the treatment of cytosine arabinoside and briefly exposing to trypsin to dislodge the fibroblasts cells was more efficient than the others.Conclusion:The method of purification for OECs through combining the differing rates of attachment of the various harvested cell types and with the treatment of cytosine arabinoside and briefly exposing to trypsin to dislodge the fibroblasts cells is an effective for the enrichment of OECs. Partâ…¡: Effect of basic fibroblast growth factor, Bovine Pituitary Extract and Forskolin on proliferation and dose-effect relationship of olfactory ensheathing cells.Objcctive: To investigate the effect of basic fibroblast growth factor, Bovine Pituitary Extract and Forskolin on proliferation and dose-effect relationship of olfactory ensheathing cells,and providing the seed cells for transplantation olfactory ensheathing cells for spinal cord injury.Methods:The OECs were dissociated from the neonatal rat olfactory bulb. The primarily cultured OECs were purified based on the differing rates of attachment of the various harvested cell types and with the treatment of cytosine arabinoside and briefly exposing to trypsin.The OECs were identified by NGFR p75 immunocytochemistry. Basic fibroblast growth factor, Bovine Pituitary Extract and Forskolin and combination of both growth factor at different concentration respectively was appended in the culture.MTT method was used for observing their proliferation in vitro.The purity of the OECs was evaluated according to the percentage of NGFR p75 immunostaining cells and calculate the ratio of increment.Results: Bovine Pituitary Extract could facilitate OECs growth after purified in vitro,but did not increase along with concentration enhanced by BPE. bFGF could facilitate OECs growth and presented direct ratio dose-effect relationship with concentration. Forskolin alone couldnot facilitate OECs growth. The combined use of bFGF and Forskolin in a proper concentration significantly enhanced cell proliferation of olfactory ensheathing cells cultured in vitro.Conclusions: Sequent use of combined growth factors in a proper concentration would be a reasonable strategy to effectively enhance cell proliferation and this might be an ideal way in dealing with seed cell scarcity for treatment with spinal cord injury.